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Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development
Abstract
Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought.
... To ask how TAp73 regulates neural precursor maintenance, we turned to a more experimentally amenable system, developing cortical radial precursors [23][24][25][26][27], which ultimately contribute to the adult SVZ stem cell pool [28]. RT-PCR ( Figure 3A) and immunostaining ( Figure 3B) showed that TAp73 was expressed in the nuclei of nestin-positive precursors of the embryonic cortical ventricular zone (Figure 3B). ...
... The E12-E13 cortical precursors from CD1 mice were cultured in 40 ng/mL FGF2 as described [5,[23][24][25][26][27] at a density of 125,000w150,000 cells/well in four-well chamber slides. For transfections, 1 hr after plating, 1 mg total DNA (J and K) Quantitative real-time PCR for Hey1, Hey2, Bmi1, and p21 mRNAs in TAp73 +/+ and TAp73 2/2 E13.5 cortex (J) or P0-P3 hippocampal neurospheres (K). ...
... Immunocytochemical analysis of cultured cells was performed as described [23][24][25][26][27]. Neurospheres were plated for immunocytochemistry by cytospins (Thermo Shandon). ...
Increasing evidence suggests that deficits in adult stem cell maintenance cause aberrant tissue repair and premature aging [1]. While the mechanisms regulating stem cell longevity are largely unknown, recent studies have implicated p53 and its family member p63. Both proteins regulate organismal aging [2-4] as well as survival and self-renewal of tissue stem cells [5-9]. Intriguingly, haploinsufficiency for a third family member, p73, causes age-related neurodegen-eration [10]. While this phenotype is at least partially due to loss of the DNp73 isoform, a potent neuronal prosurvival protein [11-16], a recent study showed that mice lacking the other p73 isoform, TAp73, have perturbations in the hippocampal dentate gyrus [17], a major neurogenic site in the adult brain. These findings, and the link between the p53 family, stem cells, and aging, suggest that TAp73 might play a previously unanticipated role in maintenance of neural stem cells. Here, we have tested this hypothesis and show that TAp73 ensures normal adult neurogenesis by promoting the long-term maintenance of neural stem cells. Moreover, we show that TAp73 does this by transcriptionally regulating the bHLH Hey2, which itself promotes neural precursor maintenance by preventing premature differentiation. Results TAp73 Is Necessary to Promote Maintenance of Postnatal Dentate Gyrus Precursors TAp73 2/2 [17] mice display an aberrant hippocampal dentate gyrus (DG), a phenotype of unknown etiology. To understand this, we characterized TAp73 expression and developmental onset of the phenotype. RT-PCR (Figure 1A) and immunostain-ing (Figure 1B) demonstrated that TAp73 is expressed in the newborn mouse hippocampus, where it is predominantly localized to nuclei of cells that coexpress Tbr2, a marker for type 2a precursors [18] (Figure 1B). To determine when the DG first became aberrant, we Nissl stained the postnatal hippocampus (Figure 1C and Figure S1A, available online); TAp73 2/2 and TAp73 +/+ hippocampi were morphologically similar at birth, started to show some differences at postnatal day 6 (P6), and by P16 the dorsal lower blade of the TAp73 2/2 DG was missing. Confirmation that the DG was similar at earlier time points was obtained by immunostaining newborn sections for nestin and prox1, markers for precursors and DG neurons, respectively (Figure S1B). Because the region that becomes aberrant postnatally is comprised of the last-born DG neurons [19], this suggests that TAp73 is necessary for postnatal neurogenesis. To test this idea, we studied ongoing neurogenesis in the adult hippo-campus [20]. Adult TAp73 2/2 and TAp73 +/+ mice were injected with BrdU, and hippocampi were analyzed immunocytochem-ically 24 hr later (Figure 1D). Quantification showed an almost 2-fold decrease in proliferating, BrdU-positive precursors in the TAp73 2/2 subgranular zone (SGZ; the location of the DG precursors) of both the lower and upper DG blades (Figures 1D and 1E). Similarly, doublecortin-positive newly born neurons (Figure 1D) were also reduced approximately 2-fold in the TAp73 2/2 DG (Figure 1F). Thus, TAp73 loss depletes adult DG precursors and decreases neurogenesis. To ask whether this phenotype reflected a cell-intrinsic precursor deficit, we cultured TAp73 +/+ and TAp73 2/2 P3 hippocampal cells in FGF2 and EGF to generate neurospheres [21]. RT-PCR demonstrated that TAp73, DNp73, and p53 mRNAs were expressed in DG neurospheres and that DNp73 and p53 mRNA levels were unaltered by loss of TAp73 (Fig-ure 1G). Immunostaining confirmed that the majority of TAp73 +/+ but not TAp73 2/2 neurosphere cells expressed nuclear TAp73 (Figure 1H). Quantitative analysis at clonal density demonstrated that TAp73 +/+ and TAp73 2/2 neonatal hippocampi contained similar numbers of neurosphere-generating precursors (Figure 1I), consistent with the lack of an in vivo phenotype at birth. However, when sequentially passaged, the TAp73 2/2 neurosphere-forming cells were progressively depleted (Figure 1J), indicating that TAp73 is required for long-term precursor maintenance. In contrast, mean neurosphere diameter (Figure 1K) and Ki67-positive proliferating cells (Figures 1L and 1M) were unchanged, suggesting that TAp73 is not necessary for proliferation of biased progenitors, which comprise the majority of cells in the spheres. Loss of TAp73 Depletes Adult SVZ Precursors and Decreases Olfactory Neurogenesis To ask whether TAp73 is required for maintenance of other adult neural precursors, we examined olfactory neurogenesis, which is ongoing for the life of the animal [20]. Adult TAp73 +/+ and TAp73 2/2 mice were injected five times with BrdU over a 12 hr period. Quantitative immunocytochemical analysis of their olfactory bulbs 30 days later demonstrated an almost 2-fold decrease in BrdU-positive newly born neurons expressing the neuron-specific protein NeuN (Figures 2A and 2B and Figure S2A) in TAp73 2/2 mice. To ask whether this was due to depletion of precursors, we generated clonal neurospheres *Correspondence: fredam@sickkids.ca (F.D.M.), dkaplan@sickkids.ca (D.R.K.) 8 These authors contributed equally to this work
... Moreover, cell-specific α, β and γ were inferred on each branch. For instance, neurotrophic tyrosine kinase receptor type 2 (Ntrk2) 31 has two major branches: the upper branch corresponds to astrocytes and OPCs, and the lower branch corresponds to dentate gyrus granule neurons and pyramidal neurons (Fig. 3b). Astrocytes and OPCs have high α and low β, resulting in high expression of unspliced Ntrk2 on the upper branch. ...
... 35), Ncald 26 and Ntrk2 (ref. 31), show increasing expression in all branches at different rates (Extended Data Fig. 4). ...
RNA velocity provides an approach for inferring cellular state transitions from single-cell RNA sequencing (scRNA-seq) data. Conventional RNA velocity models infer universal kinetics from all cells in an scRNA-seq experiment, resulting in unpredictable performance in experiments with multi-stage and/or multi-lineage transition of cell states where the assumption of the same kinetic rates for all cells no longer holds. Here we present cellDancer, a scalable deep neural network that locally infers velocity for each cell from its neighbors and then relays a series of local velocities to provide single-cell resolution inference of velocity kinetics. In the simulation benchmark, cellDancer shows robust performance in multiple kinetic regimes, high dropout ratio datasets and sparse datasets. We show that cellDancer overcomes the limitations of existing RNA velocity models in modeling erythroid maturation and hippocampus development. Moreover, cellDancer provides cell-specific predictions of transcription, splicing and degradation rates, which we identify as potential indicators of cell fate in the mouse pancreas.
... Source data 2. Related to Figure 3N. one of the major RTKs expressed in cortical precursors, regulates proliferation and differentiation into neurons by activating the MEK-ERK-C/EBP pathway (Barnabé-Heider and Miller, 2003;Bartkowska et al., 2007;Ménard et al., 2002). Thus, we examined the activity of TrkB-MEK-ERK-C/EBP pathway by western blot with antibodies for phosphorylated activated MEK1 and C/EBPβ at E15.5 of Dhcr7 -/cortices ( Figure 5J-K). ...
... Interestingly, 6-oxo-cholestan-3β,5α-diol (OCDO), a cholesterol-derived oxysterol that is structurally similar to DHCEO, is shown to bind and activate glucocorticoid receptor (GR) (Voisin et al., 2017;Figure 8-figure supplement 1A). Additionally, GR activation has been shown to activate TrkB (Jeanneteau et al., 2008), which leads to further activation of the RTK-mediated MEK-ERK pathway (Barnabé-Heider and Miller, 2003;Bartkowska et al., 2007;Ménard et al., 2002). TrkB and RTK-mediated MEK-ERK pathway are necessary for neurogenesis during embryonic cortical development. ...
Defective 3b-hydroxysterol-D ⁷ -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis from murine or human cortical neural precursors, and depletion of the cortical precursor pool, both in vitro and in vivo . We found that a major oxysterol, 3b,5a-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7 . These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.
... Neurotrophin-mediated Trk signaling plays an essential and cell-autonomous role in regulating neuronal proliferation and differentiation, axon outgrowth, and synapse development and plasticity in the nervous system (Ammendrup-Johnsen et al., 2015). Ntrk2 and Ntrk3 are expressed in the cortical ventricular/ subventricular zones (VZ/SVZ) during the period of cortical neurogenesis (Fukumitsu et al., 2006), inhibition of TrkB/C caused a delay in the generation of neurons, and ultimately perturbed the postnatal localization of the cortical neurons (Bartkowska et al., 2007). Ntrk3 in brain not only as a catalytic isoform but also as a noncatalytic isoform, and there were two Ntrk3 receptor isoforms exist in the CNS, the full length Ntrk3 (Ntrk3-FL) and the truncated Ntrk3 isoform (Ntrk3-TR) lacking the kinase domain (Barbacid, 1994). ...
... NT3− /− and Ntrk3− /− mice exhibit profound deficits in peripheral and central nervous system development (Klein et al., 1994;Ma et al., 2002). Pharmaceutical inhibition of Ntrk3 signaling in mice caused a decrease in embryonic precursor cell proliferation (Bartkowska et al., 2007). The role of Ntrk3 in neurodevelopment is depended on the relative levels of NT-3/Ntrk3 signaling (Joo et al., 2014). ...
The precise control of proliferation and differentiation of neural progenitors is crucial for the development of the central nervous system. Fused in sarcoma (FUS) is an RNA-binding protein pathogenetically linked to Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) disease, yet the function of FUS on neurodevelopment is remained to be defined. Here we report a pivotal role of FUS in regulating the human cortical brain and spinal cord development via the human iPSCs-derived organoids. We found that depletion of FUS via CRISPR/CAS9 leads to an enhancement of neural proliferation and differentiation in cortical brain-organoids, but intriguingly an impairment of these phenotypes in spinal cord-organoids. In addition, FUS binds to the mRNA of a Trk tyrosine kinase receptor of neurotrophin-3 (Ntrk3) and regulates the expression of the different isoforms of Ntrk3 in a tissue-specific manner. Finally, alleviated Ntrk3 level via shRNA rescued the effects of FUS-knockout on the development of the brain- and spinal cord-organoids, suggesting that Ntrk3 is involved in FUS-regulated organoids developmental changes. Our findings uncovered the role of FUS in the neurodevelopment of the human CNS.
... BDNF signalling through trkB in glial cells induces proliferation and differentiation of astrocytes and neuroblasts that migrate to the lesion region to contribute to tissue repair and help form glial scar tissue. the recovery role of neu-roblasts also involves the secretion of BDNF to the microenvironment of the damaged tissue [23,24], wherein neuronal activation of trkB by BDNF promotes survival via the ca 2+ balance against glutamate excitotoxicity, reducing inflammation and apoptosis and increasing Bcl-2 expression [25][26][27][28]. the anti-inflammatory role of BDNF thus prevails in the final phase of recovery. ...
Purpose
Stroke is the second most common cause of mortality worldwide and the third most common cause of motor disability. From another perspective, brain-derived neurotrophic factor (BDNF) is a metabolite that plays several neuroprotective roles. While cardiometabolic diseases are the leading cause of stroke, BDNF represents a target factor in the prevention and/or recovery from stroke. Aim: In this narrative review, I have summarised the clinical evidence of BDNF participation in the recovery from a stroke and discuss the potential use of exercise as a rehabilitation tool.
Methods
Multiple combinations of the terms ‘brain infarction’, ‘cerebral infarction’, ‘hemorrhagic stroke’, ‘ischemic stroke’, ‘embolic stroke’ or ‘thrombotic stroke’ AND ‘BDNF’ or ‘pro-BDNF’ were used in PubMed databases. Studies not available in the English language or addressing animal experiments were excluded.
Results and prospects
Seventeen clinical studies published up to June 30th of 2023 were included in this review. Changes in the patients circulating BDNF levels represent their capability of recovery from the stroke outcomes. A subtle, but consistent, negative influence of the presence of the 66Met-allele in BDNF on motor and cognitive competencies is seen in patients recovering from a stroke throughout the studies – an effect that is not reportedly detectable in other neuropathological conditions. Exercise exerts a positive modulation on BDNF levels that accompanies improvements in stroke recovery and might exert a preventive role against the severity of stroke outcomes.
... The p38 pathway becomes active when it encounters diverse external cues, including proin ammatory cytokines, growth factors, and environmental stressors. Within the cell signaling network, the p38 pathway holds a pivotal role in regulating in ammation by facilitating the generation of in ammatory agents like cytokines and chemokines [70,71]. Notably, inhibiting the p38 pathway presents a therapeutic approach for addressing in ammatory diseases. ...
Oxidative stress is a key driver of neurodegeneration, and the Nrf2/HO-1 pathway serves as a primary defense mechanism against this stress. Melittin, derived from bee venom, has shown promise in mitigating cognitive decline in mild cognitive impairment. This study for the first time investigates whether melittin can revive the compromised Nrf2/HO-1 pathway in neurodegenerative animals’ brains and whether this pathway is the initial target of melittin's action. In a scopolamine-induced neurodegeneration model in mice, melittin administration led to its significant accumulation in the hippocampus, indicating its direct interaction with neural tissues. Comprehensive analysis revealed that melittin's earliest effect was the restoration of the Nrf2/HO-1 system, reinforcing its role as an antioxidant defense against oxidative stress. In vitro studies with mouse hippocampus HT22 cells showed that melittin triggered the translocation of Nrf2 from the cytosol to the nucleus. Notably, the most significant inhibition of melittin's protective effects was observed with an HO-1 inhibitor, suggesting a close association between melittin's action and the HO-1 pathway. In summary, this study demonstrates for the first time melittin's ability to upregulate the compromised Nrf2/HO-1 pathway in neurodegenerative animals, with evidence pointing to its primary action through this pathway. The direct effect of melittin on the Keap-1/Nrf2/HO-1 pathway were further solidified with invitro evidences. These findings enhance our understanding of melittin's neuroprotective mechanisms and its potential as a therapeutic agent for neurodegenerative disorders, warranting further clinical exploration. This evidence strongly corroborates the prevailing trend of harnessing the activation of cellular antioxidation as a potent therapeutic strategy against neurodegeneration.
