CCSer2 gates dynein activity at the cell periphery.

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dynein's trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

Sodium nitroprusside as an adjunctive treatment for schizophrenia reduces the Ndel1 oligopeptidase activity.

OBJECTIVE: Schizophrenia (SCZ) is a disabling disorder that continues to defy clinicians and researchers. We investigated the effects of sodium nitroprusside (sNP) in an animal model of SCZ and as an add-on therapy in patients and the relationship between treatment with sNP and activity of the nDel1 enzyme, whose involvement in the pathophysiology of the disorder has been suggested earlier. METHODS: Ndel1 activity was measured following sNP infusions in spontaneously hypertensive rats (SHR; 2.5 or 5.0 mg/kg) and in a double-blind trial with SCZ patients (0.5 µg/kg/min). RESULTS: Ndel1 activity was significantly reduced after sNP infusion in blood of SHR compared to controls, and in patients receiving sNP (t = 7.756, df = 97, p < 0.0001, dcohen = 1.44) compared to placebo. Reduced Ndel1 activity between baseline and the end of the infusion was only seen in patients after treatment with sNP. CONCLUSION: Our findings suggest that SCZ patients may benefit from adjunctive therapy with sNP and that the Ndel1 enzyme is a candidate biomarker of psychopathology in the disorder. Future research should look into the role of Ndel1 in SCZ and the potential effects of sNP and drugs with similar profiles of action in both animals and patients.

Identification of an ex vivo inhibitor of the schizophrenia biomarker Ndel1 by high throughput screening.

Ndel1 oligopeptidase activity shows promise as a potential biomarker for diagnosing schizophrenia (SCZ) and monitoring early-stage pharmacotherapy. Ndel1 plays a pivotal role in critical aspects of brain development, such as neurite outgrowth, neuronal migration, and embryonic brain formation, making it particularly relevant to neurodevelopmental disorders like SCZ. Currently, the most specific inhibitor for Ndel1 is the polyclonal anti-Ndel1 antibody (NOAb), known for its high specificity and efficient anti-catalytic activity. NOAb has been vital in measuring Ndel1 activity in humans and animal models, enabling the prediction of pharmacological responses to antipsychotics in studies with patients and animals. To advance our understanding of in vivo Ndel1 function and develop drugs for mental disorders, identifying small chemical compounds capable of specifically inhibiting Ndel1 oligopeptidase is crucial, including within living cells. Due to challenges in obtaining Ndel1's three-dimensional structure and its promiscuous substrate recognition, we conducted a high-throughput screening (HTS) of 2,400 small molecules. Nine compounds with IC50-values ranging from 7 to 56 µM were identified as potent Ndel1 inhibitors. Notably, one compound showed similar efficacy to NOAb and inhibited Ndel1 within living cells, although its in vivo use may pose toxicity concerns. Despite this, all identified compounds hold promise as candidates for further refinement through rational drug design, aiming to enhance their inhibitory efficacy, specificity, stability, and biodistribution. Our ultimate goal is to develop druggable Ndel1 inhibitors that can improve the treatment and support the diagnosis of psychiatric disorders like SCZ.

Assessing the role of Ndel1 oligopeptidase activity in congenital Zika syndrome: Potential predictor of congenital syndrome endophenotype and treatment response.

