Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

Optimized protocol for translatome analysis of mouse brain endothelial cells.

Brain endothelial cells (BECs) are important conduits that deliver oxygen and nutrients, protect parenchyma cells from toxins, and drain wastes to maintain brain homeostasis. Impairment of BECs has been implicated in diverse neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, molecular analysis of BECs is important for understanding the molecular pathogenesis of these neurological diseases. Even though many transcriptome analyses for BECs have been developed, mRNA levels do not necessarily correlate with the levels of actively translated proteins. Translatome analysis using RiboTag mice, in which Rpl22, a ribosomal component, is tagged by the hemagglutinin epitope under Cre recombinase activation, could serve as an excellent tool that overcomes these caveats. However, implementation of this technique is limited by high noise-to-signal ratios as well as the low yield of mRNAs from BECs, which limits bulk gene expression analysis. In this study, we established a protocol to isolate highly pure mRNAs from BECs in the cortex of eight- to twelve-week-old male Tie2-Cre; Rpl22HA/HA mice by using a cell strainer to trap blood vessels prior to immunoprecipitation. According to the results of RT-PCR, the specificity of the mRNA pools isolated by our protocol was much higher than that of the pools isolated by the standard protocol. We were also able to generate a high-quality cDNA library for RNA-seq with the small amount of mRNA isolated with our protocol. Thus, this optimized method will be useful for future studies of BECs at the molecular level.

Autophagy activity contributes to the impairment of social recognition in Epac2-/- mice.

Autophagy is a lysosomal degradation pathway that regulates cellular homeostasis. It is constitutively active in neurons and controls the essential steps of neuronal development, leading to its dysfunction in neurodevelopmental disorders. Although mTOR-associated impaired autophagy has previously been reported in neurodevelopmental disorders, there is lack of information about the dysregulation of mTOR-independent autophagy in neurodevelopmental disorders. In this study, we investigated whether the loss of Epac2, involved in the mTOR-independent pathway, affects autophagy activity and whether the activity of autophagy is associated with social-behavioral phenotypes in mice with Epac2 deficiencies. We observed an accumulation of autophagosomes and a significant increase in autophagic flux in Epac2-deficient neurons, which had no effect on mTOR activity. Next, we examined whether an increase in autophagic activity contributed to the social behavior exhibited in Epac2-/- mice. The social recognition deficit observed in Epac2-/- mice recovered in double transgenic Epac2-/-: Atg5+/- mice. Our study suggests that excessive autophagy due to Epac2 deficiencies may contribute to social recognition defects through an mTOR-independent pathway.

Nonmuscle myosin IIB regulates Parkin-mediated mitophagy associated with amyotrophic lateral sclerosis-linked TDP-43.

C-terminal fragments of Tar DNA-binding protein 43 (TDP-43) have been identified as the major pathological protein in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how they affect cellular toxicity and neurodegeneration, including the modulation process remains unknown. This study revealed that the C-terminal fragment of TDP-43 (TDP-25) was localized primarily to mitochondria and caused abnormal mitochondrial morphology, inducing Parkin-mediated mitophagy. Also, we discovered that the knockdown of selective autophagy receptors, such as TAX1BP, Optineurin, or NDP52 caused TDP-25 accumulation, indicating that TDP-25 was degraded by mitophagy. Interestingly, myosin IIB, a nonmuscle type of myosin and actin-based motor protein, is mostly colocalized to TDP-25 associated with abnormal mitochondria. In addition, myosin IIB inhibition by siRNA or blebbistatin induced mitochondrial accumulation of insoluble TDP-25 and Tom20, and reduced neuronal cell viability. Our results suggest a novel role of myosin IIB in mitochondrial degradation of toxic TDP-25. Therefore, we proposed that regulating myosin IIB activity might be a potential therapeutic target for neurodegenerative diseases associated with TDP-43 pathology.

Sequestration of PRMT1 and Nd1-L mRNA into ALS-linked FUS mutant R521C-positive aggregates contributes to neurite degeneration upon oxidative stress.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, are associated with familial amyotrophic lateral sclerosis (ALS). However, little is known about how ALS-causing mutations alter protein-protein and protein-RNA complexes and contribute to neurodegeneration. In this study, we identified protein arginine methyltransferase 1 (PRMT1) as a protein that more avidly associates with ALS-linked FUS-R521C than with FUS-WT (wild type) or FUS-P525L using co-immunoprecipitation and LC-MS analysis. Abnormal association between FUS-R521C and PRMT1 requires RNA, but not methyltransferase activity. PRMT1 was sequestered into cytosolic FUS-R521C-positive stress granule aggregates. Overexpression of PRMT1 rescued neurite degeneration caused by FUS-R521C upon oxidative stress, while loss of PRMT1 further accumulated FUS-positive aggregates and enhanced neurite degeneration. Furthermore, the mRNA of Nd1-L, an actin-stabilizing protein, was sequestered into the FUS-R521C/PRMT1 complex. Nd1-L overexpression rescued neurite shortening caused by FUS-R521C upon oxidative stress, while loss of Nd1-L further exacerbated neurite shortening. Altogether, these data suggest that the abnormal stable complex of FUS-R521C/PRMT1/Nd1-L mRNA could contribute to neurodegeneration upon oxidative stress. Overall, our study provides a novel pathogenic mechanism of the FUS mutation associated with abnormal protein-RNA complexes upon oxidative stress in ALS and provides insight into possible therapeutic targets for this pathology.