... BDNF is one of the most widely distributed and extensively studied neurotrophic factors in the mammalian brain. It is critical for neurogenesis and the development and functional maintenance of the nervous system [15][16][17][18][19][20][21], and BDNF levels are found to be reduced in patients or animal models with neurodegenerative diseases such as AD and HD [22][23][24][25]. ...
The ankyrin repeat-rich membrane spanning (ARMS), a transmembrane neuronal scaffold protein, plays a fundamental role in neuronal physiology, including neuronal development, polarity, differentiation, survival and angiogenesis, through interactions with diverse partners. Previous studies have shown that the ARMS negatively regulates brain-derived neurotrophic factor (BDNF) secretion by interacting with Synaptotagmin-4 (Syt4), thereby affecting neurogenesis and the development and function of the nervous system. However, the molecular mechanisms of the ARMS/Syt4 complex assembly remain unclear. Here, we confirmed that the ARMS directly interacts with Syt4 through its N-terminal ankyrin repeats 1–8. Unexpectedly, both the C2A and C2B domains of Syt4 are necessary for binding with the ARMS. We then combined the predicted complex structural models from AlphaFold2 with systematic biochemical analyses using point mutagenesis to underline the molecular basis of ARMS/Syt4 complex formation and to identify two conserved residues, E15 and W72, of the ARMS, as essential residues mediating the assembly of the complex. Furthermore, we showed that ARMS proteins are unable to interact with Syt1 or Syt3, indicating that the interaction between ARMS and Syt4 is specific. Taken together, the findings from this study provide biochemical details on the interaction between the ARMS and Syt4, thereby offering a biochemical basis for the further understanding of the potential mechanisms and functional implications of the ARMS/Syt4 complex formation, especially with regard to the modulation of BDNF secretion and associated neuropathies.
... Inhibitor of the TGF-β pathway (Tojo et al., 2005), involved in cell growth, differentiation, development, immune response, tissue homeostasis and the determination of the neural fate BDNF Neurotrophic factor. Promotes the differentiation of NPCs into specific neuronal lineages and neuronal maturation (Bartkowska et al., 2007;Colucci-D'amato et al., 2020) bFGF Promotor of growth, survival, and regulation of neurogenesis BHA Antioxidant used to prevent cell damage due to oxidative stress during the induction process (Delanghe et al., 2021) cAMP Intracellular signaling molecule. Induces neuronal differentiation, maturation, and survival (Hansen et al., 2000;Li et al., 2000;Lepski et al., 2013) CHIR99021 Inhibitor of GSK3β, whose inactivation leads to the activation of the canonical Wnt signaling pathway, which plays a key role in neural development and differentiation (Huang et al., 2017) EGF Promotor of cell expansion, survival and differentiation FGF10 Promotor of neurogenesis initiation; regulator of cell proliferation (Sahara and O'Leary, 2009;Guillemot and Zimmer, 2011) Forskolin Activator of adenylate cyclase (Alasbahi and Melzig, 2012), promoting gene expression modulation and neuronal differentiation (Thompson et al., 2019) GDNF Neurotrophic factor inducing NPCs differentiation into specific neuronal lineages, damaged neurons regeneration, and neuronal maturation (Cortés et al., 2017) GO6983 ...
Stem cells have potential applications in the field of neurological diseases, as they allow for the development of new biological models. These models can improve our understanding of the underlying pathologies and facilitate the screening of new therapeutics in the context of precision medicine. Stem cells have also been applied in clinical tests to repair tissues and improve functional recovery. Nevertheless, although promising, commonly used stem cells display some limitations that curb the scope of their applications, such as the difficulty of obtention. In that regard, urine-derived cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, their obtaining can be challenging due to the low yield and complexity of the multi-phased and typically expensive differentiation protocols. As an alternative, urine-derived stem cells (UDSCs), included within the population of urine-derived cells, present a mesenchymal-like phenotype and have shown promising properties for similar purposes. Importantly, UDSCs have been differentiated into neuronal-like cells, auspicious for disease modeling, while overcoming some of the shortcomings presented by other stem cells for these purposes. Thus, this review assesses the current state and future perspectives regarding the potential of UDSCs in the ambit of neurological diseases, both for disease modeling and therapeutic applications.
... In the brain, BDNF is expressed by microglia (Parkhurst et al., 2013), glutamatergic neurons (Andreska et al., 2014) and glial cells, such as astrocytes isolated from the hippocampus and cortex but not from the striatum (Clarke et al., 2018). In addition to the nervous system, BDNF is also detected in several non-neuronal tissues, such as megakaryocytes neurons (Bartkowska et al., 2007). It has been reported that the BDNF-TrkB signaling pathway is implicated in adult neurogenesis in the hippocampus and that BDNF-TrkB signaling has distinct roles in the subventricular zone and dentate gyrus of the hippocampus (Vilar & Mira, 2016). ...
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which is a pulmonary manifestation of a systemic reactive inflammatory syndrome, is a serious disease with high mortality, and sepsis is an important risk factor in the development of ALI. Brain‐derived neurotrophic factor (BDNF) is a member of the nerve growth factor family. It plays an essential role in the regulation of the modification of synaptic efficacy and brain metabolic activity and enhances neuronal survival. However, the role and underlying mechanism of BDNF in sepsis‐induced ALI remain unclear. Here, we sought to observe the expression of BDNF in the lung tissues of mice. C57BL/6J mice were divided randomly into two groups: saline (n = 4) and lipopolysaccharide (LPS) (n = 4). We found that BDNF expression was elevated in the lung tissues of septic mice. Furthermore, we found that BDNF colocalized with aquaporin 5, a marker for type I alveolar epithelial cells, by immunofluorescence staining. In addition, we also found that tropomyosin‐related kinase B, the specific receptor of BDNF, colocalized with surfactant protein C, a marker for type II alveolar epithelial cells, by immunofluorescence staining. Finally, the present study indicated that BDNF may alleviate excessive LPS‐induced autophagy in alveolar epithelial cells. Overall, we hypothesize that BDNF expression increases in the lung tissues of septic mice as a compensatory mechanism to ameliorate sepsis‐induced ALI by inhibiting excessive alveolar epithelial cell autophagy.
... Taking advantage of a knock-out mouse model, we further demonstrated that NTRK3 receptor is crucial to ensure the colonization of the pituitary gland by gonadotrope cells during embryogenesis. NTRK3 deletion also led to a reduced number of gonadotrope cells that might be explained by a defect in migration, proliferation, or differentiation, in agreement with NTRK3 known functions [37][38][39][40]. ...
Pituitary gonadotrope cells are essential for the endocrine regulation of reproduction in vertebrates. These cells emerge early during embryogenesis, colonize the pituitary glands and organize in tridimensional networks, which are believed to be crucial to ensure proper regulation of fertility. However, the molecular mechanisms regulating the organization of gonadotrope cell population during embryogenesis remain poorly understood. In this work, we characterized the target genes of NEUROD1 and NEUROD4 transcription factors in the immature gonadotrope αT3-1 cell model by in silico functional genomic analyses. We demonstrated that NEUROD1/4 regulate genes belonging to the focal adhesion pathway. Using CRISPR/Cas9 knock-out approaches, we established a double NEUROD1/4 knock-out αT3-1 cell model and demonstrated that NEUROD1/4 regulate cell adhesion and cell motility. We then characterized, by immuno-fluorescence, focal adhesion number and signaling in the context of NEUROD1/4 insufficiency. We demonstrated that NEUROD1/4 knock-out leads to an increase in the number of focal adhesions associated with signaling abnormalities implicating the c-Src kinase. We further showed that the neurotrophin tyrosine kinase receptor 3 NTRK3, a target of NEUROD1/4, interacts physically with c-Src. Furthermore, using motility rescue experiments and time-lapse video microscopy, we demonstrated that NTRK3 is a major regulator of gonadotrope cell motility. Finally, using a Ntrk3 knock-out mouse model, we showed that NTRK3 regulates gonadotrope cells positioning in the developing pituitary, in vivo. Altogether our study demonstrates that the Neurod1/4-Ntrk3-cSrc pathway is a major actor of gonadotrope cell mobility, and thus provides new insights in the regulation of gonadotrope cell organization within the pituitary gland.
... Thus, TRKA is activated by the nerve growth factor, TRKB is activated by the neurotrophin-4 and the brain derived growth factor, while TRKC is activated by the neurotrophin-3 [2,4,5]. The activated TRK enzymes trigger several signal transduction pathways (such as PI3K-AKT and RAS-MAPK) that regulate the cell cycle, cell growth, apoptosis, survival and differentiation of the nervous system cells and neurons [4,[6][7][8][9]. ...
The tropomyosin receptor tyrosine kinases (TRKs) control the cell proliferation mainly in the nervous system and are encoded by NTRK genes. Fusion and mutation of NTRK genes were detected in various types of cancers. Many small molecules TRK inhibitors have been discovered during the last two decades and some of them have entered clinical trials. Moreover, two of these inhibitors; larotrectinib and entrectinib; were approved by FDA for the treatment of TRK-fusion positive solid tumors. However, mutation of TRK enzymes resulted in resistance to both drugs. Therefore, next generation TRK inhibitors were discovered to overcome the acquired drug resistance. Additionally, the off-target and on-target adverse effects on the brain initiated the need for selective TRK subtype inhibitors. Indeed, some molecules were recently reported as selective TRKA or TRKC inhibitors with minimal CNS side effects. The current review highlighted the efforts done during the last three years in the design and discovery of novel TRK inhibitors.
... BDNF is the most studied and well-characterized neurotrophic factor in the CNS. In mammals, BDNF plays a role in neuronal activity, including proper growth, development, synaptic plasticity [184], neuronal differentiation [185], and neurotransmission [186], through Trk B and the low-affinity p75NTR [187]. In addition, BDNF protein is detectable in several non-neural tissues, such as endothelial cells [188] and vascular smooth muscle cells [189]. ...
It is well established that innervation is one of the updated hallmarks of cancer and that psychological stress promotes the initiation and progression of cancer. The breast tumor environment includes not only fibroblasts, adipocytes, endothelial cells, and lymphocytes but also neurons, which is increasingly discovered important in breast cancer progression. Peripheral nerves, especially sympathetic, parasympathetic, and sensory nerves, have been reported to play important but different roles in breast cancer. However, their roles in the breast cancer progression and treatment are still controversial. In addition, the brain is one of the favorite sites of breast cancer metastasis. In this review, we first summarize the innervation of breast cancer and its mechanism in regulating cancer growth and metastasis. Next, we summarize the neural-related molecular markers in breast cancer diagnosis and treatment. In addition, we review drugs and emerging technologies used to block the interactions between nerves and breast cancer. Finally, we discuss future research directions in this field. In conclusion, the further research in breast cancer and its interactions with innervated neurons or neurotransmitters is promising in the clinical management of breast cancer.
... Au niveau central, ces facteurs neurotrophiques peuvent être responsables de remaniements neuronaux impactant la fonction cognitive. Le BDNF est en effet impliqué dans le couplage structurefonction des neurones en favorisant la synaptogénèse et la stabilisation des synapses formées(Hu, Nikolakopoulou, et Cohen-Cory 2005), participe aux mécanismes d'amélioration de la cognition suite à l'enrichissement de l'environnement(Novkovic, Mittmann, et Manahan-Vaughan 2015) et participe à la neurogénèse(Bartkowska et al. 2007). Le NGF quant à lui est plus impliqué dans la croissance et le maintien de la fonction neuronale, en particulier dans la fonction et la survie des neurones cholinergiques du prosencéphale basal(Cuello, Pentz, et Hall 2019). ...
Les agressions périphériques, c'est à dire ne touchant pas primairement le système nerveux central (SNC) par définition, peuvent être à l'origine de séquelles neurologiques secondaires invalidantes dont la prévention, la reconnaissance et la prise en charge demeurent encore aujourd'hui très incomplètes. Une défaillance d'organe comme celle rencontrée lors d'un sepsis, mais également une blessure périphérique ou une chirurgie, peuvent induire des séquelles neurologiques parfois sévères. La recherche actuelle met en évidence de plus en plus de similitudes dans les troubles neurologiques secondaires issus d'agressions périphériques variées, tant sur le plan épidémiologique, clinique, mais aussi physiopathologique. Mieux préciser ces caractéristiques communes permettrait d'améliorer la récupération des patients par une prise en charge adaptée avec la mise en place d'une thérapeutique préventive. Notre objectif était de décrypter une partie de ces phénomènes dans deux modèles complémentaires. Dans un modèle murin, nous avons précisé l'influence de la destruction musculaire dans l'apparition des troubles cognitifs. La cryolésion du muscle Tibialis anterior (blessure physique avec répercussion modérée sur l'état général et régénération musculaire complète en moins d'un mois) chez la souris CX3CR1-GFP/+ (dont les cellules microgliales expriment la GFP), nous a permis de mettre en évidence une réactivité microgliale précoce dans l'ensemble de l'encéphale (notamment l'hippocampe qui est impliquée dans de nombreux processus de mémorisation). Par une étude comportementale réalisée après régénération musculaire, nous avons montré une altération de la mémoire à court et long terme. Cette dernière était également caractérisée par une perturbation des niveaux de neurotrophines dans l'encéphale, à savoir une augmentation du brain derived neurotrophic factor (BDNF) et une diminution du nerve growth factor (NGF). Dans un second temps, nous nous sommes intéressés à l'impact du sepsis sur le SNC, chez le cheval. Le recrutement d'une cohorte clinique dans un centre spécialisé nous a permis de mettre en évidence de nombreuses similitudes cliniques et biocliniques entre la pathologie équine et celle de l'homme. Nos résultats suggèrent fortement une susceptibilité et une réactivité au sepsis comparables. Nous avons également observé une importante réactivité morphologique de la microglie dans de nombreuses régions de l'encéphale lors de la phase aigüe du sepsis. En conclusion, nos résultats montrent une réactivité centrale de la microglie dans deux types d'agressions périphériques (trauma musculaire et sepsis) chez deux espèces différentes (souris et cheval). La modulation de cet acteur cellulaire commun pourrait être une piste thérapeutique préventive concernant l'apparition des troubles cognitifs secondaires à ce type d'agressions. Les deux modèles que nous avons utilisés présentent un avantage scientifique et technique certain (i) dans la mise en évidence d'une voie de modulation impliquant la microglie (modèle murin mutant), et (ii) l'évaluation de l'efficacité d'une éventuelle thérapeutique médicamenteuse avant la mise en place d'essais cliniques chez l'homme (pathologie spontanée équine).