Maternal infections are among the main risk factors for cognitive impairments in the offspring. Zika virus (ZIKV) can be transmitted vertically, causing a set of heterogeneous birth defects, such as microcephaly, ventriculomegaly and corpus callosum dysgenesis. Nuclear distribution element like-1 (Ndel1) oligopeptidase controls crucial aspects of cerebral cortex development underlying cortical malformations. Here, we examine Ndel1 activity in an animal model for ZIKV infection, which was associated with deregulated corticogenesis. We observed here a reduction in Ndel1 activity in the forebrain associated with the congenital syndrome induced by ZIKV isolates, in an in utero and postnatal injections of different inoculum doses in mice models. In addition, we observed a strong correlation between Ndel1 activity and brain size of animals infected by ZIKV, suggesting the potential of this measure as a biomarker for microcephaly. More importantly, the increase of interferon (IFN)-beta signaling, which was used to rescue the ZIKV infection outcomes, also recovered Ndel1 activity to levels similar to those of uninfected healthy control mice, but with no influence on Ndel1 activity in uninfected healthy control animals. Taken together, we demonstrate for the first time here an association of corticogenesis impairments determined by ZIKV infection and the modulation of Ndel1 activity. Although further studies are still necessary to clarify the possible role(s) of Ndel1 activity in the molecular mechanism(s) underlying the congenital syndrome induced by ZIKV, we suggest here the potential of monitoring the Ndel1 activity to predict this pathological condition at early stages of embryos or offspring development, during while the currently employed methods are unable to detect impaired corticogenesis leading to microcephaly. Ndel1 activity may also be possibly used to follow up the positive response to the treatment, such as that employing the IFN-beta that is able to rescue the ZIKV-induced brain injury.

Ndel1 disfavors dynein-dynactin-adaptor complex formation in two distinct ways.

Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex." The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1-Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.

Effects of representative point mutations on dynamic behavior of the DISC1-Ndel1 complex: a molecular dynamics study.

It has been found that the development of schizophrenia and some other psychiatric disorders is related to defects in the normal functioning of Disrupted-In-Schizophrenia 1 (DISC1). It is a large-sized protein containing 855 residues and acts as an active hub at the core of many interactions with various proteins. On the other hand, NudE Neurodevelopment Protein 1 Like 1 (Ndel1) plays a role in nervous system development via interaction with the DISC1. It was shown that some point mutations on DISC1 have clinical implications. In line with these reports, here we have used the NMR structure of the wild-type (WT) C-terminal tail of DISC1 in complex with the N-terminal fragment of Ndel1, and have constructed the three-dimensional structures of L62Q and L29Q mutants, as the pathologic variants of the complex. The time-dependent interaction of DISC1 with Ndel1 in the WT complex and mutants was simulated by performing molecular dynamics (MD) simulation using programs in the GROMACS package. It was found that the flexibility of residues in some regions of the protein chains increases, and secondary structural changes from ordered toward unordered one leads to destabilizing of the complex in mutants. Destabilization of the complex upon substitution of Leu by Gln was also confirmed by analysis of the contact map plot.Communicated by Ramaswamy H. Sarma.

Dynein at the kinetochore.

The microtubule minus-end-directed motility of cytoplasmic dynein 1 (dynein), arguably the most complex and versatile cytoskeletal motor, is harnessed for diverse functions, such as long-range organelle transport in neuronal axons and spindle assembly in dividing cells. The versatility of dynein raises a number of intriguing questions, including how is dynein recruited to its diverse cargo, how is recruitment coupled to activation of the motor, how is motility regulated to meet different requirements for force production and how does dynein coordinate its activity with that of other microtubule-associated proteins (MAPs) present on the same cargo. Here, these questions will be discussed in the context of dynein at the kinetochore, the supramolecular protein structure that connects segregating chromosomes to spindle microtubules in dividing cells. As the first kinetochore-localized MAP described, dynein has intrigued cell biologists for more than three decades. The first part of this Review summarizes current knowledge about how kinetochore dynein contributes to efficient and accurate spindle assembly, and the second part describes the underlying molecular mechanisms and highlights emerging commonalities with dynein regulation at other subcellular sites.

LRRK1-mediated NDEL1 phosphorylation promotes cilia disassembly via dynein-2-driven retrograde intraflagellar transport.

