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![Phosphorylation state of DCX at Ser332 in developing mouse brain and cultured cortical neurons. A, B: Coronal sections of mouse brain at E14.5 (A, a–e) and E16.5 (A, f–j) and sagittal sections of P12 mouse brain (B, a–e) were immunostained with anti-DCX antibody, anti-pDCXS332 antibody, active JNK (pJNK), and DAPI (blue). Higher immunoreactivities of pDCXS332 and pJNK were found in the intermediate (IZ) of cerebral cortex and premigratory zone of cerebellar cortex (arrows in b'–e'). Images A a'–j' and B a'–e' are higher magnifications of A, a–j and B, a–e, respectively. Scale bar in A is 500 μm, and scale bar in B is 250 μm. CP (cortical plate) and IZ are indicated in the cerebral cortex. External granule cell layer (EGL) in B indicates its position in the cerebellar cortex. C: Phosphorylation of DCX at Ser332 in cortical neurons. The cultured primary cortical neurons at DIV2 were immunostained with anti-DCX antibody, anti-pDCXS332 antibody, anti-Tuj1 antibody, and DAPI (blue). Scale bar is 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]](profile/Junghee-Jin/publication/45661431/figure/fig6/AS:267964234596379@1440899060620/Phosphorylation-state-of-DCX-at-Ser332-in-developing-mouse-brain-and-cultured-cortical.png)
Phosphorylation state of DCX at Ser332 in developing mouse brain and cultured cortical neurons. A, B: Coronal sections of mouse brain at E14.5 (A, a–e) and E16.5 (A, f–j) and sagittal sections of P12 mouse brain (B, a–e) were immunostained with anti-DCX antibody, anti-pDCXS332 antibody, active JNK (pJNK), and DAPI (blue). Higher immunoreactivities of pDCXS332 and pJNK were found in the intermediate (IZ) of cerebral cortex and premigratory zone of cerebellar cortex (arrows in b'–e'). Images A a'–j' and B a'–e' are higher magnifications of A, a–j and B, a–e, respectively. Scale bar in A is 500 μm, and scale bar in B is 250 μm. CP (cortical plate) and IZ are indicated in the cerebral cortex. External granule cell layer (EGL) in B indicates its position in the cerebellar cortex. C: Phosphorylation of DCX at Ser332 in cortical neurons. The cultured primary cortical neurons at DIV2 were immunostained with anti-DCX antibody, anti-pDCXS332 antibody, anti-Tuj1 antibody, and DAPI (blue). Scale bar is 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of D...
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... and neuronal migration defects of cortical neurons in the cerebrum ( Hirai et al., 2006). Three JNK phosphorylation sites on DCX have been identi- fied ( Gdalyahu et al., 2004, namely Thr321, Thr331, and Ser334 in human DCX, and those residues are cor- responded to Thr326, Thr336, and Ser339 in mouse DCX, as shown in Supporting Information Fig. 1), and phosphorylation levels at these sites were decreased in the brain of DLKÀ/À mice (Hirai et al., ...
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... of amino acids corresponds to that of mouse DCX. Mouse DCX Ser332 is equivalent to human DCX Ser327 (Supporting Information Fig. 1). The antise- rum was purified with protein A beads, and this antiserum was affinity-purified by sequentially passing it through nonphosphopeptide and phosphopeptide columns. ...
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... Vitro and In Vivo DCX is a MAP and its carboxyl-terminal (C-termi- nal) is phosphorylated by several kinases. A previous study showed that JNK phosphorylates DCX at three sites (Supporting Information Fig. 1) in vitro and in mouse brain ( Gdalyahu et al., 2004). Graham et al. (2004) reported that Cdk5 phosphorylates multiple sites within the DCX C-terminal region in vitro, including Ser332 (S332). However, among them, only Ser297 was reported to be phosphorylated in the mouse brain ( Tanaka et al., 2004). We generated phosphospecific ...