... Similarly, transgenic mice that either completely lack TrkB (Ntrk2 -/-) (Rohrer et al., 1999) or harbour a TrkB with disabled kinase domain (trkBTK -/-) (Klein et al., 1993) develop to birth but display severe abnormalities including reduced body weight, behavioural anomalies, problems to right themselves and irregular breathing, finally leading to death during the early postnatal period (Klein et al., 1993;Rohrer et al., 1999). Moreover, TrkB-deficient mice have severe neuronal deficiencies in the CNS and the PNS (Klein et al., 1993;Lush et al., 2005;Perez-Pinera et al., 2008), In addition, TrkB deficiency is associated with impaired development of the striatum , decreased embryonic precursor cell proliferation (Bartkowska et al., 2007), and disturbed structure and function of NMJs (Kulakowski et al., 2011). Interestingly, primary cultured motoneurons from trkBTK -/-E13 mouse embryos that were cultured on laminin-221/211 show marked differentiation defects including smaller growth cones and disturbed axon elongation, accompanied by reduced b-actin mRNA and impaired ...
Spinal muscular atrophy (SMA) is a genetic pediatric condition that affects lower motoneurons leading to their degeneration and muscle weakness. It is caused by homozygous loss or mutations in the Survival Motor Neuron 1 (SMN1) gene; however, the pathomechanism leading to motoneuron degeneration is not fully resolved. Cultured embryonic SMA motoneurons display axon elongation and differentiation defects accompanied by collapsed growth cones with a disturbed actin cytoskeleton. Intriguingly, motoneurons cultured from mice deficient for the Tropomyosin-kinase receptor B (TrkB), exhibit similar pathological features. Thus, the question arises whether SMA motoneurons suffer from defective Brain-derived neurotrophic factor (BDNF)/TrkB signaling and whether there is a link to the disturbed actin cytoskeleton. In the recent years, modifier genes such as Plastin 3 (PLS3) were shown to beneficially interfere with SMA pathology. Nevertheless, the mechanism of how the actin-bundler PLS3 counteracts SMN deficiency is not well understood. In this study, we investigated TrkB localization and its activation in cultured SMA motoneurons and neuromuscular junctions (NMJs). While TrkB levels are only mildly affected locally in axon terminals, BDNF-mediated TrkB phosphorylation was massively disturbed. The activity-dependent TrkB translocation to the cell surface and its activation via BDNF were shown to be Pls3-dependent processes, that can be abolished by knockdown of Pls3. In contrast, PLS3 overexpression in SMA motoneurons rescued the defects on morphological and functional level. In particular, the relocation of TrkB to the cell surface after BDNF-induced internalization is disturbed in SMA, which is based on an actin-dependent TrkB translocation defect from intracellular stores. Lastly, AAV9-mediated PLS3 overexpression in vivo in neonatal SMA mice provided further evidence for the capacity of PLS3 to modulate actin dynamics necessary for accurate BDNF/TrkB signaling. In conclusion, we provide a novel role for PLS3 in mediating proper alignment of transmembrane proteins as prerequisite for their appropriate functioning. Hence, PLS3 is required for a key process indispensable for the development and function of motoneurons even beyond the context of SMA.
... One source of weakness in this study, which could have affected the measurements, was the usage of ELISA kits, which could not distinguish between proBDNF and mature BDNF. Mature BDNF-TrkB signaling is the process which regulates dendritic growth, cell survival, proliferation, differentiation, and enhancement of synaptic plasticity [76], while proBDNF has opposing effects [7]. Therefore, further research using ELISA assays that can differentiate between pro and mature BDNF is required to establish a greater degree of accuracy on this matter. ...
Brain-derived neurotrophic factor (BDNF) is a key modulator of neuroplasticity and has an important role in determining the susceptibility to severe psychiatric disorder with a significant neurodevelopmental component such as major psychoses. Indeed, a potential association between BDNF serum levels and schizophrenia (SCZ) and schizoaffective disorder (SAD) has been tested in diverse studies and a considerable amount of them found reduced BDNF levels in these disorders. Here, we aimed at testing the association of BDNF serum levels with several demographic, clinical, and psychometric measures in 105 patients with SCZ and SAD, assessing the moderating effect of genetic variants within the BDNF gene. We also verified whether peripheral BDNF levels differed between patients with SCZ and SAD. Our findings revealed that BDNF serum levels are significantly lower in patients affected by SCZ and SAD presenting more severe depressive symptomatology. This finding awaits replication in future independent studies and points to BDNF as a possible prognostic indicator in major psychoses.
... Tyrosine receptor kinase B (TrkB; Ntrk2), canonically bound by brain derived neurotrophic factor (BDNF), has been linked to embryonic cortical neuroblast migration (Bartkowska et al., 2007;Behar et al., 1997) (Fig. 3). BDNF stimulates calcium transients and increases cortical neuroblast migration, which can be blocked by application of Trk inhibitors or calcium chelators (Behar et al., 1997). ...
Calcium influx can be stimulated by various intra- and extracellular signals to set coordinated gene expression programs into motion. As such, the precise regulation of intracellular calcium represents a nexus between environmental cues and intrinsic genetic programs. Mounting genetic evidence points to a role for the deregulation of intracellular calcium signaling in neuropsychiatric disorders of developmental origin. These findings have prompted renewed enthusiasm for understanding the roles of calcium during normal and dysfunctional prenatal development. In this Review, we describe the fundamental mechanisms through which calcium is spatiotemporally regulated and directs early neurodevelopmental events. We also discuss unanswered questions about intracellular calcium regulation during the emergence of neurodevelopmental disease, and provide evidence that disruption of cell-specific calcium homeostasis and/or redeployment of developmental calcium signaling mechanisms may contribute to adult neurological disorders. We propose that understanding the normal developmental events that build the nervous system will rely on gaining insights into cell type-specific calcium signaling mechanisms. Such an understanding will enable therapeutic strategies targeting calcium-dependent mechanisms to mitigate disease.
... Moreover, cell-specific , , and were inferred on each branch. For instance, neurotrophic tyrosine kinase receptor type 2 (Ntrk2) 26 has two major branches: the upper branch corresponds to astrocytes and OPCs, and the lower branch corresponds to dentate gyrus granule neurons and pyramidal neurons (Fig. 3B). Astrocytes and OPCs have high and low , resulting in high expression of unspliced Ntrk2 on the upper branch. ...
RNA velocity has provided a promising approach for inferring cellular state transition from single-cell RNA sequencing (scRNA-seq) data. Conventional RNA velocity models infer universal kinetics from all cells in a scRNA-seq experiment, resulting in unpredictable performance in the experiments with multi-stage and/or -lineage transition of cell states. Here, we present cellDancer, a scalable Deep Neural Network (DNN) framework, to locally infer velocity for each cell from its neighbors on gene space and then relay cell-dependent velocities of all cells. We showed that cellDancer is efficient to overcome the fundamental limitation of existing RNA velocity models in multi-stage transition during gastrulation erythroid maturation and the multi-lineage differentiation in hippocampus development. Moreover, cellDancer provides the cell-specific prediction of transcription, splicing, and degradation rates which illuminates mechanisms of transcriptome regulation.
... These results demonstrate the importance of this neurotrophin for a baseline level of neurogenesis. Several researchers confirm these findings and also demonstrate that in mice with TrkB deletion, the differentiation and proliferation of NSCs is attenuated, thus concluding the existence of a functional relationship between BDNF that is essential for TrkB for neurogenesis [119,122]. ...
Neurogenesis is a biological process characterized by new neurons formation from stem cells. For decades, it was believed that neurons only multiplied during development and in the postnatal period but the discovery of neural stem cells (NSCs) in mature brain promoted a revolution in neuroscience field. In mammals, neurogenesis consists of migration, differentiation, maturation, as well as functional integration of newborn cells into the pre-existing neuronal circuit. Actually, NSC density drops significantly after the first stages of development, however in specific places in the brain, called neurogenic niches, some of these cells retain their ability to generate new neurons and glial cells in adulthood. The subgranular (SGZ), and the subventricular zones (SVZ) are examples of regions where the neurogenesis process occurs in the mature brain. There, the potential of NSCs to produce new neurons has been explored by new advanced methodologies and in neuroscience for the treatment of brain damage and/or degeneration. Based on that, this review highlights endogenous factors and drugs capable of stimulating neurogenesis, as well as the perspectives for the use of NSCs for neurological and neurodegenerative diseases.
Graphical abstract
... VEGF can regulate axonal growth, neuronal survival, and neovascularization (Greenberg and Jin, 2005). BDNF is a neurotrophin that is widely expressed in the central nervous system and plays a key role in memory, the differentiation and survival of neurons and synaptic plasticity (Bartkowska et al., 2007;Balkaya and Cho, 2019). Low-intensity pulsed ultrasound stimulation (LIPUS) has been proven to have a neuroprotective effect on brain injury (Bretsztajn and Gedroyc, 2018). ...
Ischemic stroke is a serious medical condition that is caused by cerebral vascular occlusion and leads to neurological dysfunction. After stroke, patients suffer from long-term sensory, motor and cognitive impairment. Non-invasive neuromodulation technology has been widely studied in the field of stroke rehabilitation. Transcranial ultrasound stimulation (TUS), as a safe and non-invasive technique with deep penetration ability and a tiny focus, is an emerging technology. It can produce mechanical and thermal effects by delivering sound waves to brain tissue that can induce the production of neurotrophic factors (NFs) in the brain, and reduce cell apoptosis and the inflammatory response. TUS, which involves application of an acoustic wave, can also dissolve blood clots and be used to deliver therapeutic drugs to the ischemic region. TUS has great potential in the treatment of ischemic stroke. Future advancements in imaging and parameter optimization will improve the safety and efficacy of this technology in the treatment of ischemic stroke.
... As our E12.0 scRNA-seq data successfully revealed genes enriched in different oral epithelial appendages, it provides a useful platform to uncover novel regulators of epithelial morphogenesis. As a proof of principle, we focused on the developing tooth and its newly characterized marker Ntrk2, which encodes the neurotrophic receptor tyrosine kinase 2. NTRK2 is a receptor for brain-derived neurotrophic factor (BDNF) and neurotrophin 5 (NTF5), and its signaling activation regulates proliferation and differentiation in other contexts (Bartkowska et al., 2007;Li et al., 2008). Because cell proliferation is important for early tooth morphogenesis (Li et al., 2016a), we tested whether signaling via NTRK2 promotes tooth growth. ...
During craniofacial development, the oral epithelium begins as a morphologically homogeneous tissue that gives rise to locally complex structures, including the teeth, salivary glands, and taste buds. How the epithelium is initially patterned and specified to generate diverse cell types remains largely unknown. To elucidate the genetic programs that direct the formation of distinct oral epithelial populations, we mapped the transcriptional landscape of embryonic day (E) 12 mouse mandibular epithelia at single cell resolution. Our analysis identified key transcription factors and gene regulatory networks that define different epithelial cell types. By examining the spatiotemporal patterning process along the oral-aboral axis, our results inform a model where the dental field is progressively confined to its position by the formation of the aboral epithelium anteriorly and the non-dental oral epithelium posteriorly. Using our data, we also identified Ntrk2 as a proliferation driver in the forming incisor, contributing to its invagination. Together, our results provide a detailed transcriptional atlas of the embryonic mandibular epithelium and unveil new genetic markers and regulators that are present during the specification of various oral epithelial structures.
... NRG1 is involved in diverse functions such as proliferation, differentiation, and apoptosis of glial and neuronal cells (Liu et al., 2005;Lu et al., 2021). The NTRK2 gene encodes a member of the neurotrophic tyrosine receptor kinase (NTRK) family and signaling through this kinase (which is a receptor for BDNF and neurotrophin-4) leads to cell differentiation (Bartkowska et al., 2007). PLP1 encodes a transmembrane proteolipid protein vital for oligodendrocyte development and axonal survival and is used to show presence of oligodendrocyte precursor cells (Venkatesh et al., 2015). ...
Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1×10⁻⁸ – 3×10⁻³ M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1×10⁻⁷ - 1×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14 to 28 days of differentiation. IC50 for cell death of AA and GA was 5.2×10-3 M and 5.8×10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10⁻⁷ M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by βIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.
... Another influential growth factor BDNF, has been found to produce an effect glioblastoma. Neuronal expression and secretion of BDNF influences diverse processes ranging from proliferation, cell survival, neural circuit development, and long term potentiation (Bartkowska, Paquin, Gauthier, Kaplan, & Miller, 2007;Hong, McCord, & Greenberg, 2008). In glioblastoma tumour cells, BDNF mutations promote proliferation and survival of tumour cells through TrkB mediated signaling (Lawn et al., 2015). ...
Medulloblastoma (MB) is the most prevalent, metastatic pediatric brain tumor and has the highest mortality rate for childhood cancers. In Canada alone, the 25-50 children that are diagnosed with MB annually account for 15-25% of all pediatric brain tumours. This research focuses on the SHH MB subgroup which is prevalent in 30% of MB tumors. The cells of origin for SHH MB are cerebellar granule neuron precursors (cGNPs) in the external granule cell layer (EGL) on the surface of the cerebellum. During brain development, cGNPs migrate tangentially and actively proliferate in response to SHH, then migrate radially towards the internal granule cell layer (IGL) to differentiate. Our lab's previous work demonstrated that a glutamate gradient, originating from the differentiated cells of the IGL may be diffusing towards undifferentiated cGNPs to initiate membrane depolarization, cell cycle exit, and differentiation. We hypothesized that neural activity impairs oncogenic transformation of cerebral granule neuron precursors and is tumor suppressive in SHH MB. We began by characterising the effects of neurotransmitters such as Glutamate and Gaba on cGNPs from P7 mice. The addition of Glutamate and GABA to cGNP cultures demonstrated an anti-proliferative effect despite the presence of a Shh agonist (SAG). Following this, glutamate production in P7 cerebellar cultures in vitro was measured to delineate the effect of glutamate on differential neural activity at different times of postnatal mouse cerebellum development. The results displayed a significant increase in glutamate production by P15 and P21 while simultaneously displaying glutamate's presence in the post-natal brain as early as P5. Lastly, we measured spontaneous neural activity during development using RNA in-situ hybridization with c-fos and math1/atoh1 probes to understand whether that the in vivo glutamate gradient translates into increased neural activity. Early expression of the c-fos marker in undifferentiated cells demonstrated the presence of depolarising events in the EGL before synapses are formed in the IGL. Co-expression of math1 and c-fos in the int-EGL 3 indicates the expression and manifestation of neural activity is an early event in differentiation in granule cells and the quantification of fluorescence using ImageJ compounded these results. Results from this thesis indicate that a glutamate gradient drives spontaneous neural activity in the cerebellum to counterbalance Shh induced proliferation and drive cell-cycle exit and differentiation.
