Emerging role of senescent microglia in brain aging-related neurodegenerative diseases.

Brain aging is a recognized risk factor for neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), but the intricate interplay between brain aging and the pathogenesis of these conditions remains inadequately understood. Cellular senescence is considered to contribute to cellular dysfunction and inflammaging. According to the threshold theory of senescent cell accumulation, the vulnerability to neurodegenerative diseases is associated with the rates of senescent cell generation and clearance within the brain. Given the role of microglia in eliminating senescent cells, the accumulation of senescent microglia may lead to the acceleration of brain aging, contributing to inflammaging and increased vulnerability to neurodegenerative diseases. In this review, we propose the idea that the senescence of microglia, which is notably vulnerable to aging, could potentially serve as a central catalyst in the progression of neurodegenerative diseases. The senescent microglia are emerging as a promising target for mitigating neurodegenerative diseases.

Progranulin haploinsufficiency mediates cytoplasmic TDP-43 aggregation with lysosomal abnormalities in human microglia.

BACKGROUND: Progranulin (PGRN) haploinsufficiency due to progranulin gene (GRN) variants can cause frontotemporal dementia (FTD) with aberrant TAR DNA-binding protein 43 (TDP-43) accumulation. Despite microglial burden with TDP-43-related pathophysiology, direct microglial TDP-43 pathology has not been clarified yet, only emphasized in neuronal pathology. Thus, the objective of this study was to investigate TDP-43 pathology in microglia of patients with PGRN haploinsufficiency. METHODS: To design a human microglial cell model with PGRN haploinsufficiency, monocyte-derived microglia (iMGs) were generated from FTD-GRN patients carrying pathogenic or likely pathogenic variants (p.M1? and p.W147*) and three healthy controls. RESULTS: iMGs from FTD-GRN patients with PGRN deficiency exhibited severe neuroinflammation phenotype and failure to maintain their homeostatic molecular signatures, along with impaired phagocytosis. In FTD-GRN patients-derived iMGs, significant cytoplasmic TDP-43 aggregation and accumulation of lipid droplets with profound lysosomal abnormalities were observed. These pathomechanisms were mediated by complement C1q activation and upregulation of pro-inflammatory cytokines. CONCLUSIONS: Our study provides considerable cellular and molecular evidence that loss-of-function variants of GRN in human microglia can cause microglial dysfunction with abnormal TDP-43 aggregation induced by inflammatory milieu as well as the impaired lysosome. Elucidating the role of microglial TDP-43 pathology in intensifying neuroinflammation in individuals with FTD due to PGRN deficiency and examining consequential effects on microglial dysfunction might yield novel insights into the mechanisms underlying FTD and neurodegenerative disorders.

TGFß4 alleviates the phenotype of Charcot-Marie-Tooth disease type 1A.

The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFß4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFß4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFß4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFß4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.

Role of NCKAP1 in the Defective Phagocytic Function of Microglia-Like Cells Derived from Rapidly Progressing Sporadic ALS.

Microglia plays a key role in determining the progression of amyotrophic lateral sclerosis (ALS), yet their precise role in ALS has not been identified in humans. This study aimed to identify a key factor related to the functional characteristics of microglia in rapidly progressing sporadic ALS patients using the induced microglia model, although it is not identical to brain resident microglia. After confirming that microglia-like cells (iMGs) induced by human monocytes could recapitulate the main signatures of brain microglia, step-by-step comparative studies were conducted to delineate functional differences using iMGs from patients with slowly progressive ALS [ALS(S), n = 14] versus rapidly progressive ALS [ALS(R), n = 15]. Despite an absence of significant differences in the expression of microglial homeostatic genes, ALS(R)-iMGs preferentially showed defective phagocytosis and an exaggerated pro-inflammatory response to LPS stimuli compared to ALS(S)-iMGs. Transcriptome analysis revealed that the perturbed phagocytosis seen in ALS(R)-iMGs was closely associated with decreased NCKAP1 (NCK-associated protein 1)-mediated abnormal actin polymerization. NCKAP1 overexpression was sufficient to rescue impaired phagocytosis in ALS(R)-iMGs. Post-hoc analysis indicated that decreased NCKAP1 expression in iMGs was correlated with the progression of ALS. Our data suggest that microglial NCKAP1 may be an alternative therapeutic target in rapidly progressive sporadic ALS.

Proteostasis and Ribostasis Impairment as Common Cell Death Mechanisms in Neurodegenerative Diseases.