Primary cilia are antenna-like organelles that regulate growth and development via extracellular signals. However, the molecular mechanisms underlying cilia dynamics, particularly those regulating their disassembly, are not well understood. Here, we show that leucine-rich repeat kinase 1 (LRRK1) plays a role in regulating cilia disassembly. The depletion of LRRK1 impairs primary cilia resorption following serum stimulation in cultured cells. Polo-like kinase 1 (PLK1) plays an important role in this process. During ciliary resorption, PLK1 phosphorylates LRRK1 at the primary cilia base, resulting in its activation. We identified nuclear distribution protein nudE-like 1 (NDEL1), which is known to positively regulate cilia disassembly, as a target of LRRK1 phosphorylation. Whereas LRRK1 phosphorylation of NDEL1 on Ser-155 promotes NDEL1 interaction with the intermediate chains of cytoplasmic dynein-2, it is also crucial for triggering ciliary resorption through dynein-2-driven retrograde intraflagellar transport. These findings provide evidence that a novel PLK1-LRRK1-NDEL1 pathway regulates cilia disassembly.

Nde1 and Ndel1: Outstanding Mysteries in Dynein-Mediated Transport.

Cytoplasmic dynein-1 (dynein) is the primary microtubule minus-end directed molecular motor in most eukaryotes. As such, dynein has a broad array of functions that range from driving retrograde-directed cargo trafficking to forming and focusing the mitotic spindle. Dynein does not function in isolation. Instead, a network of regulatory proteins mediate dynein's interaction with cargo and modulate dynein's ability to engage with and move on the microtubule track. A flurry of research over the past decade has revealed the function and mechanism of many of dynein's regulators, including Lis1, dynactin, and a family of proteins called activating adaptors. However, the mechanistic details of two of dynein's important binding partners, the paralogs Nde1 and Ndel1, have remained elusive. While genetic studies have firmly established Nde1/Ndel1 as players in the dynein transport pathway, the nature of how they regulate dynein activity is unknown. In this review, we will compare Ndel1 and Nde1 with a focus on discerning if the proteins are functionally redundant, outline the data that places Nde1/Ndel1 in the dynein transport pathway, and explore the literature supporting and opposing the predominant hypothesis about Nde1/Ndel1's molecular effect on dynein activity.

LIS1 and NDEL1 Regulate Axonal Trafficking of Mitochondria in Mature Neurons.

Defective mitochondrial dynamics in axons have been linked to both developmental and late-onset neurological disorders. Axonal trafficking is in large part governed by the microtubule motors kinesin-1 and cytoplasmic dynein 1 (dynein). Dynein is the primary retrograde transport motor in axons, and mutations in dynein and many of its regulators also cause neurological diseases. Depletion of LIS1, famous for linking dynein deregulation to lissencephaly (smooth brain), in adult mice leads to severe neurological phenotypes, demonstrating post-developmental roles. LIS1 stimulates retrograde transport of acidic organelles in cultured adult rat dorsal root ganglion (DRG) axons but findings on its role in mitochondrial trafficking have been inconsistent and have not been reported for adult axons. Here we report that there is an increased number of mitochondria in cross-sections of sciatic nerve axons from adult LIS1+/- mice. This is probably related to reduced dynein activity as axons from adult rat nerves exposed to the dynein inhibitor, ciliobrevin D also had increased numbers of mitochondria. Moreover, LIS1 overexpression (OE) in cultured adult rat DRG axons stimulated retrograde mitochondrial transport while LIS1 knockdown (KD) or expression of a LIS1 dynein-binding mutant (LIS1-K147A) inhibited retrograde transport, as did KD of dynein heavy chain (DHC). These findings are consistent with our report on acidic organelles. However, KD of NDEL1, a LIS1 and dynein binding protein, or expression of a LIS1 NDEL1-binding mutant (LIS1-R212A) also dramatically impacted retrograde mitochondrial transport, which was not the case for acidic organelles. Manipulations that disrupted retrograde mitochondrial transport also increased the average length of axonal mitochondria, suggesting a role for dynein in fusion or fission events. Our data point to cargo specificity in NDEL1 function and raise the possibility that defects in the LIS1/NDEL1 dynein regulatory pathway could contribute to mitochondrial diseases with axonal pathologies.

miR-103-3p targets Ndel1 to regulate neural stem cell proliferation and differentiation.