... It has been suggested that neuronal factors, such as neurotrophins, are involved not only with the balance of lipid and glucose levels but are also for the regulation of energy expenditure and cardiovascular homeostasis [12,13]. Among these, the brain-derived neurotrophic factor (BDNF) and its catalytic receptor tropomyosin-related kinase B receptor (TrkB), which are widely expressed throughout the brain, seem to play a crucial role on synaptic transmission and plasticity events during both the developmental and adulthood stages [14,15]. Altered expression levels of neurotrophins and Trk receptors resulted to be modified by the ingestion of flavonoids. ...
Current evidence supports the beneficial role of phytoestrogens in metabolic diseases, but their influences on spontaneous motor and anxiety behaviors plus neuroprotective effects have still not been completely elucidated. With the present study, neuro-behavioral activities were correlated to daidzein (DZ)-dependent expression changes of a high affinity catalytic receptor for several neurotrophins, and namely tropomyosin-related kinase B receptor (TrkB) in the cerebellar cortex of high-fat diet (HFD) hamsters (Mesocricetus auratus). Indeed, these changes appear to be tightly linked to altered plasma lipid profiles as shown by reduced low-density lipoproteins plus total cholesterol levels in DZ-treated obesity hamsters accounting for increased spontaneous locomotor together with diminished anxiety activities in novel cage (NCT) and light/dark box (LDT) tests. For this latter case, the anxiolytic-like hamsters spent more time in the light compartment, which was retained the aversive area of the LDT box. As for the evaluation of the neurotrophin receptor site, significantly elevated TrkB levels were also detected, for the first time, in the cerebellum of obese hamsters treated with DZ. In this condition, such a treatment widely led to an overall improvement of HFD-induced neurodegeneration damages, above all in the Purkinje and granular layers of the cerebellum. In this context, the notably active TrkB signaling events occurring in a DZ-dependent manner may turn out to be a key neuroprotective element capable of restoring normal emotional and spontaneously linked locomotor behaviors regulated by cerebellar cortical areas especially in obesity-related conditions.
... BDNF concentrations in the amniotic fluid influence its levels in the fetus [56][57][58][59][60][61]. Thus, fetal brain development is a strictly regulated process, as evidenced by pathological changes occurring with the rapid increase of BDNF in the fetal brain [62,63]. For example, injection of the protein into the brain ventricles of the mouse embryo in vitro leads to premature neurogenesis and abnormal cell proliferation [62]. ...
The purpose of this review is to summarize the current knowledge regarding the reciprocal associations between brain-derived neurotrophic factor (BDNF) and immune-inflammatory pathways and how these links may explain the involvement of this neurotrophin in the immune pathophysiology of mood disorders and schizophrenia. Toward this end, we delineated the protein–protein interaction (PPI) network centered around BDNF and searched PubMed, Scopus, Google Scholar, and Science Direct for papers dealing with the involvement of BDNF in the major psychosis, neurodevelopment, neuronal functions, and immune-inflammatory and related pathways. The PPI network was built based on the significant interactions of BDNF with neurotrophic (NTRK2, NTF4, and NGFR), immune (cytokines, STAT3, TRAF6), and cell–cell junction (CTNNB, CDH1) DEPs (differentially expressed proteins). Enrichment analysis shows that the most significant terms associated with this PPI network are the tyrosine kinase receptor (TRKR) and Src homology region two domain-containing phosphatase-2 (SHP2) pathways, tyrosine kinase receptor signaling pathways, positive regulation of kinase and transferase activity, cytokine signaling, and negative regulation of the immune response. The participation of BDNF in the immune response and its interactions with neuroprotective and cell–cell adhesion DEPs is probably a conserved regulatory process which protects against the many detrimental effects of immune activation and hyperinflammation including neurotoxicity. Lowered BDNF levels in mood disorders and schizophrenia (a) are associated with disruptions in neurotrophic signaling and activated immune-inflammatory pathways leading to neurotoxicity and (b) may interact with the reduced expression of other DEPs (CTNNB1, CDH1, or DISC1) leading to multiple aberrations in synapse and axonal functions.
... As astrocytes are an important source of BDNF, the decreased BDNF in IL-1β group may reflect that the function of astrocytes is inhibited in the inflammatory environment to a certain extent. It is well known that BDNF plays a critical role in the survival of neurons as well as the growth and differentiation of new neurons and synapses by binding to TrkB, while the binding to p75 receptors leads to the activation of NF-kB receptors and signal cell apoptosis [41,42]. Thus, in the present study, the changes of BDNF and its receptors in IL-1β group may result in cell death via apoptosis. ...
Background:
Activated microglia can trigger pro-inflammatory cytokine releases and neuroinflammation, which may inhibit astrocytes to produce neurotrophins and anti-inflammatory factors. Both eventually lead to neuron apoptosis or death. Furthermore, effective antidepressant or anti-dementia treatments can reduce pro-inflammatory cytokines, while enhance interleukin (IL)-10 production. However, the underline mechanism by which IL-10 modulates glial cell function, hence improves cognitive impairment or depression-like behavior is unknown. This study evaluated whether and how IL-10 attenuated chronic IL-1β administration-induced behavioral changes and the possible involved mechanisms.
Methods:
Rats received intracerebroventricular injection of IL-1β and/or IL-10 for 14 days. Then animal memory and depression-like behavior, pro-inflammatory cytokines, glial activities, expression of brain-derived neurotrophic factor (BDNF), Trk B, p75, and apoptosis-related genes were studied.
Results:
Compared to controls, significantly increased latent time and swimming distance in the Morris-water-maze, decreased sucrose consumption, and decreased locomotor and center zone entries in the open-field were found in rats administrated with IL-1β. These changes were associated with the reduction of GFAP expression, and concentrations of BDNF and anti-inflammatory cytokine IL-10, but the increase in the expressions of CD11b, TrkB, p75, and Caspase-3, the ratio of Bax/Bcl-2, and the concentrations of IL-1β, tumor necrosis factor-α, and IL-6. IL-10 treatment markedly attenuated IL-1β-induced above changes, except for the expressions of neurotrophin receptors.
Conclusion:
IL-10-improved behavioral changes may be through suppressing microglia activity and inflammation, while restoring astrocyte function and BDNF expression.
... During nervous system development, the release and interaction of BDNF with TrkB receptors initiates several pathway cascades that regulate local transcription of proteins in neural precursor cells to promote cell proliferation and differentiation [65,66]. During this process, the pro-BDNF molecule helps define the strong neuronal connections by depressing weak connections and neuronal growth processes through p75NTR signaling interaction [67][68][69]. ...
Unlabelled:
Brain-derived neurotrophic factor (BDNF) is the brain's most-produced neurotrophin during the lifespan, essentially involved in multiple mechanisms of nervous system development and function. The production/release of BDNF requires multi-stage processing that appears to be regulated at various stages in which the presence of a polymorphism "Val66Met" can exert a critical influence.
Aim:
To synthesize the knowledge on the BDNF Val66Met polymorphism on intracellular processing and function of BDNF.
Methods:
We performed a systematic review and collected all available studies on the post-translation processes of BDNF, regarding the Val66Met polymorphism. Searches were performed up to 21st March 2021.
Results:
Out of 129 eligible papers, 18 studies addressed or had findings relating to BDNF post-translation processes and were included in this review.
Discussion:
Compilation of experimental findings reveals that the Val66Met polymorphism affects BDNF function by slightly altering the processing, distribution, and regulated release of BDNF. Regarding the critical role of pro-BDNF as a pro-apoptotic factor, such alteration might represent a risk for the development of neuropsychiatric disorders.
... BDNF is a crucial player in cortical and hippocampal GABAergic interneuron maturation [59,107,126,127] and has a regulatory role in the closure of the critical period of cortical plasticity and PNN maturation [43][44][45]76]. ...
Perineuronal nets (PNNs) are specialised extracellular matrix structures which preferentially enwrap fast-spiking (FS) par-valbumin interneurons and have diverse roles in the cortex. PNN maturation coincides with closure of the critical period of cortical plasticity. We have previously demonstrated that BDNF accelerates interneuron development in a c-Jun-NH 2-terminal kinase (JNK)-dependent manner, which may involve upstream thousand-and-one amino acid kinase 2 (TAOK2). Chondroitinase-ABC (ChABC) enzymatic digestion of PNNs reportedly reactivates 'juvenile-like' plasticity in the adult CNS. However, the mechanisms involved are unclear. We show that ChABC produces an immature molecular phenotype in cultured cortical neurons, corresponding to the phenotype prior to critical period closure. ChABC produced different patterns of PNN-related, GABAergic and immediate early (IE) gene expression than well-characterised modulators of mature plasticity and network activity (GABA A-R antagonist, bicuculline, and sodium-channel blocker, tetrodotoxin (TTX)). ChABC downregulated JNK activity, while this was upregulated by bicuculline. Bicuculline, but not ChABC, upregulated Bdnf expression and ERK activity. Furthermore, we found that BDNF upregulation of semaphorin-3A and IE genes was TAOK mediated. Our data suggest that ChABC heightens structural flexibility and network disinhibition, potentially contributing to 'juvenile-like' plasticity. The molecular phenotype appears to be distinct from heightened mature synaptic plasticity and could relate to JNK signalling. Finally, we highlight that BDNF regulation of plasticity and PNNs involves TAOK signalling.
... BDNF concentrations in the amniotic fluid influence its levels in the fetus [55][56][57][58][59][60]. Thus, fetal brain development is a strictly regulated process, as evidenced by pathological changes occurring with the rapid increase of BDNF in the fetal brain [61,62]. For example, injection of the protein into the brain ventricles of the mouse embryo in vitro leads to premature neurogenesis and abnormal cell proliferation [61]. ...
The purpose of this review is to summarize the current knowledge regarding the reciprocal associations between BDNF and immune-inflammatory pathways and how these links may explain the involvement of this neurotrophin in the immune pathophysiology of mood disorders and schizophrenia. Toward this end, we delineated the protein-protein interaction (PPI) network centered around BDNF and searched PubMed, Scopus, Google Scholar and Science Direct for papers dealing with the involvement of BDNF in the major psychosis, neurodevelopment, neuronal functions, and immune-inflammatory and related pathways. The PPI network was built based on the significant interactions of BDNF with neurotrophic (NTRK2, NTF4, and NGFR), immune (cytokines, STAT3, TRAF6), and cell-cell junction (CTNNB, CDH1) DEPs (differentially expressed proteins). Enrichment analysis shows that the most significant terms associated with this PPI network are the tyrosine kinase receptor (TRKR) and Src homology region two domain-containing phosphatase-2 (SHP2) pathways, tyrosine kinases receptor signaling pathways, positive regulation of kinase and transferase activity, cytokine signaling, and negative regulation of the immune response. The participation of BDNF in the immune response and its interactions with neuroprotective and cell–cell adhesion DEPs is probably a conserved regulatory process which protects against the many detrimental effects of immune activation and hyperinflammation including neurotoxicity. Lowered BDNF levels in mood disorders and schizophrenia a) are associated with disruptions in neurotrophic signaling and activated immune-inflammatory pathways leading to neurotoxicity; and b) may interact with the reduced expression of other DEPs (CTNNB1, CDH1, or DISC1) leading to multiple aberrations in synapse and axonal functions.
... Remarkably, bee venom pre-treatment stimulated the production of p-TrkB and p-CREB, and in turn, increased the expression of BDNF [63]. Moreover, BDNF has been shown to bind to subsets of TrkB receptors, thus further upregulates neural proliferation and enhanced neurogenesis, increasing the neuronal cell's ability to combat oxidative stress [64,65]. As mentioned, in previous studies of bee venom in cell models which seem to lack the significance of BDNF/TrkB/CREB, such as studies on steatohepatitis [36], wound healing [37], and hepatitis [38], bee venom also showed a remarkable ability to upregulate the Nrf2/HO-1 pathway. ...
Honeybee venom has recently been considered an anti-neurodegenerative agent, primarily due to its anti-inflammatory effects. The natural accumulation of amyloid-beta (Aβ) in the brain is reported to be the natural cause of aging neural ability downfall, and oxidative stress is the main route by which Aβ ignites its neural toxicity. Anti-neural oxidative stress is considered an effective approach for neurodegenerative therapy. To date, it is unclear how bee venom ameliorates neuronal cells in oxidative stress induced by Aβ. Here, we evaluated the neuroprotective effect of bee venom on Aβ-induced neural oxidative stress in both HT22 cells and an animal model. Our results indicate that bee venom protected HT22 cells against apoptosis induced by Aβ1–42. This protective effect was explained by the increased nuclear translocation of nuclear factor erythroid 2-like 2 (Nrf2), consequently upregulating the production of heme oxygenase-1 (HO-1), a critical cellular instinct antioxidant enzyme that neutralizes excessive oxidative stress. Furthermore, bee venom treatment activated the tropomyosin-related kinase receptor B (TrkB)/cAMP response element-binding (CREB)/brain-derived neurotrophic factor (BDNF), which is closely related to the promotion of cellular antioxidant defense and neuronal functions. A mouse model with cognitive deficits induced by Aβ1–42 intracerebroventricular (ICV) injections was also used. Bee venom enhanced animal cognitive ability and enhanced neural cell genesis in the hippocampal dentate gyrus region in a dose-dependent manner. Further analysis of animal brain tissue and serum confirmed that bee venom reduced oxidative stress, cholinergic system activity, and intercellular neurotrophic factor regulation, which were all adversely affected by Aβ1–42. Our study demonstrates that bee venom exerts antioxidant and neuroprotective actions against neural oxidative stress caused by Aβ1–42, thereby promoting its use as a therapeutic agent for neurodegenerative disorders.