The cellular homeostasis of proteins (proteostasis) and RNA metabolism (ribostasis) are essential for maintaining both the structure and function of the brain. However, aging, cellular stress conditions, and genetic contributions cause disturbances in proteostasis and ribostasis that lead to protein misfolding, insoluble aggregate deposition, and abnormal ribonucleoprotein granule dynamics. In addition to neurons being primarily postmitotic, nondividing cells, they are more susceptible to the persistent accumulation of abnormal aggregates. Indeed, defects associated with the failure to maintain proteostasis and ribostasis are common pathogenic components of age-related neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, the neuronal deposition of misfolded and aggregated proteins can cause both increased toxicity and impaired physiological function, which lead to neuronal dysfunction and cell death. There is recent evidence that irreversible liquid-liquid phase separation (LLPS) is responsible for the pathogenic aggregate formation of disease-related proteins, including tau, α-synuclein, and RNA-binding proteins, including transactive response DNA-binding protein 43, fused in sarcoma, and heterogeneous nuclear ribonucleoprotein A1. Investigations of LLPS and its control therefore suggest that chaperone/disaggregase, which reverse protein aggregation, are valuable therapeutic targets for effective treatments for neurological diseases. Here we review and discuss recent studies to highlight the importance of understanding the common cell death mechanisms of proteostasis and ribostasis in neurodegenerative diseases.

Clinical and genetic characteristics of amyotrophic lateral sclerosis patients with ANXA11 variants.

Increasing genetic evidence supports the hypothesis that variants in the annexin A11 gene (ANXA11) contribute to amyotrophic lateral sclerosis pathogenesis. Therefore, we studied the clinical aspects of sporadic amyotrophic lateral sclerosis patients carrying ANXA11 variants. We also implemented functional experiments to verify the pathogenicity of the hotspot variants associated with amyotrophic lateral sclerosis-frontotemporal dementia. Korean patients diagnosed with amyotrophic lateral sclerosis (n = 882) underwent genetic evaluations through next-generation sequencing, which identified 16 ANXA11 variants in 26 patients. We analysed their clinical features, such as the age of onset, progression rate, initial symptoms and cognitive status. To evaluate the functional significance of the ANXA11 variants in amyotrophic lateral sclerosis-frontotemporal dementia pathology, we additionally utilized patient fibroblasts carrying frontotemporal dementia-linked ANXA11 variants (p.P36R and p.D40G) to perform a series of in vitro studies, including calcium imaging, stress granule dynamics and protein translation. The frequency of the pathogenic or likely pathogenic variants of ANXA11 was 0.3% and the frequency of variants classified as variants of unknown significance was 2.6%. The patients with variants in the low-complexity domain presented unique clinical features, including late-onset, a high prevalence of amyotrophic lateral sclerosis-frontotemporal dementia, a fast initial progression rate and a high tendency for bulbar-onset compared with patients carrying variants in the C-terminal repeated annexin homology domains. In addition, functional studies using amyotrophic lateral sclerosis-frontotemporal dementia patient fibroblasts revealed that the ANXA11 variants p.P36R and p.D40G impaired intracellular calcium homeostasis, stress granule disassembly and protein translation. This study suggests that the clinical manifestations of amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia spectrum patients with ANXA11 variants could be distinctively characterized depending upon the location of the variant.

Therapeutic modulation of GSTO activity rescues FUS-associated neurotoxicity via deglutathionylation in ALS disease models.

Fused in sarcoma (FUS) is a DNA/RNA-binding protein that is involved in DNA repair and RNA processing. FUS is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the molecular mechanisms underlying FUS-mediated neurodegeneration are largely unknown. Here, using a Drosophila model, we showed that the overexpression of glutathione transferase omega 2 (GstO2) reduces cytoplasmic FUS aggregates and prevents neurodegenerative phenotypes, including neurotoxicity and mitochondrial dysfunction. We found a FUS glutathionylation site at the 447th cysteine residue in the RanBP2-type ZnF domain. The glutathionylation of FUS induces FUS aggregation by promoting phase separation. GstO2 reduced cytoplasmic FUS aggregation by deglutathionylation in Drosophila brains. Moreover, we demonstrated that the overexpression of human GSTO1, the homolog of Drosophila GstO2, attenuates FUS-induced neurotoxicity and cytoplasmic FUS accumulation in mouse neuronal cells. Thus, the modulation of FUS glutathionylation might be a promising therapeutic strategy for FUS-associated neurodegenerative diseases.

ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival.

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. Here, we demonstrate that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). We further demonstrate that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.

ANXA11 mutations in ALS cause dysregulation of calcium homeostasis and stress granule dynamics.