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... Ser332 (S332). However, among them, only Ser297 was reported to be phosphorylated in the mouse brain ( Tanaka et al., 2004). We generated phosphospecific antibody against DCXS332 and stained embryonic mouse brain. High-level immuno- reactivity of pDCXS332 was observed in the interme- diate zone (IZ) of the cerebral cortex at E14.5 and E16.5 [ Fig. 1(A)]. In the postnatal developing , pDCXS332 was stained in granule cells, which resided in the premigratory or migrating state [Fig. 1(B)]. These results indicate that DCX is phos- phorylated at S332 in developing mouse brain. To determine whether S332 is phosphorylated by Cdk5 in vivo, we examined the phosphorylation level of DCX ...
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... the phosphorylation level of DCX at S332 (pDCXS332/ DCX) in the Cdk5À/À brain was elevated by 2.7-fold as compared with levels in Cdk5+/+ mice [ Fig. 2(B)]. This study also revealed slight elevation of DCX protein in Cdk5À/À mice [ Fig. 2(B)]. Further, pDCXS332 was detected in neuronal soma and neu- rites of cultured primary cortical neurons [ Fig. 1(C)]. pDCXS332 was found at high levels in the cultured neurons at DIV2 to DIV4 (data not shown). When cortical neurons with the Cdk inhibitor roscovitine were incubated at DIV4, the level of pDCXS322 was not altered [Fig. 3(A)]. These results indicate that ki- nase(s) other than Cdk5 phosphorylate DCX at S332 in ...
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... studies have reported that phosphorylation of DCX by JNK at other three sites, which correspond to Thr326, Thr336, and Ser339 in mouse DCX (Sup- porting Information Fig. 1), affected neurite outgrowth and neuronal migration ( Gdalyahu et al., 2004). To investigate the role of phosphorylation of DCX at S332, we introduced GFP, GFP-DCX-WT, or GFP- DCX-S332A into SH-SY5Y neuroblastoma cells. As shown in Figure 4(A), SH-SY5Y cells expressing GFP-DCX-WT increased the incidence and length of neurites extending ...
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... phosphorylation. Localization of pDCXS332 immunoreactivities in developing brain correlated with those of active JNK [ Fig. 1(A)]. Mice deficient in JNK1, JNK2, and JNK3 were generated, and these single-knockout (KO) mice exhibit residual activity of JNK. Unfortunately, JNK1/JNK2 double- Figure 7 Phosphorylation of DCX, including phosphorylation at S332, by JNK reduces its binding to tubulin. A-C: Cultured primary cortical neurons at DIV4 were treated with or ...
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... activity in the IZ and IZ-CP in the cerebral cor- tex was high at E14-16 [Hirai et al., 2002;Kawauchi et al., 2003; Fig. 1(A)] as well as in the premigratory zone in developing cerebellar cortex [ Fig. 1(B)], and high levels of DCX phosphorylation at JNK sites were observed in these areas ( Gdalyahu et al., 2004 and pres- ent study). On the basis of the expression patterns of its activating subunits p35 and p39, Cdk5 activity was shown to be high in the IZ-CP ...
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... activity in the IZ and IZ-CP in the cerebral cor- tex was high at E14-16 [Hirai et al., 2002;Kawauchi et al., 2003; Fig. 1(A)] as well as in the premigratory zone in developing cerebellar cortex [ Fig. 1(B)], and high levels of DCX phosphorylation at JNK sites were observed in these areas ( Gdalyahu et al., 2004 and pres- ent study). On the basis of the expression patterns of its activating subunits p35 and p39, Cdk5 activity was shown to be high in the IZ-CP and CP ( Ohshima et al., 2002). The phosphorylation state of DCX by several ki- ...
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... However, S332 is probably not a GSK3b primed site because treatment of GSK3b inhibitors did not affect the phosphorylation level of DCXS332 [ Fig. 3(B)]. Because high levels of active JNK have been observed in the premigratory and migrating neurons in the developing cerebral and cer- ebellar cortices (Hirai et al., 2002;Kawauchi et al., 2003; Fig. 1), immature neurons may exhibit high JNK activity. DCX is also highly expressed in imma- ture-stage neurons and levels of DCX are elevated in the Cdk5À/À embryonic brain [ Fig. 2(A)]. This find- ing suggests a delay in neuronal maturation in the Cdk5À/À embryonic brain. The results of the immu- nohistochemical study of the embryonic ...