... As our E12.0 scRNA-seq data successfully revealed genes enriched in different oral epithelial appendages, it provides a useful platform to uncover novel regulators of epithelial morphogenesis. As a proof of principle, we focused on the developing tooth and its newly characterized marker Ntrk2, which encodes the Neurotrophic receptor tyrosine Kinase 2. NTRK2 is a receptor for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NTF4), and its signaling activation regulates proliferation and differentiation in other contexts [112][113][114][115]. Because cell proliferation is critical for epithelial stratification during early tooth morphogenesis [28], and the relatively shallow dental lamina begins to grow rapidly in size at E12.0, signaling via NTRK2 may play a role in promoting these processes. ...
During vertebrate craniofacial development, the oral epithelium begins as a simple and morphologically homogeneous tissue. It then gives rise to locally complex structures, including the developing teeth, salivary glands, and taste buds. While there is significant knowledge about the molecular mechanisms regulating the morphogenesis of these organs at later stages, how the epithelium is initially patterned and specified to generate diverse cell types and organs remains largely unknown. To elucidate the genetic programs that direct the formation of distinct oral epithelial populations, we mapped the transcriptional landscape of embryonic day (E) 12 mouse mandibular epithelia at single cell resolution. Our analysis identified key transcription factors and gene regulatory networks that define different epithelial cell types as well as regions patterned along the oral-aboral axis. By examining the spatiotemporal expression of region-specific markers in embryonic mandibles, our results pointed to a model where the dental field is progressively confined to its position by the formation of the aboral epithelium anteriorly and the non-dental oral epithelium posteriorly. Using our data, we also identified Ntrk2 as a promoter of cell proliferation in the forming incisor, contributing to its invagination. Together, our results provide a detailed transcriptional atlas of the developing mandibular epithelium and unveil new genetic markers and regulators that are present during the specification of various oral epithelial structures.
... BDNF regulates many different cellular processes involved in the maintenance and development of normal brain function, by binding and activating the TrkB, which is a member of the larger family of Trk receptors [88]. For example, during embryogenesis, BDNF-TrkB signaling promotes the differentiation of cortical progenitor cells and then promotes differentiation of cortical progenitor cells into neurons (i.e., neurogenesis) [89]. The single nucleotide polymorphism of BDNF most likely affects memory through long-term potentiation (LTP), which is important for memory persistence [90]. ...
Humans and other primates have memory, and the hippocampus plays a critical role in this process. The neural circuitry is one of the structural foundations for the hippocampus in exerting memory function. To understand the relationship between the hippocampus and memory, we need to understand neural circuits. Past research has identified several classical neural circuits involved in memory. Although there are challenges with the study of hippocampal neural circuits, research on this topic has continued, and some progress has been made. Here, we discuss recent advances in our understanding of hippocampal neural circuit mechanisms and some of the newly discovered factors that affect memory. Substantial progress has been made regarding hippocampal memory circuits and Alzheimer’s disease. However, it is unclear whether these novel findings regarding hippocampal memory circuits hold promise for human memory studies. Additional research on this topic is needed.
... Ras activation results in MAPK-mediated neuronal regeneration and growth, while PI3-K results in Akt-mediated cell survival. These pathways further promote prosurvival by regulating genetic transcription to promote precursor cell development, cell genesis and proliferation resulting in neuronal and synaptic plasticity (Bartkowska et al., 2007;Houlton et al., 2019). ...
Neurotrophin is a growth factor that regulates the development and repair of the nervous system. From all factors, two pioneer groups, the nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), have been widely explored for their role in disease pathogenesis and potential use as therapeutic agents. Nonetheless, neurotrophin-3 (NT3) and neurotrophin-4 (NT4) also have promising potential, albeit less popular than their counterparts. This review focuses on the latter two factors and their roles in the pathogenesis of brain disorders and potential therapies. An extensive literature search of NT3 and NT4 with their receptors, the TrkB and TrkC on the nervous system were extracted and analyzed. We found that NT3 and NT4 are not only involved in the pathogenesis of some neurodegenerative diseases, but also have promising therapeutic potential on injury- and vascular-related nervous system disease, neuropsychiatry, neurodegeneration and peripheral nerve diseases. In conclusion, the role of NT3 and NT4 should be further emphasized, and more studies could be explored on the potential use of these neurotrophins in the human study.
... These cortical neurons exhibit shorter axons with fewer branches. Activated HERV-K LTR also robustly upregulates neurotrophic tyrosine receptor kinase 3 (NTRK3), critical in cortical neuron development (Bartkowska et al., 2007). The knock-down of NTRK3 in HERV-K LTR-activated cortical neurons reverts the observed phenotypes, suggesting that HERV-K activation impairs NTRK3-dependent cortical neuron development, which ultimately results in abnormal brain development (Padmanabhan Nair et al., 2021). ...
The human genome contains many retroviral elements called human endogenous retroviruses (HERVs), resulting from the integration of retroviruses throughout evolution. HERVs once were considered inactive junk because they are not replication-competent, primarily localized in the heterochromatin, and silenced by methylation. But HERVs are now clearly shown to actively regulate gene expression in various physiological and pathological conditions such as developmental processes, immune regulation, cancers, autoimmune diseases, and neurological disorders. Recent studies report that HERVs are activated in patients suffering from coronavirus disease 2019 (COVID-19), the current pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection. In this review, we describe internal and external factors that influence HERV activities. We also present evidence showing the gene regulatory activity of HERV LTRs (long terminal repeats) in model organisms such as mice, rats, zebrafish, and invertebrate models of worms and flies. Finally, we discuss several molecular and cellular pathways involving various transcription factors and receptors, through which HERVs affect downstream cellular and physiological events such as epigenetic modifications, calcium influx, protein phosphorylation, and cytokine release. Understanding how HERVs participate in various physiological and pathological processes will help develop a strategy to generate effective therapeutic approaches targeting HERVs.
Intracellular calcium plays a pivotal role in central nervous system (CNS) development by regulating various processes such as cell proliferation, migration, differentiation, and maturation. However, understanding the involvement of calcium (Ca2+) in these processes during CNS development is challenging due to the dynamic nature of this cation and the evolving cell populations during development. While Ca2+ transient patterns have been observed in specific cell processes and
molecules responsible for Ca2+ homeostasis have been identified in excitable and non-excitable cells, further research into Ca2+ dynamics and the underlying mechanisms in neural stem cells (NSCs) is required. This review focuses on molecules involved in Ca2+ entrance expressed in NSCs in vivo and in vitro, which are crucial for Ca2+ dynamics and signaling. It also discusses how these molecules
might play a key role in balancing cell proliferation for self-renewal or promoting differentiation. These processes are finely regulated in a time-dependent manner throughout brain development, influenced by extrinsic and intrinsic factors that directly or indirectly modulate Ca2+ dynamics. Furthermore, this review addresses the potential implications of understanding Ca2+ dynamics in NSCs for treating neurological disorders. Despite significant progress in this field, unraveling the elements contributing to Ca2+ intracellular dynamics in cell proliferation remains a challenging puzzle that requires further investigation.
Neurodevelopmental disorders are characterized by alterations in the development of the cerebral cortex, including aberrant changes in the number and function of neural cells. Although neurogenesis is one of the most studied cellular processes in these pathologies, little evidence is known about glial development. Genetic association studies have identified several genes associated with neurodevelopmental disorders. Indeed, variations in the PTPRD gene have been associated with numerous brain disorders, including autism spectrum disorder, restless leg syndrome, and schizophrenia. We previously demonstrated that constitutive loss of PTPRD expression induces significant alterations in cortical neurogenesis, promoting an increase in intermediate progenitors and neurons in mice. However, its role in gliogenesis has not been evaluated. To assess this, we developed a conditional knockout mouse model lacking PTPRD expression in telencephalon cells. Here, we found that the lack of PTPRD in the mouse cortex reduces glial precursors, astrocytes, and oligodendrocytes. According to our results, this decrease in gliogenesis resulted from a reduced number of radial glia cells at gliogenesis onset and a lower gliogenic potential in cortical neural precursors due to less activation of the JAK/STAT pathway and reduced expression of gliogenic genes. Our study shows PTPRD as a regulator of the glial/neuronal balance during cortical neurodevelopment and highlights the importance of studying glial development to understand the etiology of neurodevelopmental diseases.
It has been reported both in clinic and rodent models that beyond spinal cord injury directly induced symptoms, such as paralysis, neuropathic pain, bladder/bowel dysfunction, and loss of sexual function, there are a variety of secondary complications, including memory loss, cognitive decline, depression, and Alzheimer’s disease. The large-scale longitudinal population-based studies indicate that post-trauma depression is highly prevalent in spinal cord injury patients. Yet, few basic studies have been conducted to address the potential molecular mechanisms. One of possible factors underlying the depression is the reduction of adult hippocampal neurogenesis which may come from less physical activity, social isolation, chronic pain, and elevated neuroinflammation after spinal cord injury. However, there is no clear consensus yet. In this review, we will first summarize the alteration of hippocampal neurogenesis post-spinal cord injury. Then, we will discuss possible mechanisms underlie this important spinal cord injury consequence. Finally, we will outline the potential therapeutic options aimed at enhancing hippocampal neurogenesis to ameliorate depression.
Alterations in adult neurogenesis are a common hallmark of neurodegenerative diseases. Therefore, understanding the molecular mechanisms that control this process is an indispensable requirement for designing therapeutic interventions addressing neurodegeneration. Neurotrophins have been implicated in multiple functions including proliferation, survival, and differentiation of the neural stem cells (NSCs), thereby being good candidates for therapeutic intervention. Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been proven to promote neurogenesis in the subgranular zone. However, the effects of BDNF in the adult subventricular zone (SVZ) still remain unclear due to contradictory results. Using in vitro cultures of adult NSCs isolated from the mouse SVZ, we show that low concentrations of BDNF are able to promote self-renewal and proliferation in these cells by activating the tropomyosin-related kinase B (TrkB) receptor. However, higher concentrations of BDNF that can bind the p75 neurotrophin receptor (p75NTR) potentiate TrkB-dependent self-renewal and proliferation and promote differentiation of the adult NSCs, suggesting different molecular mechanisms in BDNF-promoting proliferation and differentiation. The use of an antagonist for p75NTR reduces the increment in NSC proliferation and commitment to the oligodendrocyte lineage. Our data support a fundamental role for both receptors, TrkB and p75NTR, in the regulation of NSC behavior.
Depression is characterized by prominent indicators and manifestations, such as anhedonia, which refers to the inability to experience pleasure, and persistent feelings of hopelessness. In clinical practice, the primary treatment approach involves the utilization of selective serotonin reuptake inhibitors (SSRIs) and related pharmacological interventions. Nevertheless, it is crucial to recognize that these agents are associated with significant adverse effects. Traditional Chinese medicine (TCM) adopts a multifaceted approach, targeting diverse components, multiple targets, and various channels of action. TCM has potential antidepressant effects. Anomalies in adult hippocampal neurogenesis (AHN) constitute a pivotal factor in the pathology of depression, with the regulation of AHN emerging as a potential key measure to intervene in the pathogenesis and progression of this condition. This comprehensive review presented an overview of the pharmacological mechanisms underlying the antidepressant effects of active ingredients found in TCM. Through examination of recent studies, we explored how these ingredients modulated AHN. Furthermore, we critically assessed the current limitations of research in this domain and proposed novel strategies for preclinical investigation and clinical applications in the treatment of depression in future.
The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.
Transactivation of Tropomyosin receptor kinase B (TrkB) by EGF leads to cell surface transport of TrkB, promoting its signaling responsiveness to brain-derived neurotrophic factor (BDNF), a critical process for proper cortical plate development. However, the mechanisms that regulate the transport of TrkB to the cell surface are not fully understood. Here, we identified Calnexin as a regulator for targeting TrkB either to the cell surface or toward autophagosomal processing. Calnexin-deficient mouse embryos show impaired cortical plate formation and elevated levels of transactivated TrkB. In Calnexin-depleted mouse neuronal precursor cells, we detected an impaired cell surface transport of TrkB in response to EGF and an impaired delivery to autophagosomes. Mechanistically, we show that Calnexin facilitates the interaction of TrkB with the ER-phagy receptor Fam134b, thereby targeting TrkB to ER-phagy. This mechanism appears as a critical process for fine-tuning the sensitivity of neurons to BDNF.
Currently, heterocycles have occupied an important position in the fields of drug design. Among them, azaindole moiety is regarded as one privileged scaffold to develop therapeutic agents. Since two nitrogen atoms of azaindole increase the possibility to form hydrogen bonds in the adenosine triphosphate (ATP)-binding site, azaindole derivatives are important sources of kinase inhibitors. Moreover, some of them have been on the market or in clinical trials for the treatment of some kinase-related diseases (e.g., vemurafenib, pexidartinib, decernotinib). In this review, we focused on the recent development of azaindole derivatives as potential kinase inhibitors based on kinase targets, such as adaptor-associated kinase 1 (AAK1), anaplastic lymphoma kinase (ALK), AXL, cell division cycle 7 (Cdc7), cyclin-dependent kinases (CDKs), dual-specificity tyrosine (Y)-phosphorylation regulated kinase 1A (DYRK1A), fibroblast growth factor receptor 4 (FGFR4), phosphatidylinositol 3-kinase (PI3K) and proviral insertion site in moloney murine leukemia virus (PIM) kinases. Meanwhile, the structure-activity relationships (SARs) of most azaindole derivatives were also elucidated. In addition, the binding modes of some azaindoles complexed with kinases were also investigated during the SARs elucidation. This review may offer an insight for medicinal chemists to rationally design more potent kinase inhibitors bearing the azaindole scaffold.
Brain-derived Neurotrophic Factor (BDNF) promotes neuronal differentiation and survival and is implicated in the pathogenesis of many neurological disorders. Here, we identified a novel intergenic enhancer located 170 kb from the Bdnf gene, which promotes the expression of Bdnf transcript variants during mouse neuronal differentiation and activity. Following Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Bdnf enhancer activity is necessary for neuronal clustering and dendritogenesis in vitro, and for cortical development in vivo. Our findings provide the first evidence of a regulatory mechanism whereby the activation of a distal enhancer promotes Bdnf expression during brain development.
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
Tropomyosin receptor kinases (TRKs) are associated with the proliferation and differentiation of cells, and thus their continuous activation and overexpression cause cancer. Herein, based on scaffold hopping and computer-aid drug design, 38 pyrazolo[3,4-b]pyridine derivatives were synthesised. Further, we evaluated their activities to inhibit TRKA. Among them, compound C03 showed acceptable activity with an IC50 value of 56 nM and it inhibited the proliferation of the Km-12 cell line with an IC50 value of 0.304 μM together with obvious selectivity for the MCF-7 cell line and HUVEC cell line. Furthermore, compound C03 possessed good plasma stability and low inhibitory activity to a panel of cytochrome P450 isoforms except CYP2C9. Overall, C03 has potential for further exploration.