The regulation of adult neural stem cells (NSCs) is critical for lifelong neurogenesis. MicroRNAs (miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, miR-103-3p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that miR-103-3p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, miR-103-3p negatively regulated NudE neurodevelopment protein 1-like 1 (Ndel1) expression by binding to the 3' untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3a, ß-catenin, phosphor-GSK-3ß, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/ß-catenin signaling pathway. These findings suggest that Ndel1 is a novel miR-103-3p target and that miR-103-3p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. 20200826-003) on August 26, 2020.

Nde1 is a Rab9 effector for loading late endosomes to cytoplasmic dynein motor complex.

Rab9 is mainly located on late endosomes and required for their intracellular transport to trans-Golgi network (TGN). The cytoplasmic dynein motor, together with its regulatory proteins Nde1/Ndel1 and Lis1, controls intracellular retrograde transport of membranous organelles along the microtubule network. How late endosomes are tethered to the microtubule-based motor dynein for their retrograde transport remains unclear. Here, we demonstrate that the guanosine triphosphate (GTP)-bound Rab9A/B specifically uses Nde1/Ndel1 as an effector to interact with the dynein motor complex. We determined the crystal structure of Rab9A-GTP in complex with the Rab9-binding region of Nde1. The functional roles of key residues involved in the Rab9A-Nde1 interaction are verified using biochemical and cell biology assays. Rab9A mutants unable to bind to Nde1 also failed to associate with dynein, Lis1, and dynactin. Therefore, Nde1 is a Rab9 effector that tethers Rab9-associated late endosomes to the dynein motor for their retrograde transport to the TGN.

Behavioral Deficits in Mice with Postnatal Disruption of Ndel1 in Forebrain Excitatory Neurons: Implications for Epilepsy and Neuropsychiatric Disorders.

Dysfunction of nuclear distribution element-like 1 (Ndel1) is associated with schizophrenia, a neuropsychiatric disorder characterized by cognitive impairment and with seizures as comorbidity. The levels of Ndel1 are also altered in human and models with epilepsy, a chronic condition whose hallmark feature is the occurrence of spontaneous recurrent seizures and is typically associated with comorbid conditions including learning and memory deficits, anxiety, and depression. In this study, we analyzed the behaviors of mice postnatally deficient for Ndel1 in forebrain excitatory neurons (Ndel1 CKO) that exhibit spatial learning and memory deficits, seizures, and shortened lifespan. Ndel1 CKO mice underperformed in species-specific tasks, that is, the nest building, open field, Y maze, forced swim, and dry cylinder tasks. We surveyed the expression and/or activity of a dozen molecules related to Ndel1 functions and found changes that may contribute to the abnormal behaviors. Finally, we tested the impact of Reelin glycoprotein that shows protective effects in the hippocampus of Ndel1 CKO, on the performance of the mutant animals in the nest building task. Our study highlights the importance of Ndel1 in the manifestation of species-specific animal behaviors that may be relevant to our understanding of the clinical conditions shared between neuropsychiatric disorders and epilepsy.

Expression and function of Ndel1 during the differentiation of neural stem cells induced by hippocampal exosomesticle.

