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Morphology of Purkinje cells in P10 rats and in culture. Purkinje cells were stained using a monoclonal antibody against calbindin28 kD protein (see Materials and Methods). Note the increased cell size and vigorous neurite outgrowth in NT-3 treated cultures. There were 58 + 4 ealbindin-28 kD positive cells per field in controls and 60 + 3 (n = 6) in NT-3-treated cultures. (A) Hypothyroid rats; (B) Control euthyroid rats; (C) Cultured Purkinje cells, controls; and (D) cells treated with 30 ng NT-3 per ml. Bar, 25 #m.
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Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridiza...
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... Purkinje cells are heavily affected by hypothy- roidism ( Legrand, 1979;Legrand and Clos, 1991) and characteristically exhibit a lower degree of dendritic arbor- ization as compared with control rots (Fig. 5). To study whether NT-3 affects Purkinje cell differentiation, cerebellar cultures enriched for these cells from embryonic rats were established. Addition of recombinant NT-3 ( G0tz et al., 1992) to these cultures led to a vigorous outgrowth of neu- rites of cultured Purkinje cells, which were identified by staining with an antibody ...
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... cell differentiation, cerebellar cultures enriched for these cells from embryonic rats were established. Addition of recombinant NT-3 ( G0tz et al., 1992) to these cultures led to a vigorous outgrowth of neu- rites of cultured Purkinje cells, which were identified by staining with an antibody against the calcium-binding pro- tein calbindin-28 kD (Fig. 5, lower panel). Likewise, NT-3 also increased Purkinje cell body size without increasing their number (Fig. ...
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... of recombinant NT-3 ( G0tz et al., 1992) to these cultures led to a vigorous outgrowth of neu- rites of cultured Purkinje cells, which were identified by staining with an antibody against the calcium-binding pro- tein calbindin-28 kD (Fig. 5, lower panel). Likewise, NT-3 also increased Purkinje cell body size without increasing their number (Fig. ...
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... levels of mRNA encoding calbindin-28 kD, exclusively ex- pressed in these cells in cerebellum ( Jande et al., 1981), were measured using a specific eDNA probe. Fig. 6 shows that NT-3 induced (2.5-fold) ealbindin-mRNA in these cultures whereas T3 did not. Thus, mRNA data corroborate those obtained by staining of the cells with a specific antibody (Fig. 5) and demonstrate that NT-3 promotes the differentia- tion of Purkinje cells, whereas the "1"-3 effect is an indirect one regulating the NT-3 expression in granule cells which then regulates the differentiation of Purkinje cells in an or- thograde ...
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... Importantly, the vesicle release of neurotransmitters, neuropeptides and neurotrophin-3 has a common upstream mediator in Cadps2, which showed decreased cerebellar mRNA levels [151][152][153][154][155][156]. The balance between neurotrophin support and glutamate neurotoxicity is known to be critical also for the survival of Purkinje neurons [157][158][159][160]. ...
The autosomal recessive disorder Ataxia-Telangiectasia is caused by dysfunction of the stress response protein ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumor risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies the cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits by bone-marrow transplantation, and reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated ATM depletion to trigger upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently neuropeptide machinery, e.g. Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM localized only to cytoplasm, similar to brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient / oxidative stress, not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.
... Differentiation of iPSCs into Purkinje cells was conducted following modifications of protocols established from [15,16,[24][25][26][27][28][29][30][31][32][33][34]. The iPSCs were dissociated with StemPro Accutase (Thermo Fisher Scientific), combined with 5 μM Y-27632 (Abcam), and diluted in DMEM-F12 medium added with 5 μM Y-27632 without serum. ...
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease mainly characterized by spasticity in the lower limbs and poor muscle control. The disease is caused by mutations in the SACS gene leading in most cases to a loss of function of the sacsin protein, which is highly expressed in motor neurons and Purkinje cells. To investigate the impact of the mutated sacsin protein in these cells in vitro, induced pluripotent stem cell- (iPSC-) derived motor neurons and iPSC-derived Purkinje cells were generated from three ARSACS patients. Both types of iPSC-derived neurons expressed the characteristic neuronal markers β3-tubulin, neurofilaments M and H, as well as specific markers like Islet-1 for motor neurons, and parvalbumin or calbindin for Purkinje cells. Compared to controls, iPSC-derived mutated SACS neurons expressed lower amounts of sacsin. In addition, characteristic neurofilament aggregates were detected along the neurites of both iPSC-derived neurons. These results indicate that it is possible to recapitulate in vitro, at least in part, the ARSACS pathological signature in vitro using patient-derived motor neurons and Purkinje cells differentiated from iPSCs. Such an in vitro personalized model of the disease could be useful for the screening of new drugs for the treatment of ARSACS.