Herein, we report the discovery process and antitumor activity of the TRK inhibitor CZw-124 (8o), which is a quinazoline derivative. Starting from a PAK4 inhibitor, we used various drug design strategies, including pharmacophore feature supplementation, F-scanning, and blocking metabolic sites, and finally found a TRK inhibitor CZw-124 that is effective in vitro and in vivo. Docking studies and molecular dynamics simulations revealed a possible mode of binding of CZw-124 to TRKA. Biological activity evaluation showed that CZw-124 belongs to a class of pan-TRK inhibitors with moderate kinase selectivity. It inhibited the proliferation and induced the apoptosis of Km-12 cells in vitro by interfering with the phosphorylation of TRKA. Pharmacodynamic evaluation in vivo showed that CZw-124 had a tumor inhibition rate comparable to that of larotrectinib after oral administration of 40 mg/kg/d (tumor growth inhibiton = 71%).
Abnormal development of corpus callosum is relatively common and causes a broad spectrum of cognitive impairments in humans. We use acallosal Neurod2/6-deficient mice to study callosal axon guidance within the ipsilateral cerebral cortex. Initial callosal tracts form but fail to traverse the ipsilateral cingulum and are not attracted towards the midline in the absence of Neurod2/6. We show that the restoration of Ephrin-A4 (EfnA4) expression in the embryonic neocortex of Neurod2/6-deficient embryos is sufficient to partially rescue targeted callosal axon growth towards the midline. EfnA4 cannot directly mediate reverse signaling within outgrowing axons, but it forms co-receptor complexes with TrkB (Ntrk2). The ability of EfnA4 to rescue the guided growth of a subset of callosal axons in Neurod2/6-deficient mice is abolished by the co-expression of dominant negative TrkBK571N (kinase-dead) or TrkBY515F (SHC-binding deficient) variants, but not by TrkBY816F (PLCγ1-binding deficient). Additionally, EphA4 is repulsive to EfnA4-positive medially projecting axons in organotypic brain slice culture. Collectively, we suggest that EfnA4-mediated reverse signaling acts via TrkB-SHC and is required for ipsilateral callosal axon growth accuracy towards the midline downstream of Neurod family factors.
Depression during pregnancy adversely affects fetal development. Desvenlafaxine drug is used for the treatment of gestational depression. In light of the well‐established role of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in regulating neurogenesis and neural survival, the role of S100b in nerve cell energetic metabolism, differentiation of neurons and glial cells, an aberrant increase in NGF, BDNF and S100b expression in the fetal brain may contribute to desvenlafaxine cognitive disorders by altering brain development. This study is trying to determine the effect of desvenlafaxine on brain development. Thirty timed pregnant rats (from the 5th to the 20th day) were divided into three groups: control, low dose (5.14 mg/kg/day) and high dose (10.28 mg/kg/day) of desvenlafaxine where all animals received the corresponding doses by gavage. Maternal and fetal brain samples were fixed for histological, immunohistochemical (IHC) study of NGF and evaluated for BDNF and S100b genes expression. Desvenlafaxine induced some of the histopathological alterations in maternal and fetal rat brains. Moreover, IHC analysis of maternal and fetal rat brains showed that groups treated with desvenlafaxine demonstrated a significant increase of NGF protein immunoreactivity compared with that in the controls. Gene expression results revealed upregulation of messenger RNA BDNF and S100B expression. According to developmental changes in the brain, desvenlafaxine affects neonatal growth during pregnancy, which may lead to delay of brain development. So, it is essential to survey the roles of antidepressant drugs on neonatal development during pregnancy.
The response of brain tissue to neurotrophins during rat development was examined using a novel in vitro assay for Trk/neurotrophin receptor activity. In this assay, brain tissues were exposed to neutrophins and ligand-induced Trk tyrosine phosphorylation was measured. During the perinatal period, Trk tyrosine phsphorylation in all brain area was induced very similarly by the TrkB and TrkC ligands brain-derived neurotrophic factor (BNDF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT-4/5). In the adult brain, minimal signals were observed after treatment with these three factors, despite the continued presence of full length and truncated TrikB protein. In contrast, responsiveness to the TrkA ligand NGF was absent in the ebmryo and increased during the first 2 weeks after birth in various brain areas, particularly in striatum, basal forebrain, and hippocampus. Our results, showing maximal responsiveness of brain tissue to BDNF, NT-3, and NT-4/5 during early neuronal differentiation and migration, suggest involvement of TrkB in these events. The lack of a significant response to these neurotrophins in the adult brain indicates effective posttranslational mechanisms that control the response of Trk family receptors. Our findings further demonstrate that neurons of the striatum and basal forebrain remain NGF responsive in the adult, confirming at the molecular level results obtained earlier at the cellular level for the basal forebrain cholinergic neurons.
The trkC gene is expressed throughout the mammalian nervous system and encodes a series of tyrosine protein kinase isoforms that serve as receptors for neurotrophin-3 (NT3), a member of the nerve growth factor (NGF) family of neurotrophic factors. One of these isoforms, gp145trkC/TrkC K1, mediates the trophic properties of NT3 in cultured cells. Here we show that homozygous mice defective for TrkC tyrosine protein kinase receptors lack Ia muscle afferent projections to spinal motor neurons and have fewer large myelinated axons in the dorsal root and posterior columns of the spinal cord. These mice display abnormal movements and postures, indicating that NT3/TrkC-dependent sensor; neurons may play a primary role in proprioception, the sense of position and movement of the limbs.
The Trk family of tyrosine kinases encodes receptors for nerve growth factor-related neurotrophins. Here we present a developmental expression study of trkC, which encodes a receptor for neurotrophin-3 (NT-3). Like the related genes, trk and trkB, trkC is expressed primarily in neural lineages although the pattern is complex and includes non-neuronal cells. Direct comparison with trk and trkB developmental expression patterns permits the following observations. (1) trkC is expressed in novel neural tissues where other Trk genes are silent. (2) Some tissues appear to coexpress trkB and trkC receptors in the embryo and in the adult. (3) trkC expression can be detected in the gastrulating embryo. These data provide insights into the role of Trk-family receptors and nerve growth factor-related neurotrophins during development and suggest that, in addition to regulating neuronal survival and differentiation, the neurotrophin/Trk receptor system may have broader physiological effects. Finally, interspecific mouse backcrosses have been used to map the location of each of the Trk genes on mouse chromosomes. Alignment with available chromosomal maps identify possible linkage between the Trk genes and known neurological mutations.
Mice lacking neurotrophin-3 (NT-3) have been shown previously to be born with severe sensory deficits. This study characterizes the developmental course of this deficit in the trigeminal sensory ganglion, which in NT-3 homozygous mutants contains only 35% of the normal number of neurons at birth. At embryonic day 10.5 (E10.5), normal numbers of neurons, as assessed by expression of neurofilament protein and of total cells, are present in the ganglia of mutant homozygotes. During the next 3 d (E10.5–E13.5), virtually all of the deficit develops, after which mutant animals retain only ∼30% the normal number of neurons. Quantification of neuronal and neuronal precursor numbers in normal and mutant animals reveals that neurons are specifically depleted in the absence of NT-3. A deficiency in precursor proliferation is only seen after most of the neuronal deficit has developed. Numbers of apoptotic cells in the ganglia of mutant animals are elevated during this same interval, indicating that the neuronal deficit is caused, in large part, by increased cell death of embryonic neurons.
To determine sources of NT-3 in the trigeminal system, we examined the expression pattern of β-galactosidase in mice, in which lacZ has replaced the NT-3 coding exon. E10.5–E11.5 embryos exhibit intense reporter expression throughout the mesenchyme and epithelia of the first branchial arch. β-galactosidase expression in E13.5 embryos is largely confined to the oral epithelium and the mesenchyme underlying the skin. Throughout the E10.5–E13.5 interval, the trigeminal ganglion and its targets in the CNS do not express reporter activity.
We conclude that NT-3 acts principally as a peripherally derived survival factor for early trigeminal neurons.
Newborn mice carrying targeted mutations in genes encoding neurotrophins or their signaling Trk receptors display severe neuronal deficits in the peripheral nervous system but not in the CNS. In this study, we show that trkB (-/-) mice have a significant increase in apoptotic cell death in different regions of the brain during early postnatal life. The most affected region in the brain is the dentate gyrus of the hippocampus, although elevated levels of pyknotic nuclei were also detected in cortical layers II and III and V and VI, the striatum, and the thalamus. Furthermore, axotomized hippocampal and motor neurons of trkB (-/-) mice have significantly lower survival rates than those of wild-type littermates. These results suggest that neurotrophin signaling through TrkB receptors plays a role in the survival of CNS neurons during postnatal development. Moreover, they indicate that TrkB receptor signaling protects subpopulations of CNS neurons from injury- and axotomy-induced cell death.
Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.
Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trk B (−/−) and trk C (−/−) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10–P16) did not reveal major differences in the topographic patterns of hippocampal connections.
In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21–49%) and decreased densities of axonal varicosities (8–17%) in both trk B (−/−) and trk C (−/−) mice. In addition, electron microscopic analyses showed that trk B (−/−) and trk C (−/−) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trk B (−/−) mice. We conclude that neither trk B nor trk C genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo . The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.
The retinoblastoma tumor suppressor protein (pRb) family is essential for cortical progenitors to exit the cell cycle and survive. In this report, we test the hypothesis that pRb collaborates with basic helix-loop-helix (bHLH) transcription factors to regulate cortical neurogenesis, taking advantage of the naturally occurring dominant-inhibitory HLH protein Id2. Overexpression of Id2 in cortical progenitors completely inhibited the induction of neuron-specific genes and led to apoptosis, presumably as a consequence of conflicting differentiation signals. Both of these phenotypes were rescued by coexpression of a constitutively activated pRb mutant. In contrast, Id2 overexpression in postmitotic cortical neurons affected neither neuronal gene expression nor survival. Thus, pRb collaborates with HLHs to ensure the coordinate induction of terminal mitosis and neuronal gene expression as cortical progenitors become neurons.
Many neurons die as the normal brain develops. How this is regulated and whether the mechanism involves neurotrophic molecules from target cells are unknown. We found that cultured neurons from a key forebrain structure, the dorsal thalamus, develop a need for survival factors including brain-derived neurotrophic factor (BDNF) from their major target, the cerebral cortex, at the age at which they innervate it. Experiments in vivo have shown that rates of dorsal thalamic cell death are reduced by increasing cortical levels of BDNF and are increased in mutant mice lacking functional BDNF receptors or thalamocortical projections; these experiments have also shown that an increase in the rates of dorsal thalamic cell death can be achieved by blocking BDNF in the cortex. We suggest that the onset of a requirement for cortex-derived neurotrophic factors initiates a competitive mechanism regulating programmed cell death among dorsal thalamic neurons.
During telencephalic development, cells from the medial ganglionic eminence (MGE) are thought to migrate to the neocortex to give rise to a majority of cortical GABAergic interneurons. By combining time-lapse video-microscopy, immunofluorescence and pharmacological perturbations in a new in vitro migration assay, we find that MGE-derived cells migrate through the entire extent of the cortex and into the CA fields of the hippocampus, but avoid the dentate gyrus. Migrating neurons initially travel within the marginal zone and intermediate zone, and can enter the cortical plate from either location. Tangential migration is strongly stimulated by BDNF and NT4 and attenuated by the Trk-family inhibitor, K252a, suggesting that migration is regulated by TrkB signaling. Furthermore, TrkB-null mice show a significant decrease in the number of calbindin-positive neurons migrating tangentially in the embryonic cortex. BDNF and NT4 cause rapid activation of PI3-kinase in MGE cells, and inhibition of PI3-kinase (but not of MAP kinase or PLCgamma) dramatically attenuates tangential migration. These observations suggest that TrkB signaling, via PI3-kinase activation, plays an important role in controlling interneuron migration in the developing cerebral cortex.
Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.
Trk receptors are a family of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT3 and NT4). Neurotrophin signaling through these receptors regulates cell survival, proliferation, the fate of neural precursors, axon and dendrite growth and patterning, and the expression and activity of functionally important proteins, such as ion channels and neurotransmitter receptors. In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. The cytoplasmic domains of Trk receptors contain several sites of tyrosine phosphorylation that recruit intermediates in intracellular signaling cascades. As a result, Trk receptor signaling activates several small G proteins, including Ras, Rap-1, and the Cdc-42-Rac-Rho family, as well as pathways regulated by MAP kinase, PI 3-kinase and phospholipase-C-gamma (PLC-gamma). Trk receptor activation has different consequences in different cells, and the specificity of downstream Trk receptor-mediated signaling is controlled through expression of intermediates in these signaling pathways and membrane trafficking that regulates localization of different signaling constituents. Perhaps the most fascinating aspect of Trk receptor-mediated signaling is its interplay with signaling promoted by the pan-neurotrophin receptor p75NTR. p75NTR activates a distinct set of signaling pathways within cells that are in some instances synergistic and in other instances antagonistic to those activated by Trk receptors. Several of these are proapoptotic but are suppressed by Trk receptor-initiated signaling. p75NTR also influences the conformations of Trk receptors; this modifies ligand-binding specificity and affinity with important developmental consequences.
Nerve growth factor and neurotrophin-3 (NT-3) are processed within the constitutive secretory pathway of neurons and neuroendocrine
cells and are released continuously in an activity-independent fashion. In contrast, brain-derived neurotrophic factor (BDNF)
is processed in the regulated secretory pathway, stored in vesicles, and released in response to neuronal activity, consistent
with its role in modulating synaptic plasticity. In this study, we used vaccinia virus infection and transfection methods
to monitor the processing and sorting of neurotrophin-4 (NT-4) in AtT-20 cells, which have been used as a model for the sorting
of secretory proteins in neurons. Our data show that NT-4 is processed in the constitutive secretory pathway. The molecule
is diffusely distributed within the cells and released, soon after being synthesized, in a manner that is not affected by
cell depolarization. We further show that NT-4 and BDNF, when co-expressed, can form heterodimers that are constitutively
released. In contrast, heterodimers of NT-3 and BDNF have been shown to be released through the regulated secretory pathway.
Thus, NT-4, alone or when co-expressed with BDNF, is processed within and secreted by the constitutive secretory pathway.