BACKGROUND: In the brain of adult mammals, neural stem cells persist in the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus, which are specialized niches with proliferative capacity. Most neural stem cells are in a quiescent state, but in response to extrinsic stimuli, they can exit from quiescence and become reactivated to produce new neurons, so neural stem cells are considered to be a potential source for cell replacement therapy of many nervous system diseases. We characterized the expression of Ndel1 during the differentiation of neural stem cells induced by hippocampus exosomes, and assessed the effect of Ndel1 on neural stem cells differentiation. METHODS: Hippocampal exosomes were isolated and extracted, and co-cultured exosomes with neural stem cells. Western blot, flow cytometry, and immunofluorescence analyses were used to analyze expression of neuronal markers. Further, utilizing high-throughput RNA sequencing technology, we found that nudE neurodevelopment protein 1-like 1 was significantly upregulated in exosomes derived from denervated hippocampus, and then characterized its mechanism and function during neural stem cells differentiation by qRT-PCR, western blot, flow cytometry, and immunofluorescence analyses. RESULTS: Our results revealed that exosomes of denervated hippocampus promoted the differentiation of neural stem cells into neuron. Hence, we identified that nudE neurodevelopment protein 1-like 1 was significantly upregulated and highly expressed in the nervous system. In addition, we found that miR-107-3p may regulate neural stem cell differentiation by targeting Ndel1. CONCLUSIONS: Our results revealed that deafferentation of the hippocampal exosomes co-cultured with neural stem cells could promote them to differentiate into neurons. Hence, we found that miR-107-3p may regulate neural stem cells differentiation by targeting Ndel1. Importantly, Ndel1 enhanced spatial learning and hippocampal neurogenesis in rats after fimbria fornix transection in vivo. These findings set the stage for a better understanding of neurogenesis, a process that 1 day may inspire new treatments for central nervous system diseases.

The Cytoplasmic Dynein Associated Protein NDE1 Regulates Osteoclastogenesis by Modulating M-CSF and RANKL Signaling Pathways.

Cytoskeleton organization and lysosome secretion play an essential role in osteoclastogenesis and bone resorption. The cytoplasmic dynein is a molecular motor complex that regulates microtubule dynamics and transportation of cargos/organelles, including lysosomes along the microtubules. LIS1, NDE1, and NDEL1 belong to an evolutionary conserved pathway that regulates dynein functions. Disruption of the cytoplasmic dynein complex and deletion of LIS1 in osteoclast precursors arrest osteoclastogenesis. Nonetheless, the role of NDE1 and NDEL1 in osteoclast biology remains elusive. In this study, we found that knocking-down Nde1 expression by lentiviral transduction of specific shRNAs markedly inhibited osteoclastogenesis in vitro by attenuating the proliferation, survival, and differentiation of osteoclast precursor cells via suppression of signaling pathways downstream of M-CSF and RANKL as well as osteoclast differentiation transcription factor NFATc1. To dissect how NDEL1 regulates osteoclasts and bone homeostasis, we generated Ndel1 conditional knockout mice in myeloid osteoclast precursors (Ndel1ΔlysM) by crossing Ndel1-floxed mice with LysM-Cre mice on C57BL/6J background. The Ndel1ΔlysM mice developed normally. The µCT analysis of distal femurs and in vitro osteoclast differentiation and functional assays in cultures unveiled the similar bone mass in both trabecular and cortical bone compartments as well as intact osteoclastogenesis, cytoskeleton organization, and bone resorption in Ndel1ΔlysM mice and cultures. Therefore, our results reveal a novel role of NDE1 in regulation of osteoclastogenesis and demonstrate that NDEL1 is dispensable for osteoclast differentiation and function.

Postnatal Role of the Cytoskeleton in Adult Epileptogenesis.

Mutations in cytoskeletal proteins can cause early infantile and childhood epilepsies by misplacing newly born neurons and altering neuronal connectivity. In the adult epileptic brain, cytoskeletal disruption is often viewed as being secondary to aberrant neuronal activity and/or death, and hence simply represents an epiphenomenon. Here, we review the emerging evidence collected in animal models and human studies implicating the cytoskeleton as a potential causative factor in adult epileptogenesis. Based on the emerging evidence, we propose that cytoskeletal disruption may be an important pathogenic mechanism in the mature epileptic brain.

Arsenic trioxide disturbs the LIS1/NDEL1/dynein microtubule dynamic complex by disrupting the CLIP170 zinc finger in head and neck cancer.

