Arf6 GTPase deficiency leads to porcine oocyte quality decline during aging.

Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.

CD24 promotes metastasis and chemoresistance by directly targeting Arf6-ERK pathway in esophageal squamous cell carcinoma.

Increasing evidence suggests the importance of CD24 in tumor progression, but its role and mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. The present study aims to explore the potential of CD24 as a novel predictive biomarker in ESCC, as well as its mechanism and therapeutic implications in metastasis and 5-FU chemoresistance. By using tissue microarray and immunohistochemistry, we found that CD24 expression was higher in ESCC tumor tissues than paired non-tumor tissues, further indicating that CD24 was markedly associated with poor prognosis. CD24 significantly promoted metastasis and 5-FU chemoresistance in vitro and in vivo. Mechanistically, CD24 competes with GIT2 to bind to Arf6, and stabilizes Arf6-GTP to activate the subsequent ERK pathway, thus promoting cancer progression. In addition, a significant positive correlation between CD24 and p-ERK was observed in clinical ESCC tissues. In summary, this study not only reveals CD24 as a regulatory factor for Arf6 activity, but also uncovers CD24-Arf6-ERK signaling axis as a novel mechanism of ESCC progression. Our findings suggest CD24 as a promising biomarker and therapeutic target in ESCC.

The Arf-GAPs, AoAge1 and AoAge2, regulate diverse cellular processes, conidiation, trap formation, and secondary metabolism in Arthrobotrys oligospora.

Guanine nucleotide-binding proteins of the ADP ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) are essential for diverse biological processes. Here, two homologous Arf-GAPs, Age1 (AoAge1) and Age2 (AoAge2), were identified in the widespread nematode-trapping fungus Arthrobotrys oligospora. Our results demonstrated that AoAge1, especially AoAge2, played crucial roles in mycelial growth, sporulation, trap production, stress response, mitochondrial activity, DNA damage, endocytosis, reactive oxygen species production, and autophagy. Notably, transcriptome data revealed that approximately 62.7% of the genes were directly or indirectly regulated by AoAge2, and dysregulated genes in Aoage2 deletion were enriched in metabolism, ribosome biogenesis, secondary metabolite biosynthesis, and autophagy. Furthermore, Aoage2 inactivation caused a substantial reduction in several compounds compared to the wild-type strain. Based on these results, a regulatory network for AoAge1 and AoAge2 was proposed and verified using a yeast two-hybrid assay. Based on our findings, AoAge1 and AoAge2 are essential for vegetative growth and mycelial development. Specifically, AoAge2 is required for sporulation and trapping morphogenesis. Our results demonstrated the critical functions of AoAge1 and AoAge2 in mycelial growth, diverse cellular processes, and pathogenicity, offering deep insights into the functions and regulatory mechanisms of Arf-GAPs in nematode-trapping fungi.

EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3T31A and ARL3T31A/C118F mutations.

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.

Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin.

Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.

Differential expression of circular RNAs in human umbilical cord mesenchymal stem cells treated with icariin.

Human umbilical cord mesenchymal stem cells (hUMSCs) belong to a multipotent stem cell population. Transplantation of icariin (ICA)-treated hUMSCs have better tissue repairing function in chronic liver injury. This study was to investigate whether the tissue-repairing effects and migration of hUMSCs after ICA treatment were regulated by circular RNAs (circRNAs). ICA was used to treat hUMSCs in vitro for 1 week and the expression profiles of circRNAs were generated using RNA sequencing. Differentially expressed circRNAs in hUMSCs after ICA intervention were screened. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were carried out to predict the potential function of dysregulated circRNAs. There were 52 differentially expressed circRNAs (32 circRNAs up-regulated and 20 circRNAs down-regulated) with fold change ≥2.0 before and after ICA treatment. ADP-ribosylation factors were associated with the dysregulated circRNAs among Gene Ontology analysis. Kyoto Encyclopedia of Genes and Genomes analysis showed that only endocytosis pathway was associated with up-regulated circRNAs, whereas 4 pathways including homologous recombination, RNA transport, axon guidance, and proteoglycans in cancer were related to down-regulated circRNAs. Fifty-two differentially expressed circRNAs and 238 predicted microRNAs were included in circRNAs-microRNAs network. The mechanism of ICA inducing hUMSCs migration may be through regulating circRNAs expression which affects ADP-ribosylation factors protein signal pathways.

Novel compound heterozygous variants in ARL13B lead to Joubert syndrome.

Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.

Expression analysis of type I ARF small GTPases ARF1-3 during mouse brain development.