Dysregulation of calcium ion homeostasis and abnormal protein aggregation have been proposed as major pathogenic hallmarks underpinning selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Recently, mutations in annexin A11 (ANXA11), a gene encoding a Ca2+-dependent phospholipid-binding protein, have been identified in familial and sporadic ALS. However, the physiological and pathophysiological roles of ANXA11 remain unknown. Here, we report functions of ANXA11 related to intracellular Ca2+ homeostasis and stress granule dynamics. We analyzed the exome sequences of 500 Korean patients with sALS and identified nine ANXA11 variants in 13 patients. The amino-terminal variants p.G38R and p.D40G within the low-complexity domain of ANXA11 enhanced aggregation propensity, whereas the carboxyl-terminal ANX domain variants p.H390P and p.R456H altered Ca2+ responses. Furthermore, all four variants in ANXA11 underwent abnormal phase separation to form droplets with aggregates and led to the alteration of the biophysical properties of ANXA11. These functional defects caused by ALS-linked variants induced alterations in both intracellular Ca2+ homeostasis and stress granule disassembly. We also revealed that p.G228Lfs*29 reduced ANXA11 expression and impaired Ca2+ homeostasis, as caused by missense variants. Ca2+-dependent interaction and coaggregation between ANXA11 and ALS-causative RNA-binding proteins, FUS and hnRNPA1, were observed in motor neuron cells and brain from a patient with ALS-FUS. The expression of ALS-linked ANXA11 variants in motor neuron cells caused cytoplasmic sequestration of endogenous FUS and triggered neuronal apoptosis. Together, our findings suggest that disease-associated ANXA11 mutations can contribute to ALS pathogenesis through toxic gain-of-function mechanisms involving abnormal protein aggregation.

BMP-dependent synaptic development requires Abi-Abl-Rac signaling of BMP receptor macropinocytosis.

Retrograde BMP trans-synaptic signaling is essential for synaptic development. Despite the importance of endocytosis-regulated BMP receptor (BMPR) control of this developmental signaling, the mechanism remains unknown. Here, we provide evidence that Abelson interactor (Abi), a substrate for Abl kinase and component of the SCAR/WAVE complex, links Abl and Rac1 GTPase signaling to BMPR macropinocytosis to restrain BMP-mediated synaptic development. We find that Abi acts downstream of Abl and Rac1, and that BMP ligand Glass bottom boat (Gbb) induces macropinocytosis dependent on Rac1/SCAR signaling, Abl-mediated Abi phosphorylation, and BMPR activation. Macropinocytosis acts as the major internalization route for BMPRs at the synapse in a process driven by Gbb activation and resulting in receptor degradation. Key regulators of macropinocytosis (Rabankyrin and CtBP) control BMPR trafficking to limit BMP trans-synaptic signaling. We conclude that BMP-induced macropinocytosis acts as a BMPR homeostatic mechanism to regulate BMP-mediated synaptic development.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling.

In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. We show that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. We further show that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. We also show that Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Finally, we demonstrate that loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.

Graf regulates hematopoiesis through GEEC endocytosis of EGFR.

GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. We show that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. We also find that loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on our findings, we propose a model in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation.

Genetic and functional analysis of TBK1 variants in Korean patients with sporadic amyotrophic lateral sclerosis.

The TANK-binding kinase 1 (TBK1) gene has recently been identified as a novel causative gene of amyotrophic lateral sclerosis (ALS). This study aims to determine the frequency and spectrum of TBK1 variants and their functional implications in Korean patients with sporadic ALS (sALS). TBK1 sequences were analyzed in 129 consecutive patients with sALS using either multigene panel or exome sequencing. One frameshift (c.1414delA) and 3 missense variants of uncertain significance in TBK1 were found in 4 patients each. In vitro functional studies revealed that the c.1414delA (p.Ile472Serfs*8) variant was associated with reduced mRNA expression of TBK1. Moreover, protein expression of this variant in patient-derived fibroblasts disrupted binding to autophagy adapter proteins and inhibited the function of TBK1 in HEK293T cells. In contrast, the 3 other missense variants of uncertain significance showed normal mRNA expression and no abnormalities in protein function. Based on these findings, the frequency of pathogenic TBK1 variants in Korean sALS patients was estimated to be 0.8% (1/129). In conclusion, pathogenic variants in TBK1 are rare but could be responsible for sALS in a small number of Korean patients.

Patient fibroblasts-derived induced neurons demonstrate autonomous neuronal defects in adult-onset Krabbe disease.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by defective ß-galactosylceramidase (GALC), a lysosomal enzyme responsible for cleavage of several key substrates including psychosine. Accumulation of psychosine to the cytotoxic levels in KD patients is thought to cause dysfunctions in myelinating glial cells based on a comprehensive study of demyelination in KD. However, recent evidence suggests myelin-independent neuronal death in the murine model of KD, thus indicating defective GALC in neurons as an autonomous mechanism for neuronal cell death in KD. These observations prompted us to generate induced neurons (iNeurons) from two adult-onset KD patients carrying compound heterozygous mutations (p.[K563*];[L634S]) and (p.[N228_S232delinsTP];[G286D]) to determine the direct contribution of autonomous neuronal toxicity to KD. Here we report that directly converted KD iNeurons showed not only diminished GALC activity and increased psychosine levels, as expected, but also neurite fragmentation and abnormal neuritic branching. The lysosomal-associated membrane proteins 1 (LAMP1) was expressed at higher levels than controls, LAMP1-positive vesicles were significantly enlarged and fragmented, and mitochondrial morphology and its function were altered in KD iNeurons. Strikingly, we demonstrated that psychosine was sufficient to induce neurite defects, mitochondrial fragmentation, and lysosomal alterations in iNeurons derived in healthy individuals, thus establishing the causal effect of the cytotoxic GALC substrate in KD and the autonomous neuronal toxicity in KD pathology.