The generation of complex neuronal structures, such as the neocortex, requires accurate positioning of neurons and glia within the structure, followed by differentiation, formation of neuronal connections, and myelination. To understand the importance of TrkB signaling during these events, we have used conditional and knockin mutagenesis of the TrkB neurotrophin receptor, and we now show that this tyrosine kinase receptor, through docking sites for the Shc/FRS2 adaptors and phospholipase Cgamma (PLCgamma), coordinates these events in the cerebral cortex by (1) controlling cortical stratification through the timing of neuronal migration during cortex formation, and (2) regulating both neuronal and oligodendrocyte differentiation. These results provide genetic evidence that TrkB regulates important functions throughout the formation of the cerebral cortex via recruitment of the Shc/FRS2 adaptors and PLCgamma.
Although most cells in the embryonic mouse cortex express the serine-threonine kinase Akt-1, a small population of progenitors expresses Akt-1 protein at a higher level. To determine the functional significance of this difference, we used a retrovirus to increase Akt-1 expression in cortical progenitors. Increased Akt expression enhanced Akt activation after growth factor stimulation of progenitors. In vivo, it promoted retention in progenitor layers, the ventricular zone and subventricular zone. In vitro, it enhanced proliferation and survival, but did not impair migration. Moreover, it increased the proportion of stem cells, defined by a self-renewal assay. These effects did not depend on the Akt substrate p21(Cip1). In contrast, rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), altered effects of elevated Akt-1 selectively: it eliminated the increase in stem cells and reduced the proliferative response, but had no effect on survival. The ability of elevated Akt-1 to increase the self-renewing population therefore depends on a rapamycin-sensitive mechanism (presumably inhibition of mTOR activity) but not on p21(Cip1), and can be distinguished from its effects on the proliferation and survival of other types of progenitors. Our findings suggest that expression of a high level of Akt-1 by a subpopulation of cortical progenitors biases their responses to extrinsic signals to increase their survival, proliferation, and/or self-renewal. Heterogeneity in Akt-1 level among progenitors could therefore allow cells that share a microenvironment to respond differently to the same extrinsic signals.
The intracellular mechanisms that bias mammalian neural precursors to generate neurons versus glial cells are not well understood. We demonstrated previously that the growth factor-regulated mitogen-activated protein kinase kinase (MEK) and its downstream target, the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, are essential for neurogenesis in cultured cortical precursor cells (Ménard et al., 2002). Here, we examined a role for this pathway during cortical cell fate determination in vivo using in utero electroporation of the embryonic cortex. These studies demonstrate that inhibition of the activity of either MEK or the C/EBPs inhibits the genesis of neurons in vivo. Moreover, the MEK pathway mediates phosphorylation of C/EBPbeta in cortical precursors, and expression of a C/EBPbeta construct in which the MEK pathway phosphorylation sites are mutated inhibits neurogenesis. Conversely, expression of a C/EBPbeta construct, in which the same sites are mutated to glutamate and therefore are "constitutively" phosphorylated, enhances neurogenesis in the early embryonic cortex. A subpopulation of precursors in which C/EBP activity is inhibited are maintained as cycling precursors in the ventricular/subventricular zone of the cortex until early in postnatal life, when they have an enhanced propensity to generate astrocytes, presumably in response to gliogenic signals in the neonatal environment. Thus, activation of an MEK-C/EBP pathway in cortical precursors in vivo biases them to become neurons and against becoming astrocytes, thereby acting as a growth factor-regulated switch.
Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.
Multipotent cortical progenitor cells differentiate into neurons and glial cells during development; however, mechanisms governing the specification of progenitors to a neuronal fate are not well understood. Although both extrinsic and intrinsic factors regulate this process, little is known about kinase signaling mechanisms that direct neuronal fate. Here, we report that extracellular signal-regulated kinase (ERK) 5 is expressed and active in proliferating cortical progenitors. Lentiviral gene delivery of a dominant negative ERK5 or dominant negative MAP kinase kinase 5 reduced the number of neurons generated from rat cortical progenitor cells in culture, whereas constitutive activation of ERK5 increased the production of neurons. Furthermore, when cortical progenitor cells were treated with ciliary neurotrophic factor, which induces precocious glial differentiation, ERK5 activation still promoted neuronal fate while suppressing glial differentiation. Our data also indicate that ERK5 does not directly regulate proliferation or apoptosis of cultured cortical progenitors. We conclude that ERK5 is necessary and sufficient to stimulate the generation of neurons from cortical progenitors. These results suggest a previously uncharacterized function for ERK5 signaling during brain development and raise the interesting possibility that extrinsic factors may instruct cortical progenitors to become neurons by activating the ERK5 pathway.
• neural progenitor cell
• neural stem cell
• neurogenesis
Lamina formation in the developing cerebral cortex requires precisely regulated generation and migration of the cortical progenitor cells. To test the possible involvement of brain-derived neurotrophic factor (BDNF) in the formation of the cortical lamina, we investigated the effects of BDNF protein and anti-BDNF antibody separately administered into the telencephalic ventricular space of 13.5-d-old mouse embryos. BDNF altered the position, gene-expression properties, and projections of neurons otherwise destined for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex, whereas anti-BDNF antibody changed some of those of neurons of upper layers II/III. Additional analysis revealed that BDNF altered the laminar fate of neurons only if their parent progenitor cells were exposed to it at approximately S-phase and that it hastened the timing of the withdrawal of their daughter neurons from the ventricular proliferating pool by accelerating the completion of S-phase, downregulation of the Pax6 (paired box gene 6) expression, an essential transcription factor for generation of the upper layer neurons, and interkinetic nuclear migration of cortical progenitors in the ventricular zone. These observations suggest that BDNF participates in the processes forming the neuronal laminas in the developing cerebral cortex. BDNF can therefore be counted as one of the key extrinsic factors that regulate the laminar fate of cortical neurons.
To identify production sites and action targets of neurotrophins during neurogenesis, we investigated immunoreactivities of neurotrophins and their tyrosine kinase receptors in the cerebral cortex of rat embryos. Two sets of ligand-receptor systems, brain-derived neurotrophic factor/TrkB and neurotrophin-3/TrkC, were expressed simultaneously in Cajal-Retzius, subplate neurons and ventricular multipotent stem cells at embryonic days 13 and 15. Intraventricular administration of brain-derived neurotrophic factor or neurotrophin-3 at embryonic day 16 markedly modulated microtubule-associated protein II and/or Hu protein expression in different ways in the cortical plate cells by embryonic day 20. These observations indicate the involvement of autocrine and/or local paracrine action of brain-derived neurotrophic factor and/or neurotrophin-3 during formation of the cerebral cortex.
During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al, 1991; Jacobson, 1991). At this time (E15-E21), reverse transcriptase-polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high-affinity receptor, TrkB. Immunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15-E21) using a quantitative in vitro chemotaxis assay. High-affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT-3, a low-affinity TrkB ligand, only stimulated significant migration at high concentrations (> or =100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF-induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+-chelator, BAPTA-AM, suggesting that BDNF-induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+-dependent mechanisms. BDNF-stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.
To investigate the role of the gp140trk receptor tyrosine kinase in nerve growth factor (NGF)-induced differentiation, we have overexpressed gp140trk in the NGF-responsive PC12 cell line. Here we demonstrate that overexpression of gp140trk results in marked changes in NGF-induced differentiation. Whereas PC12 cells elaborated neurites after 2 days of continuous exposure to NGF, PC12 cells overexpressing gp140trk by 20-fold(trk-PC12) began this process within hours. Compared with wild-type PC12 cells, trk-PC12 exhibited an increase in both high and low affinity NGF-binding sites. Furthermore, trk-PC12 cells displayed an enhanced level of NGF-dependent gp140trk autophosphorylation, and this activity was sustained for many hours following ligand binding. The tyrosine phosphorylation or activity of several cellular proteins, such as PLC-gamma 1, PI-3 kinase, and Erk1 and the expression of the mRNA for the late response gene transin were also sustained as a consequence of gp140trk overexpression. The data indicate that overexpression of gp140trk in PC12 cells markedly accelerates NGF-induced differentiation pathways, possibly through the elevation of gp140trk tyrosine kinase activity.
To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.
Neurotrophin-3-deficient (NT-3-deficient) mice were generated by gene targeting. Mutant mice displayed severe movement defects of the limbs, and most died shortly after birth. Substantial portions of peripheral sensory and sympathetic neurons were lost while motor neurons were not affected. Significantly, spinal proprioceptive afferents and their peripheral sense organs (muscle spindles and Golgi tendon organs) were completely absent in homozygous mutant mice. This correlated with a loss of parvalbumin and carbonic anhydrase-positive neurons in the dorsal root ganglion. No gross abnormalities were seen in Pacinian corpuscles, cutaneous afferents containing substance P and calcitonin gene-related peptide, and deep nerve fibers in the joint capsule and tendon. Importantly, the number of muscle spindles in heterozygous mutant mice was half of that in control mice, indicating that NT-3 is present at limiting concentrations in the embryo.
Interactions of the trk family of tyrosine kinase receptors with neurotrophins result in growth and maturational changes in neuronal cells. The continued progression, maturation, or regression of neuroblastoma, an embryonal, sympathetic nervous system-derived tumor of infants and children, might be governed by neurotrophic influences. Immunocytochemistry was utilized to evaluate TrkA, TrkB, and TrkC protein expression at the cellular level in the developing human fetal sympathetic nervous system and in a selection of neuroblastoma tumor specimens. TrkA and TrkC expression was identified in sympathetic ganglia and within the adrenal medulla, with intense TrkB expression restricted to paraganglia, of the normal developing human sympathetic nervous system. In neuroblastoma, pp140trkA expression correlated positively with favorable tumor stage (P = 0.0027) and favorable outcome (P = 0.026). No statistically significant correlation of TrkC expression with outcome was evident; however, both TrkA and TrkC expression was most apparent in tumor cells of increased differentiation. TrkB expression was primarily localized to cells within the fibrovascular tumor stroma. A model of neurotrophin receptor expression and neurotrophin reactivity with differentiation is proposed. The existence and spatial distribution of neurotrophin receptors in neuroblastoma lend supportive evidence that neurotrophic influences may be involved in tumor persistence or regression.
To identify molecules that regulate the transition of dividing neuroblasts to terminally differentiated neurons in the CNS, conditions have been developed that allow the neuronal differentiation of cortical precursor cells to be examined in vitro. In these cultures, the proliferation of undifferentiated precursor cells is controlled by basic fibroblast growth factor (bFGF). The proliferative effects of bFGF do not preclude the action of signals that promote differentiation, since addition of neurotrophin-3 (NT-3) antagonizes the proliferative effects of bFGF and enhances neuronal differentiation. In addition, blocking NT-3 function with neutralizing antibodies leads to a marked decrease in the number of differentiated neurons, without affecting the proliferation of cortical precursors or the survival of postmitotic cortical neurons. These observations suggest that bFGF and NT-3, by their distinct effects on cell proliferation and differentiation, are key regulators of neurogenesis in the CNS.
Neurotrophin 3 (NT-3) is one of four related polypeptide growth factors that share structural and functional homology to nerve growth factor (NGF). NT-3 and its receptor, called neurotrophic tyrosine kinase receptor type 3 (Ntrk3; also called TrkC), are expressed early and throughout embryogenesis. We have inactivated the NT-3 gene in embryonic stem (ES) cells by homologous recombination. The mutated allele has been transmitted through the mouse germ line, and heterozygote intercrosses have yielded homozygous mutant newborn pups. The NT-3-deficient mutants fail to thrive and exhibit severe neurological dysfunction. Analysis of mutant embryos uncovers loss of Ntrk3/TrkC-expressing sensory neurons and abnormalities at early stages of sensory neuronal development. NT-3-deficient mice will permit further study of the role of this neurotrophin in neural development.
Brain-derived neurotrophic factor (BDNF), a neurotrophin, enhances the survival and differentiation of several classes of neurons in vitro. To determine its essential functions, we have mutated the BDNF gene. Most homozygote mutants die within 2 days after birth, but a fraction live for 2-4 weeks. These develop symptoms of nervous system dysfunction, including ataxia. The BDNF mutant homozygotes have substantially reduced numbers of cranial and spinal sensory neurons. Although their central nervous systems show no gross structural abnormalities, expression of neuropeptide Y and calcium-binding proteins is altered in many neurons, suggesting they do not function normally. In contrast with mice lacking the BDNF receptor TrkB, motor neurons appear normal in the BDNF mutant.
The Trk family of protein tyrosine kinases (TrkA/B/C) are receptors for neurotrophins, a family of closely related proteins that are important physiological regulators of the survival of specific neurons within the peripheral nervous system (PNS) of vertebrates. In contrast to the PNS, brains of mutant mice deficient in a single neurotrophin or Trk receptor species do not show signs of major cell loss. However, in double mutant mice, we now show that reducing the expression of both TrkB and TrkC causes massive cell death of postnatal hippocampal and cerebellar granule neurons. Kinetic analysis of neuronal death in the hippocampus showed that dentate gyrus granule neurons become dependent on TrkB and TrkC after the first postnatal week, shortly after the period of naturally occurring cell death, indicating a role of these receptors in supporting postmitotic neurons. Correlating with the loss of granule cells, the number of mossy fibers projecting to CA3 pyramidal neurons was markedly reduced in mice carrying mutant trkB/trkC alleles, demonstrating impairment of excitatory pathways in the hippocampus. In the cerebellum, TrkB and TrkC receptors were specifically required for premigratory granule neurons located in the external granule layer. In contrast, cerebellar Purkinje cells were found to be poorly differentiated, but showed no signs of increased cell death. These results provide in vivo evidence that neurotrophins are essential physiological survival factors for specific central neurons. Moreover, they suggest that central, in contrast to peripheral, neurons are capable of using more than one neurotrophin/Trk receptor signaling pathway to stay alive.
Cajal-Retzius (CR) cells of the cerebral cortex express receptors for the neurotrophin brain-derived neurotrophic factor (BDNF) and downregulate expression of the extracellular matrix protein Reelin during early postnatal development, coincident with the onset of cortical BDNF expression. During this period, mice lacking BDNF have elevated levels of Reelin in CR cells. Acute BDNF stimulation of cortical neuron cultures and overexpression of BDNF in the developing brain of transgenic mice prior to the onset of endogenous production causes a profound, dose-dependent reduction of Reelin expression in CR cells. In addition, overexpression of BDNF produces gaps and heterotopias in the marginal zone and disorganization and aggregation of cortical CR cells and induces several other malformations, including aberrant cortical lamination, similar to the phenotype of reeler mutant mice, which lack Reelin. These results demonstrate a role for BDNF on cortical CR cells and identify Reelin as a direct effector of this neurotrophin during brain development.