BACKGROUND: ARF (ADP-ribosylation factor) GTPases are major regulators of intracellular trafficking, and classified into 3 groups (Type I - III), among which the type I group members, ARF1 and 3, are responsible genes for neurodevelopmental disorders. METHODS: In this study, we analysed the expression of Type I ARFs ARF1-3 during mouse brain development using biochemical and morphological methods. RESULTS: Western blotting analyses revealed that ARF1-3 are weakly expressed in the mouse brain at embryonic day 13 and gradually increase until postnatal day 30. ARF1-3 appear to be abundantly expressed in various telencephalon regions. Biochemical fractionation studies detected ARF1-3 in the synaptosome fraction of cortical neurons containing both pre- and post-synapses, however ARF1-3 were not observed in post-synaptic compartments. In immunohistochemical analyses, ARF1-3 appeared to be distributed in the cytoplasm and dendrites of cortical and hippocampal neurons as well as in the cerebellar molecular layer including dendrites of Purkinje cells and granule cell axons. Immunofluorescence in primary cultured hippocampal neurons revealed that ARF1-3 are diffusely distributed in the cytoplasm and dendrites with partial colocalization with a pre-synaptic marker, synaptophysin. CONCLUSIONS: Overall, our results support the notion that ARF1-3 could participate in vesicle trafficking both in the dendritic shaft (excluding spines) and axon terminals (pre-synaptic compartments).

ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

Distinct ADP-ribosylation factor-GTP exchange factors govern the opposite polarity of 2 receptor kinases.

Polarity of plasma membrane proteins is essential for cell morphogenesis and control of cell division and, thus, influences organ and whole plant development. In Arabidopsis (Arabidopsis thaliana) root endodermal cells, 2 transmembrane kinases, INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) and KINASE ON THE INSIDE (KOIN), accumulate at opposite lateral domains. Their polarization is tightly linked to their activities regulating cell division and ground tissue patterning. The polarization of IRK and KOIN relies solely on the secretion of newly synthesized protein. However, the secretion machinery by which their opposite, lateral polarity is achieved remains largely unknown. Here, we show that different sets of ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factors (ARF-GEFs) mediate their secretion. ARF-GEF GNOM-like-1 (GNL1) regulates KOIN secretion to the inner polar domain, thereby directing KOIN sorting early in the secretion pathway. For IRK, combined chemical and genetic analyses showed that the ARG-GEF GNL1, GNOM, and the BREFELDIN A-INHIBITED-GUANINE NUCLEOTIDE-EXCHANGE FACTORs 1 to 4 (BIG1-BIG4) collectively regulate its polar secretion. The ARF-GEF-dependent mechanisms guiding IRK or KOIN lateral polarity were active across different root cell types and functioned regardless of the protein's inner/outer polarity in those cells. Therefore, we propose that specific polar trafficking of IRK and KOIN occurs via distinct mechanisms that are not constrained by cell identity or polar axis and likely rely on individual protein recognition.

ARL15 and its Multiple Disease Association: Emerging Functions and Potential Therapeutic Application.

ARL15 is a member of the RAS superfamily of small GTPases and is associated with several metabolic traits, including increased risk of diabetes, rheumatoid arthritis and lipid metabolism disorders. The ARL15 gene encodes for an uncharacterized small GTP binding protein. Its precise role in human physiology remains unknown, but several genetic association studies have recognized different variants in this gene to be statistically associated with numerous traits and complex diseases. Here, we provided the unique features of ARL15 small G protein, its association with varied metabolic and lifestyle diseases, its function in vesicular and lipid trafficking, and its binding partners. We outlined this protein as a promising and emerging therapeutic target to combat metabolic disorders like cardiovascular diseases, diabetes and rheumatoid arthritis. The review provides a comprehensive description of the current advancements in ARL15 research with a perspective that focused research will position this small GTPase as a viable target for the treatment of rheumatoid arthritis.

Molecular modeling of ARF6 dysregulation caused by mutations in IQSEC2.

IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations. We integrated experimental data of IQSEC2 mutants with protein structure prediction by the RaptorX server combined with molecular modeling and molecular dynamics simulations. Normally, apocalmodulin (apoCM) binds to IQSEC2 resulting in its N-terminal fragment inhibiting access of its Sec 7 domain to ARF6. An increase in Ca2+ concentration destabilizes the interaction of IQSEC2 with apoCM and removes steric hindrance of Sec 7 binding with ARF6. Mutations at amino acid residue 350 of IQSEC2 result in loss of steric hindrance of Sec 7 binding with ARF6 leading to constitutive activation of ARF6 by Sec 7. On the other hand, a mutation at amino acid residue 359 of IQSEC2 results in constitutive hindrance of Sec 7 binding to ARF6 leading to the loss of the ability of IQSEC2 to activate ARF6. These studies provide a model for dysregulation of IQSEC2 Sec 7 activity by mutant IQSEC2 proteins.Communicated by Ramaswamy H. Sarma.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

ADP Ribosylation Factor 4 (Arf4) Regulates Radial Migration through N-Cadherin Trafficking during Cerebral Cortical Development.

During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the Class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of Class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knock-down of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate (CP). Migrating neurons with Arf4 knock-down exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knock-down. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.