Cancer mortality is mainly caused by metastasis, which requires dynamic remodeling of cytoskeletal components such as microtubules. Targeting microtubules presents a promising antimetastatic strategy that could prevent cancer spreading and recurrence. It is known that arsenic trioxide (ATO) is able to inhibit the migration and invasion of solid malignant tumors, but its exact molecular mechanism remains unclear. Here, we report a novel molecular target and antimetastatic mechanism of ATO in head and neck squamous cell carcinoma (HNSCC). We found that cytoplasmic linker protein 170 (CLIP170) was overexpressed in HNSCC tissues and cells compared to normal controls. ATO at non-cytotoxic level (1 µM) inhibited the migration and invasion of HNSCC cells by displacing zinc in the zinc finger motif of CLIP170, which is a key protein that controls microtubule dynamics. The antimetastatic effects of ATO were equivalent to those of siRNA-mediated CLIP170 knockdown. Furthermore, ATO dysregulated microtubule polymerization via the CLIP170/LIS1/NDEL1/dynein signaling pathway in Cal27 cells as a functional consequence of CLIP170 zinc finger disruption. The effect was partially reversed by zinc supplementation. Taken together, these findings reveal that CLIP170 is a novel molecular target of ATO and demonstrate the capability and underlying mechanisms of ATO as a potential antimetastatic agent for HNSCC treatment.

Role of NDEL1 and VEGF/VEGFR-2 in Mouse Hippocampus After Status Epilepticus.

Nuclear-distribution element-like 1 (NDEL1) is associated with the proliferation and migration of neurons. Vascular endothelial growth factor (VEGF) in combination with VEGF receptor-2 (VEGFR-2) regulates the proliferation and migration of neurons. This study was performed to explore undefined alterations in the expression levels of NDEL1 and VEGF/VEGFR-2 within the hippocampus after status epilepticus (SE). Following the creation of pilocarpine-induced epilepsy models using adolescent male C57BL/6 mice, Western blotting and reverse transcription quantitative polymerase chain reaction were applied to assess the levels of NDEL1, VEGF, and VEGFR-2 expression in whole hippocampi at 1, 2, 3, and 4 weeks post-SE, respectively. Immunofluorescent labeling was also employed to detect the colocalization of NDEL1 and VEGF in the hippocampus. Our results indicated that NDEL1 and VEGF have similar patterns of upregulation throughout the hippocampus. Upregulation of VEGFR-2 occurred only in the early stages, and the expression decreased shortly afterward. NDEL1 and VEGF were coexpressed in the cornu ammonis 3 pyramidal cell, granular, and polymorph layers of the dentate gyrus in the hippocampus. This study revealed that NDEL1, VEGF, and VEGFR-2 may work together and are involved in the pathophysiology in the hippocampus after SE.

Mechanistic insights into the interactions of dynein regulator Ndel1 with neuronal ankyrins and implications in polarity maintenance.

Ankyrin-G (AnkG), a highly enriched scaffold protein in the axon initial segment (AIS) of neurons, functions to maintain axonal polarity and the integrity of the AIS. At the AIS, AnkG regulates selective intracellular cargo trafficking between soma and axons via interaction with the dynein regulator protein Ndel1, but the molecular mechanism underlying this binding remains elusive. Here we report that Ndel1's C-terminal coiled-coil region (CT-CC) binds to giant neuron-specific insertion regions present in both AnkG and AnkB with 2:1 stoichiometry. The high-resolution crystal structure of AnkB in complex with Ndel1 CT-CC revealed the detailed molecular basis governing the AnkB/Ndel1 complex formation. Mechanistically, AnkB binds with Ndel1 by forming a stable 5-helix bundle dominated by hydrophobic interactions spread across 6 distinct interaction layers. Moreover, we found that AnkG is essential for Ndel1 accumulation at the AIS. Finally, we found that cargo sorting at the AIS can be disrupted by blocking the AnkG/Ndel1 complex formation using a peptide designed based on our structural data. Collectively, the atomic structure of the AnkB/Ndel1 complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel1 complex formation and for the maintenance of axonal polarity. Our study will also be valuable for future studies of the interaction between AnkB and Ndel1 perhaps at distal axonal cargo transport.