MICAL-like Regulates Fasciclin II Membrane Cycling and Synaptic Development.

Fasciclin II (FasII), the Drosophila ortholog of neural cell adhesion molecule (NCAM), plays a critical role in synaptic stabilization and plasticity. Although this molecule undergoes constitutive cycling at the synaptic membrane, how its membrane trafficking is regulated to ensure proper synaptic development remains poorly understood. In a genetic screen, we recovered a mutation in Drosophila mical-like that displays an increase in bouton numbers and a decrease in FasII levels at the neuromuscular junction (NMJ). Similar phenotypes were induced by presynaptic, but not postsynaptic, knockdown of mical-like expression. FasII trafficking assays revealed that the recycling of internalized FasII molecules to the cell surface was significantly impaired in mical-like-knockdown cells. Importantly, this defect correlated with an enhancement of endosomal sorting of FasII to the lysosomal degradation pathway. Similarly, synaptic vesicle exocytosis was also impaired in mical-like mutants. Together, our results identify Mical-like as a novel regulator of synaptic growth and FasII endocytic recycling.

CLEC4C p.K210del variant causes impaired cell surface transport in plasmacytoid dendritic cells of amyotrophic lateral sclerosis.

The type II C-type lectin CLEC4C is a transmembrane protein selectively expressed on plasmacytoid dendritic cells (PDCs). Although its mechanism of action remains unclear, triggering of the extracellular C-terminal C-type carbohydrate recognition region of CLEC4C regulates the secretion of proinflammatory cytokines and type I IFNs in PDCs. Applying whole-exome sequencing in a patient with juvenile amyotrophic lateral sclerosis (ALS) and both healthy parents, we identified a de novo CLEC4C variant (c.629_631delAGA; p.Lys210del). In this study, we report that the deletion of a lysine residue at the extracellular region of CLEC4C yields a C-terminal dilysine motif that results in endoplasmic reticulum (ER) retention of the protein in transfected HeLa and Jurkat T lymphoma cell models. As a consequence, a decrease in the surface expression of CLEC4C and the ER localization of the mutant construct were observed. Furthermore, depletion of the cell surface CLEC4C level was also observed in the patient PDCs, further suggesting that the variant p.Lys210del CLEC4C may contribute to juvenile ALS susceptibility.

Directly converted patient-specific induced neurons mirror the neuropathology of FUS with disrupted nuclear localization in amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the fused in sarcoma (FUS) gene have been linked to amyotrophic lateral sclerosis (ALS). ALS patients with FUS mutations exhibit neuronal cytoplasmic mislocalization of the mutant FUS protein. ALS patients' fibroblasts or induced pluripotent stem cell (iPSC)-derived neurons have been developed as models for understanding ALS-associated FUS (ALS-FUS) pathology; however, pathological neuronal signatures are not sufficiently present in the fibroblasts of patients, whereas the generation of iPSC-derived neurons from ALS patients requires relatively intricate procedures. RESULTS: Here, we report the generation of disease-specific induced neurons (iNeurons) from the fibroblasts of patients who carry three different FUS mutations that were recently identified by direct sequencing and multi-gene panel analysis. The mutations are located at the C-terminal nuclear localization signal (NLS) region of the protein (p.G504Wfs*12, p.R495*, p.Q519E): two de novo mutations in sporadic ALS and one in familial ALS case. Aberrant cytoplasmic mislocalization with nuclear clearance was detected in all patient-derived iNeurons, and oxidative stress further induced the accumulation of cytoplasmic FUS in cytoplasmic granules, thereby recapitulating neuronal pathological features identified in mutant FUS (p.G504Wfs*12)-autopsied ALS patient. Importantly, such FUS pathological hallmarks of the patient with the p.Q519E mutation were only detected in patient-derived iNeurons, which contrasts to predominant FUS (p.Q519E) in the nucleus of both the transfected cells and patient-derived fibroblasts. CONCLUSIONS: Thus, iNeurons may provide a more reliable model for investigating FUS mutations with disrupted NLS for understanding FUS-associated proteinopathies in ALS.

Tbc1d15-17 regulates synaptic development at the Drosophila neuromuscular junction.

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15-17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15-17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15-17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15-17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15-17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15-17 and its potential substrate Rab7 in regulating synaptic development.