Neurotrophin-3 (NT-3) and its receptor TrkC are known to be important for neuronal survival. More recently, NT-3 has been implicated as playing a role in oligodendrocyte (OL) proliferation and survival in vitro. Examination of NT-3 and TrkC knockout mice revealed a reduction in NT-3-dependent neurons. To date, no study has examined alterations in glial cell populations in these knockout mice. In this report, we demonstrate a decline in OL progenitor cell numbers within the CNS of NT-3 and TrkC knockout mice. We also observed that immature and mature OL-specific markers were attenuated in the NT-3 and TrkC knockout animals. Deficiencies in other CNS glial cells, including astrocytes and ameboid microglia, were also observed. The subventricular zone (SVZ), a highly proliferative region for progenitor glial cells, was reduced in size. Furthermore, a nuclear-specific stain revealed a decline in the numbers of pyknotic nuclei in and around the SVZ of the knockout mice. These data will support an in vivo NT-3-dependent mechanism for the normal development of CNS glial cells.
To examine functions of TrkB in the adult CNS, TrkB has been removed from neurons expressing CaMKII, primarily pyramidal neurons, using Cre-mediated recombination. A floxed trkB allele was designed so that neurons lacking TrkB express tau-beta-galactosidase. Following trkB deletion in pyramidal cells, their dendritic arbors are altered, and cortical layers II/III and V are compressed, after which there is an apparent loss of mutant neurons expressing the transcription factor SCIP but not of those expressing Otx-1. Loss of neurons expressing SCIP requires deletion of trkB within affected neurons; reduction of neuronal ER81 expression does not, suggesting both direct and indirect effects of TrkB loss. Thus, TrkB is required for the maintenance of specific populations of cells in the adult neocortex.
The spatial and temporal expression of the protein-tyrosine kinase B (TrkB) receptor and its ligands has been correlated with the development of the neocortex. Activation of the receptor has been associated with neocortical neuronal survival, differentiation, connectivity and neurotransmitter release. Although such findings suggest an important role for TrkB signaling in corticogenesis, conclusive evidence from targeted gene deletion ("knockout"; TrkB -/-) mice has been limited, due in part to the neonatal lethality of most of these mutant mice and the confounding variables associated with the poor health of those few surviving slightly longer postnatally. In the present study, the effects of TrkB signaling on the survival, differentiation and integration of neocortical neurons was directly investigated in vitro and in vivo. First, we conducted a neuron-specific immunocytochemical analysis of TrkB -/- mice to determine whether early cortical structure and patterns of histogenesis were normal or perturbed. We then employed in vitro and in vivo approaches to extend the life of TrkB -/- neocortical neurons beyond the period possible in TrkB -/- mutant mice themselves: (i) dissociated cell culture to directly compare the developmental potential of TrkB -/-, +/- and +/+ neurons; and (ii) neural transplantation into homochronic wild-type recipients to investigate the cell-autonomous effects of the receptor knockout on the differentiation, growth and integration of neocortical neurons. These latter experiments allowed, for the first time, study of the survival and differentiation potential of TrkB -/- neocortical neurons beyond the initial stages of corticogenesis. Direct comparison of brains of TrkB -/-, +/- and +/+ littermates immunocytochemically labeled with antibodies to microtubule-associated protein-2, neurofilament and beta-tubulin III revealed subtle anatomical anomalies in the mutant mice. These anomalies include abnormally diffuse microtubule-associated protein-2 positive neurons just dorsal to the corpus callosum, and heterotopic aggregations of postmitotic neurons in the subventricular zones of the ganglionic eminences, both suggesting delayed neuronal migration and differentiation. Cell culture experiments revealed substantially reduced survival by TrkB -/- neocortical neurons, and a significant reduction in neurite outgrowth by surviving TrkB -/- neurons. In experiments where prelabeled embryonic or neonatal TrkB -/- neocortical neurons were transplanted into the cerebral cortices of neonatal wild-type recipients, a similar quantitatively significant defect in the formation of dendrites, as well as reduced integration of TrkB -/- neocortical neurons, was also evident. These findings demonstrate cell-autonomous abnormalities in the development of neocortical neurons from TrkB -/- mice, and the subtle, but potentially critical, role of protein-tyrosine kinase B signaling in neocortical neuronal survival, differentiation and connectivity.
To determine how signals emanating from Trk transmit neurotrophin actions in primary neurons, we tested the ability of TrkB mutated at defined effector binding sites to promote sympathetic neuron survival or local axon growth. TrkB stimulated signaling proteins and induced survival and growth in a manner similar to TrkA. TrkB mutated at the Shc binding site supported survival and growth poorly relative to wild-type TrkB, whereas TrkB mutated at the PLC-gamma1 binding site supported growth and survival well. TrkB-mediated neuronal survival was dependent on P13-kinase and to a lesser extent MEK activity, while growth depended upon both MEK and P13-kinase activities. These results indicate that the TrkB-Shc site mediates both neuronal survival and axonal outgrowth by activating the P13-kinase and MEK signaling pathways.
In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Formation of the thalamocortical tract occurs during pre- and postnatal development, with distinct thalamic nuclei projecting to specific cortical regions. The molecular forces that underlie the invasion by axons into specific cortical layers followed by activity-dependent maturation of synapses are poorly understood. We show that genetic ablation of neurotrophin-3 (NT-3) in the mouse neocortex results in reduction of a set of anatomically distinct axonal bundles projecting from thalamus through cortical white matter. These bundles include thalamocortical axons that normally establish connections with retrosplenial and visual cortex, sites of early postnatal NT-3 expression. These results implicate neurotrophins in the critical stage of precise thalamocortical connections.
Mammalian neurogenesis is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues such as growth factors. Here we have defined a signaling cascade, a MEK-C/EBP pathway, that is essential for cortical progenitor cells to become postmitotic neurons. Inhibition of MEK or of the C/EBP family of transcription factors inhibits neurogenesis while expression of a C/EBPbeta mutant that is a phosphorylation-mimic at a MEK-Rsk site enhances neurogenesis. C/EBP mediates this positive effect by direct transcriptional activation of neuron-specific genes such as Talpha1 alpha-tubulin. Conversely, inhibition of C/EBP-dependent transcription enhances CNTF-mediated generation of astrocytes from the same progenitor cells. Thus, activation of a MEK-C/EBP pathway enhances neurogenesis and inhibits gliogenesis, thereby providing a mechanism whereby growth factors can selectively bias progenitors to become neurons during development.
GABAergic interneurons perform crucial roles in cortical development and function. These roles are executed by a diversity of interneuron subtypes, and abnormal function of particular subtypes has been implicated in a variety of neuropsychiatric diseases. However, little is known about the mechanisms that generate interneuron diversity. This paper discusses the potential origins of interneuron subtypes. Evidence is reviewed that suggests bipolar calretinin expressing interneurons may have distinct origins from those that express parvalbumin or somatostatin. In addition, evidence is presented that migratory cells from the subcortical subventricular zone (SVZ) do not proliferate after migration into the cortical SVZ.
Cultured embryonic cortical progenitor cells will mimic the temporal differentiation pattern observed in vivo, producing neurons first and then glia. Here, we investigated the role of two endogenously produced growth factors, the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 (NT-3), in the early progenitor-to-neuron transition. Cultured cortical progenitors express BDNF and NT-3, as well as their receptors TrkB (tyrosine kinase receptor B) and TrkC. Inhibition of these endogenously expressed neurotrophins using function-blocking antibodies resulted in a marked decrease in the survival of cortical progenitors, accompanied by decreased proliferation and inhibition of neurogenesis. Inhibition of neurotrophin function also suppressed the downstream Trk receptor signaling pathways, PI3-kinase (phosphatidyl inositol-3-kinase) and MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase), indicating the presence of autocrine-paracrine neurotrophin:Trk receptor signaling in these cells. Moreover, specific inhibition of these two Trk signaling pathways led to distinct biological effects; inhibition of PI3-kinase decreased progenitor cell survival, whereas inhibition of MEK selectively blocked the generation of neurons, with no effects on survival or proliferation. Thus, neurotrophins made by cortical progenitor cells themselves signal through the TrkB and TrkC receptors to mediate cortical progenitor cell survival and neurogenesis via two distinct downstream signaling pathways.
The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P3, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.
Cancer development requires the acquisition of several capabilities that include increased replicative potential, anchorage and growth-factor independence, evasion of apoptosis, angiogenesis, invasion of surrounding tissues and metastasis. One protein that has emerged as promoting many of these phenotypes when dysregulated is integrin-linked kinase (ILK), a unique intracellular adaptor and kinase that links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. The recent findings of increased levels of ILK in various cancers, and that inhibition of ILK expression and activity is antitumorigenic, makes ILK an attractive target for cancer therapeutics.
The mammalian cerebral cortex comprises six layers of neurons. Cortical progenitors in the ventricular zone generate neurons specific to each layer through successive cell divisions. Neurons of layer VI are generated at an early stage, whereas later-born neurons occupy progressively upper layers. The underlying molecular mechanisms of neurogenesis, however, are relatively unknown. In this study, we devised a system where the Notch pathway was activated spatiotemporally in the cortex by in vivo electroporation and Cre-mediated DNA recombination. Electroporation at E13.5 transferred DNA to early progenitors that gave rise to neurons of both low and upper layers. Forced expression of a constitutively active form of Notch (caNotch) at E13.5 inhibited progenitors from generating neurons and kept progenitors as proliferating radial glial cells. After subsequent transfection at E15.5 of a Cre expression vector to remove caNotch, double-transfected cells, in which caNotch was excised, migrated into the cortical plate and differentiated into neurons specific to upper layers. Bromodeoxyuridine-labeling experiments showed that the neurons were born after Cre transfection. These results indicate that cortical progenitors that had been temporarily subjected to Notch activation at an early stage generated neurons at later stages, but that the generation of low-layer neurons was skipped. Moreover, the double-transfected cells gave rise to upper-layer neurons, even after their transplantation into the E13.5 brain, indicating that the developmental state of progenitors is not halted by caNotch activity.
Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.
Brain-derived neurotrophic factor (BDNF) and other neurotrophins induce a unique prolonged activation of mitogen-activated protein kinase (MAPK) compared with growth factors. Characterization and kinetic and spatial modeling of the signaling pathways underlying this prolonged MAPK activation by BDNF will be important in understanding the physiological role of BDNF in many complex systems in the nervous system. In addition to Shc, fibroblast growth factor receptor substrate 2 (FRS2) is required for the BDNF-induced activation of MAPK. BDNF induces phosphorylation of FRS2. However, BDNF does not induce phosphorylation of FRS2 in cells expressing a deletion mutant of TrkB (TrkBDeltaPTB) missing the juxtamembrane NPXY motif. This motif is the binding site for SHC. NPXY is the consensus sequence for phosphotyrosine binding (PTB) domains, and notably, FRS2 and SHC contain PTB domains. This NPXY motif, which contains tyrosine 484 of TrkB, is therefore the binding site for both FRS2 and SHC. Moreover, the proline containing region (VIENP) of the NPXY motif is also required for FRS2 and SHC phosphorylation, which indicates this region is an important component of FRS2 and SHC recognition by TrkB. Previously, we had found that the phosphorylation of FRS2 induces association of FRS2 and growth factor receptor binding protein 2 (Grb2). Now, we have intriguing data that indicates BDNF induces association of the SH2 domain containing protein tyrosine phosphatase, Shp2, with FRS2. Moreover, the PTB association motif of TrkB containing tyrosine 484 is required for the BDNF-induced association of Shp2 with FRS2 and the phosphorylation of Shp2. These results imply that FRS2 and Shp2 are in a BDNF signaling pathway. Shp2 is required for complete MAPK activation by BDNF, as expression of a dominant negative Shp2 in cells attenuates BDNF-induced activation of MAPK. Moreover, expression of a dominant negative Shp2 attenuates Ras activation showing that the protein tyrosine phosphatase is required for complete activation of MAPKs by BDNF. In conclusion, Shp2 regulates BDNF signaling through the MAPK pathway by regulating either Ras directly or alternatively, by signaling components upstream of Ras. Characterization of MAPK signaling controlled by BDNF is likely to be required to understand the complex physiological role of BDNF in neuronal systems ranging from the regulation of neuronal growth and survival to the regulation of synapses.
Neurotrophins and their cognate trk receptors regulate key events, most notably survival and differentiation of specific neuron populations, during the development of the nervous system. Their functions in the adult and aged CNS are far less well understood. We have analysed mice aged 21-23 months with haploinsufficiencies of the trkB and/or trkC genes with regard to morphological alterations in the hippocampus and amygdala. Neuronal densities and total numbers of neurons in the dentate gyrus were significantly decreased in trkB+/-, trkC+/-, and trkB+/-/C+/- mutants. In the hippocampal area CA2/3, neuronal density and the total number of neurons were only reduced in double-heterozygous mice. Within the amygdala, neuronal densities were not altered. The lateral, basolateral and basomedial nuclei of the amygdala, as well as the dentate gyrus and area CA3, revealed significant increases in the densities of degenerated axonal fragments; the most pronounced changes were found in the double-heterozygous mice. Thus, partial impairment in trkB and/or trkC receptor expression caused region-specific neuron losses in the hippocampus and increased axonal fragmentation in both hippocampus and amygdala, which may result from degeneration of both intrinsic and extrinsic fibre systems. Together, these data indicate that endogenous ligands to the trkB and trkC receptors are essential to maintain neuronal integrity in the aged hippocampus and amygdala.
Within the developing mammalian CNS, growth factors direct multipotent precursors to generate neurons versus glia, a process that if perturbed might lead to neural dysfunction. In this regard, genetic mutations resulting in constitutive activation of the protein tyrosine phosphatase SHP-2 cause Noonan Syndrome (NS), which is associated with learning disabilities and mental retardation. Here, we demonstrate that genetic knockdown of SHP-2 in cultured cortical precursors or in the embryonic cortex inhibited basal neurogenesis and caused enhanced and precocious astrocyte formation. Conversely, expression of an NS SHP-2 mutant promoted neurogenesis and inhibited astrogenesis. Neural cell-fate decisions were similarly perturbed in a mouse knockin model that phenocopies human NS. Thus, SHP-2 instructs precursors to make neurons and not astrocytes during the neurogenic period, and perturbations in the relative ratios of these two cell types upon constitutive SHP-2 activation may contribute to the cognitive impairments in NS patients.
During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neural circuitry, and subsequent matching of glial numbers and position to that circuitry. Here, we have reviewed work addressing the mechanisms underlying this timed cell genesis, with a particular focus on the developing cortex. These studies have defined an intriguing interplay between intrinsic epigenetic status, transcription factors, and environmental cues, all of which work together to establish this fascinating and complex biological timing mechanism.