... Whilst the expression of BDNF is well known to be regulated by neuronal activity (West et al., 2002) such is not the case with NTF3 (Hernandez-Echeagaray, 2020). The genomic organisation of NTF3 is much less complex than BDNF (West et al., 2014) and the levels of NTF3 are regulated by hormones such as thyroxin, rather than by neuronal activity (Lindholm et al., 1993). Downstream of Trk activation, the results of the RNAseq experiments indicated that the transcriptional F I G U R E 8 Phosphorylation of TrkB following prolonged exposure of H9 neurons to low concentrations of BDNF can be recovered upon re-exposure to BDNF. ...
In the central nervous system, most neurons co‐express TrkB and TrkC, the tyrosine kinase receptors for brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down‐regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.
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... NGF plays an important role in the processes of neuronal survival, ischemic tolerance of the brain and it is involved in the mechanism, by which neurons can be protected from cell death (Shigeno et al. 1991). Several studies have also shown that the thyroid hormone regulates NGF expression (Lindholm et al. 1993). Thyroxine given to adult rats and mice elevates NGF levels in the cortex, hippocampus, and cerebellum (Giordano et al. 1992). ...
... NTF3, a member of the NT family, has emerged as a key mediator of neuronal development which promotes the survival/differentiation cascade during neuronal differentiation (Lin et al. 2018). The expression of NTF3 is regulated by thyroid hormones (Lindholm et al. 1993). T3 induces NTF3 mRNA expression and neuronal development in the neonatal cerebellum (Lindholm et al. 1993). ...
... The expression of NTF3 is regulated by thyroid hormones (Lindholm et al. 1993). T3 induces NTF3 mRNA expression and neuronal development in the neonatal cerebellum (Lindholm et al. 1993). Thyroxine, which is administered to adult rats and mice, elevates NTF3 levels in the cortex, hippocampus, and cerebellum (Giordano et al. 1992). ...
Objective. Thyroid hormones play an important role in the development and maturation of the central nervous symptom and their failure in the prenatal period leading to an irreversible brain damage. Their effect on the brain of adult, however, has not been fully studied. With the discovery of neurogenesis in the adult brain, many recent studies have been focused on the understanding the basic mechanisms controlling this process. Many neurogenesis regulatory genes are not only transcribed but also translated into the blood cells. The goal of our study was to analyze the transcriptional activity of neurogenesis regulatory genes in peripheral blood cells in patients with thyroid pathology. Methods. The pathway-specific PCR array (Neurotrophins and Receptors RT2 Profiler PCR Array , QIAGEN, Germany) was used to identify and validate the neurogenesis regulatory genes expression in patients with thyroid pathology and control group. Results. The results showed that GFRA3, NGFR, NRG1, NTF3, NTRK1, and NTRK2 significantly decreased their expression in patients with autoimmune thyroiditis with rising serum of autoantibodies. The patients with primary hypothyroidism, as a result of autoimmune thyroiditis and postoperative hypothyroidism, had significantly lower expression of FGF2, NGFR, NRG1, and NTF3. The mRNA level of CNTFR was markedly decreased in the group of patients with postop-erative hypothyroidism. No change in the ARTN, PSPN, TFG, MT3, and NELL1 expression was observed in any group of patients. Conclusion. The finding indicates that a decrease in thyroid hormones and a high level of au-toantibodies, such as anti-thyroglobulin antibody and anti-thyroid peroxidase antibody, affect the expression of mRNA neurogenesis-regulated genes in patients with thyroid pathology.
... Granule cells secrete neurotropic factors like BDNF to stimulate PC growth and survival. This raises the discussion that these neurotrophins are highly responsible for enhanced PC survival (Maisonpierre et al. 1990;Lindholm et al. 1993). ...
Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.
... For our purposes, we focused on the further maturation of Purkinje cells, demonstrating the presence of human calbindin-positive cells after 15 days of co-culture with mouse cerebellar progenitors. In the presence of BDNF and NT3 (known to promote survival and phenotypic differentiation of Purkinje cells [20,21]), these calbindin-positive cells could be maintained in culture for at least 55 days, during which time they continued to undergo lineage specification, producing increasingly elaborate dendritic arbours, and expressing additional markers of Purkinje cell development, including FOXP2 and PCP4. Future work will include immunostaining for markers of mature Purkinje cells, as well as detailed electrophysiology, in order to assess the continuing maturity of these cells in long-term culture. ...
The establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes for study in vitro. However, to date, only a handful of studies have generated Purkinje cells from human pluripotent stem cells, with most of these protocols proving challenging to reproduce. Here, we describe a simplified method for the reproducible generation of Purkinje cells from human iPSCs. After 21 days of treatment with factors selected to mimic the self-inductive properties of the isthmic organiser—insulin, fibroblast growth factor 2 (FGF2), and the transforming growth factor β (TGFβ)-receptor blocker SB431542—hiPSCs could be induced to form En1-positive cerebellar progenitors at efficiencies of up to 90%. By day 35 of differentiation, subpopulations of cells representative of the two cerebellar germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more accessible, in order to enable future advances in the field.
Electronic supplementary material
The online version of this article (10.1007/s12311-017-0913-2) contains supplementary material, which is available to authorized users.
... In contrast, mutant PC had a severely disrupted fiber network, with stunted, thinned dendrites and poorly branched arbors (Fig. 7D, arrowheads). PC dendritic outgrowth depends on electrical activity (Schilling et al., 1991;Baptista et al., 1994) and neurotrophins secreted from CGNPs, including thyroid hormone (Heuer and Mason, 2003), neurotrophin-3 (Lindholm et al., 1993), and BDNF (Shimada et al., 1998). Thus, the disruption in PC arborization seen in Smo cko mutants could be attributed to the loss of the EGL. ...
Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNCYFP-CreER line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNCYFP-CreER; SmoF/- (SmoCKO) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in SmoCKO CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture.
... Three major classes of NTFs regulate neuronal development, survival and maintenance: conventional NTFs, such as BDNF, NT-3 and neurotrophin-4 (NT-4); the cerebral dopamine neurotrophic factor (CDNF)-MANF family; and the glialderived neurotrophic factor (GDNF) family. Several studies have already identified protective roles for BDNF or MANFin preventing Purkinje cell degeneration (Schwartz et al., 1997;Yang et al., 2014) and for NT-3 in promoting Purkinje cell proliferation (Lindholm et al., 1993). However, researchers have not extensively determined whether the loss of Ankfy1 affects NTFs. ...
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disorder. In 2007, a novel locus, SAX2, which is located on chromosome 17p13 and contains 3 genes, ankyrin repeat and FYVE domain-containing 1 (ANKFY1), β-arrestin 2 (ARRB2) and kinesin family member 1C (KIF1C), was linked to ARSACS. We generated Ankfy1 heterozygous (Ankfy1/+) mice to establish an animal model and examine the pathophysiological basis of ARSACS. The transgenic mice displayed an abnormal gait with progressive motor and cerebellar nerve dysfunction that was highly reminiscent of ARSACS. These clinical features were accompanied by an early-onset and progressive loss of Purkinje cells, followed by gliosis. Additionally, the loss of Ankfy1 function resulted in an abnormal expression of neurotrophic factors (NTFs) in the Ankfy1/+ mouse cerebellum. Moreover, Purkinje cells cultured from neonatal Ankfy1/+ mice exhibited a shorter dendritic length and decreased numbers of dendritic spines. Importantly, cerebellar Purkinje cells from Ankfy1/+ mice and cells transfected with a lentiviral Ankfy1 shRNA underwent apoptosis. We propose that transgenic Ankfy1/+ mice are a useful model for studying the pathogenesis of ARSACS and for exploring the molecular mechanisms involved in this neurodegenerative disease.
... These neurotrophins are necessary for the Purkinje cell maturation. This transient-positive feedback loop greatly amplifies the initial response of Purkinje cells to T3. by a positive feedback loop involving the secretion of neurotrophin 3 and of other proteins secreted by neighboring cells (Fauquier et al., 2014;Lindholm et al., 1993;Neveu & Arenas, 1996;Picou et al., 2012). The in vitro response of Purkinje cells to T3 is lost after Thra, but not Thrb knockout, showing a predominant intervention of TRα1 in Purkinje cell maturation (Heuer & Mason, 2003). ...
Thyroid hormones exert a broad influence on brain development and function, which has been extensively studied over the years. Mouse genetics has brought an important contribution, allowing precise analysis of the interplay between TRα1 and TRβ1 nuclear receptors in neural cells. However, the exact contribution of each receptor, the possible intervention of nongenomic signaling, and the nature of the genetic program that is controlled by the receptors remain poorly understood.
... In this study we demonstrated for the first time that proNT3 is a negative regulator of GCP proliferation, acting via p75 NTR . NT3 expression has previously been detected in the developing cerebellum, however the mRNA was localized by in situ hybridization to granule cells (Lindholm et al., 1993) and the protein has been localized to Purkinje cells (Friedman et al., 1998;Zhou and Rush, 1994). ...
Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75 NTR) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75 NTR in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75 NTR , GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.