Citations
... In DCX, phosphorylation of S297 shifted the distribution of DCX away from MT bundles, and a phospho-mimetic S297D substitution led to impaired MT polymerization in vitro [18]. Phosphorylation of the DCX-PEST domain by JNK1 promoted its mobilization to the growth cones of the leading edge in migrating neurons, suggesting a function for these phosphorylation sites in actin cytoskeleton dynamics [105,107]. In addition, inhibition of the phosphatase PP2A resulted in a rapid loss of DCX localization to the neurite tips and DCX accumulation in the cell body [20]. ...
... Multiple (auto-)phosphorylation sites within the DC linker and PEST region have been identified for both DCLK1 and DCX and have been functionally linked to altered localization and MT polymerization activity [18,20,66,70,89,[105][106][107]. Accordingly, substitutions of any of the phosphorylated serine or threonine residues (S158W, S305G, T311I, S330L, S334L, and S337N) are anticipated to result in functional changes ( Figure S1C). ...
... Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines11030990/s1, Figure S1: In silico prediction of proteins that interact with the (A) N-terminal region, (B) DC-linker region, and (C) PEST linker region for isoforms 1 & 2 (top) and isoforms 3 & 4 (bottom); Figure S2: DCLK1 missense mutations in cancer; Table S1: Experimentally determined structures of the DC1 and DC2 domains from DCX and DCLK1, and the DCLK1 kinase domain; Table S2: Functional residues of DCLK1 and their mutation frequency; Table S3: Predicted impact of point mutations on the structural stability of DCLK1. References [18,20,48,49,[51][52][53][54]70,[73][74][75]80,83,85,86,89,95,105,107,115,120,[135][136][137][138] ...
Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αβ-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.
... DCX loss-of-function and gain-of-function increase or decrease the number of cells in the MP stage [100,101]. GSK3β and JNK phosphorylate DCX in neurons and thereby inhibit axon branching and self-contact and enhance neurite extension [102,103]. ...
The establishment and maintenance of neuronal polarity are important for neural development and function. Abnormal neuronal polarity establishment commonly leads to a variety of neurodevelopmental disorders. Over the past three decades, with the continuous development and improvement of biological research methods and techniques, we have made tremendous progress in the understanding of the molecular mechanisms of neuronal polarity establishment. The activity of positive and negative feedback signals and actin waves are both essential in this process. They drive the directional transport and aggregation of key molecules of neuronal polarity, promote the spatiotemporal regulation of ordered and coordinated interactions of actin filaments and microtubules, stimulate the specialization and growth of axons, and inhibit the formation of multiple axons. In this review, we focus on recent advances in these areas, in particular the important findings about neuronal polarity in two classical models, in vitro primary hippocampal/cortical neurons and in vivo cortical pyramidal neurons, and discuss our current understanding of neuronal polarity.
... This dynamic process is likely regulated directly by the interactions of the C-terminal and N-terminal domains or indirectly through phosphorylation of residues in the C-terminal domain. Since the C-terminal domain has several phosphorylation sites (Graham et al., 2004;Jin et al., 2010;Shmueli et al., 2006;Slepak et al., 2012;Tanaka et al., 2004), it will be interesting in future studies to determine whether phosphorylation of residues in the C-terminal domain regulates the intramolecular interactions of the C-and N-terminal domain and with it DCX's 'closed' state. In doing so, phosphorylation of the C-terminal domain could regulate whether DCX preferentially binds MTs or the DDJ complex. ...
Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport in neurons from Dcx -/y or Dcx -/y ;Dclk1 -/- mice by reducing dynein's association with MTs and by disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and by regulating the composition of the dynein motor complex.
... MAP2 phosphorylations at Thr1619, Thr1622, and Thr1625 increase the dendritic complexity and stabilize MTs in rats [89]. Conversely, doublecortin phosphorylations by JNK1/2 at Thr321, Thr331, Ser334 (Thr326, Thr336, Ser339 in human) [90] and at Ser332 favor neurite development and neuronal migration by decreasing the affinity of this stabilizing MAP for tubulin and MTs [91]. Since doublecortin can switch from MT-to F-actin-binding [92], this may explain its positive effects on neurons [93] despite its release from MTs. ...
This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.
... In addition, neuron-specific Mkk7 knockout mice displayed age-dependent motor dysfunction (Yamasaki et al., 2017). JNK phosphorylates doublecortin at S332 to modulate neurite extension and neuronal migration in vivo (Jin et al., 2010). The Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic accumulation of PSD-95 (Kim et al., 2007). ...
The c-Jun N-terminal kinase (JNK) is highly evolutionarily conserved and plays important roles in a broad range of physiological and pathological processes. The WD40-repeat protein 62 (WDR62) is a scaffold protein that recruits different components of the JNK signaling pathway to regulate several human diseases including neurological disorders, infertility, and tumorigenesis. Recent studies revealed that WDR62 regulates the process of neural stem cell mitosis and germ cell meiosis through JNK signaling. In this review we summarize the roles of WDR62 and JNK signaling in neuronal and non-neuronal contexts and discuss how JNK-dependent signaling regulates both processes. WDR62 is involved in various human disorders via JNK signaling regulation, and may represent a promising therapeutic strategy for the treatment of related diseases.
... (a) DCX decorates microtubules in the perinuclear cage and the growth cone of the leading process [36]. (b) JNK phosphorylation of Ser332, Thr326 and Thr336 in DCX reduces affinity to microtubules and causes delayed migration in cortical neuron cultures [36,114]. (c) Phospho-DCX, phosphor-SGC10 and phospho-JNK overlap in the intermediate zone in developing cortices. ...
The c-Jun N-terminal Kinases (JNKs) are a group of regulatory elements responsible for the control of a wide array of functions within the cell. In the central nervous system (CNS), JNKs are involved in neuronal polarization, starting from the cell division of neural stem cells and ending with their final positioning when migrating and maturing. This review will focus mostly on isoform JNK1, the foremost contributor of total JNK activity in the CNS. Throughout the text, research from multiple groups will be summarized and discussed in order to describe the involvement of the JNKs in the different steps of neuronal polarization. The data presented support the idea that isoform JNK1 is highly relevant to the regulation of many of the processes that occur in neuronal development in the CNS.
... Another important regulator of nucleokinesis and leading process branching is the microtubule associated protein doublecortin (Dcx; Kappeler et al., 2006;Friocourt et al., 2007), which is a downstream target of JNK signaling in neurons (Gdalyahu et al., 2004;Jin et al., 2010). Cortical interneurons lacking Dcx show a decreased duration of interstitial side branches, and significantly shorter nuclear translocation distances with no overall changes in migratory speed (Kappeler et al., 2006), similar to what we found in cTKO interneurons. ...
Aberrant migration of inhibitory interneurons can alter the formation of cortical circuitry and lead to severe neurological disorders including epilepsy, autism, and schizophrenia. However, mechanisms involved in directing the migration of interneurons remain incompletely understood. Using a mouse model, we performed live-cell confocal microscopy to explore the mechanisms by which the c-Jun NH2-terminal kinase (JNK) pathway coordinates leading process branching and nucleokinesis, two cell biological processes that are essential for the guided migration of cortical interneurons. Pharmacological inhibition of JNK signaling disrupts the kinetics of leading process branching, rate and amplitude of nucleokinesis, and leads to the rearward mislocalization of the centrosome and primary cilium to the trailing process. Genetic loss of Jnk from interneurons also impairs leading process branching and nucleokinesis, suggesting that important mechanics of interneuron migration depend on the intrinsic activity of JNK. These findings highlight key roles for JNK signaling in leading process branching, nucleokinesis, and the trafficking of centrosomes and cilia during interneuron migration, and further implicates JNK signaling as an important mediator of cortical development.
... Among the phosphorylation targets of JNK1 are regulators of microtubule and actin dynamics. JNK1 regulates neuronal architecture and neuronal migration by phosphorylating targets such as microtubule-associated protein 2 (MAP2), stathmin-2, myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), and doublecortin (DCX) [25][26][27][28][29][30][31]. Studies in Jnk1-/mice demonstrated that JNK1 controls dendritic arborisation in the cortex and cerebellum [26,30]. ...
... We first re-evaluated the JNK phosphorylation sites on DCX by phospho-blotting extracts from wild-type and Jnk knockout mouse brains (Figure 5a). This data validated the previous finding that GFP-DCX-T331 and -S334 were phosphorylated by JNKs [25,28], as phosphorylation on these sites was substantially depleted in Jnk1-/-, Jnk2-/-, and Jnk3-/brains ( Figure 5a). We next tested whether phospho-mutants would rescue spine development. ...
... Among the cytoskeletal regulatory proteins that JNK phosphorylates is DCX [25,28]. Here, we demonstrated using gene knockdown that DCX was critical for maintaining mushroom spine density in hippocampal neurons, consistent with a previous study that showed the same in the olfactory bulb [56]. ...
The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates.
... One means by which JNK could exert a cell autonomous influence on cortical interneuron migration is through modulation of the cytoskeleton. For example, doublecortin, a microtubule-binding protein known to regulate leading process branching and guided migration of cortical interneurons (Friocourt et al., 2007;Kappeler et al., 2006), is a downstream target of JNK signaling (Gdalyahu et al., 2004;Jin et al., 2010). Additionally, p27 kip1 is a microtubule-associated protein that coordinates microtubule polymerization and actomyosin contraction to attune leading process branching and nucleokinesis (Godin et al., 2012). ...
The precise migration of cortical interneurons is essential for the formation and function of cortical circuits, and disruptions to this key developmental process are implicated in the etiology of complex neurodevelopmental disorders, including schizophrenia, autism, and epilepsy. We recently identified the c-Jun N-terminal kinase (JNK) pathway as an important mediator of cortical interneuron migration, regulating the proper timing of interneuron arrival into the cortical rudiment. In the current study, we demonstrate a vital role for JNK signaling at later stages of corticogenesis, when interneurons transition from tangential to radial modes of migration. Pharmacological inhibition of JNK signaling in ex vivo slice cultures caused cortical interneurons to rapidly depart from migratory streams and prematurely enter the cortical plate. Similarly, genetic loss of JNK function led to precocious stream departure ex vivo, and stream disruption, morphological changes, and abnormal allocation of cortical interneurons in vivo. These data suggest that JNK signaling facilitates the tangential migration and laminar deposition of cortical interneurons, and further implicates the JNK pathway as an important regulator of cortical development.
... Activity of DCX is regulated through the phosphorylation of different sites controlled by several intracellular signaling cascades, including the JNK and PKA/Akt pathways. JNK phosphorylates DCX at multiple sites, contributing significantly to neurite outgrowth and migration [75,76]. PKA similarly regulates DCX at the leading edge of migrating NPCs through phosphorylation of Ser47 [77,78]. ...
Neural stem cells present in the subventricular zone (SVZ), the largest neurogenic niche of the mammalian brain, are able to self-renew as well as generate neural progenitor cells (NPCs). NPCs are highly migratory and traverse the rostral migratory stream (RMS) to the olfactory bulb, where they terminally differentiate into mature interneurons. NPCs from the SVZ are some of the few cells in the CNS that migrate long distances during adulthood. The migratory process of NPCs is highly regulated by intracellular pathway activation and signaling from the surrounding microenvironment. It involves modulation of cell volume, cytoskeletal rearrangement, and isolation from compact extracellular matrix. In malignant brain tumors including high-grade gliomas, there are cells called brain tumor stem cells (BTSCs) with similar stem cell characteristics to NPCs but with uncontrolled cell proliferation and contribute to tumor initiation capacity, tumor progression, invasion, and tumor maintenance. These BTSCs are resistant to chemotherapy and radiotherapy, and their presence is believed to lead to tumor recurrence at distal sites from the original tumor location, principally due to their high migratory capacity. BTSCs are able to invade the brain parenchyma by utilizing many of the migratory mechanisms used by NPCs. However, they have an increased ability to infiltrate the tight brain parenchyma and utilize brain structures such as myelin tracts and blood vessels as migratory paths. In this article, we summarize recent findings on the mechanisms of cellular migration that overlap between NPCs and BTSCs. A better understanding of the intersection between NPCs and BTSCs will to provide a better comprehension of the BTSCs’ invasive capacity and the molecular mechanisms that govern their migration and eventually lead to the development of new therapies to improve the prognosis of patients with malignant gliomas.