TMEM106B, a frontotemporal lobar dementia (FTLD) modifier, associates with FTD-3-linked CHMP2B, a complex of ESCRT-III.

BACKGROUND: Transmembrane protein 106B (TMEM106B) has been identified as a risk factor for frontotemporal lobar degeneration, which is the second most common form of progressive dementia in people under 65 years of age. Mutations in charged multivesicular body protein 2B (CHMP2B), which is involved in endosomal protein trafficking, have been found in chromosome 3-linked frontotemporal dementia. Despite the number of studies on both CHMP2B and TMEM106B in the endolysosomal pathway, little is known about the relationship between CHMP2B and TMEM106B in the endosomal/autophagy pathway. RESULTS: This study found that endogenous TMEM106B was partially sequestered in CHMP2B-positive structures, suggesting its possible involvement in endosomal sorting complexes required for transport (ESCRT)-associated pathways. The role of single nucleotide polymorphisms of TMEM106B (T185, S185, or S134N) in the ESCRT-associated pathways were characterized. The T185 and S185 variants were more localized to Rab5-/Rab7-positive endosomes compared with S134N, while all of the variants were more localized to Rab7-positive endosomes compared to Rab5-positive endosomes. T185 was more associated with CHMP2B compared to S185. Autophagic flux was slightly reduced in the T185-expressing cells compared to the control or S185-expressing cells. Moreover, T185 slightly enhanced the accumulation of EGFR, impairments in autophagic flux, and neurotoxicity that were caused by CHMP2B(Intron5) compared to S185-expressing cells. CONCLUSIONS: These findings suggest that the T185 variant functions as a risk factor in neurodegeneration with endolysosomal defects. This study provides a better understanding of pathogenic functions of TMEM106B, which is a risk factor for the progression of neurodegenerative diseases that are associated with endosomal defects in the aged brain.

Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal neurodegenerative disease that causes progressive muscular weakness and has overlapping clinical and pathologic characteristics with frontotemporal lobar degeneration. However, the role of autophagy in regulation of FUS-positive stress granules (SGs) and aggregates remains unclear. We found that the ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under oxidative stress, leading to a disruption in the release of FUS from SGs in cultured neurons. Autophagy controls the quality of proteins or organelles; therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs. Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However, FUS(R521C) expression did not significantly impair autophagic degradation. Moreover, autophagy activation with rapamycin reduced the accumulation of FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced neurite fragmentation and cell death in neurons expressing mutant FUS under oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS associated with a FUS mutation under oxidative stress, as well as therapeutic insight regarding FUS pathology associated with excessive SGs.

Characterization of the cellular localization of C4orf34 as a novel endoplasmic reticulum resident protein.

Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including Ca2+ homeostasis and ER stress.

Autophagy negatively regulates early axon growth in cortical neurons.

Neurite growth requires neurite extension and retraction, which are associated with protein degradation. Autophagy is a conserved bulk degradation pathway that regulates several cellular processes. However, little is known about autophagic regulation during early neurite growth. In this study, we investigated whether autophagy was involved in early neurite growth and how it regulated neurite growth in primary cortical neurons. Components of autophagy were expressed and autophagy was activated during early neurite growth. Interestingly, inhibition of autophagy by atg7 small interfering RNA (siRNA) caused elongation of axons, while activation of autophagy by rapamycin suppressed axon growth. Surprisingly, inhibition of autophagy reduced the protein level of RhoA. Moreover, expression of RhoA suppressed axon overelongation mediated by autophagy inhibition, whereas inhibition of the RhoA signaling pathway by Y-27632 recovered rapamycin-mediated suppression of axon growth. Interestingly, hnRNP-Q1, which negatively regulates RhoA, accumulated in autophagy-deficient neurons, while its protein level was reduced by autophagy activation. Overall, our study suggests that autophagy negatively regulates axon extension via the RhoA-ROCK pathway by regulating hnRNP-Q1 in primary cortical neurons. Therefore, autophagy might serve as a fine-tuning mechanism to regulate early axon extension.

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons.

Tar-DNA binding protein of 43kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons.

The functional analysis of the CHMP2B missense mutation associated with neurodegenerative diseases in the endo-lysosomal pathway.

Endosomal sorting complexes required for transport (ESCRTs) regulate a key sorting step of protein trafficking between endosomal compartments in lysosomal degradation. Interestingly, mutations in charged multivesicular body protein 2B (CHMP2B), which is a core subunit of ESCRT-III, have been identified in some neurodegenerative diseases. However, the cellular pathogenesis resulting from CHMP2B missense mutations is unclear. Furthermore, little is known about their functional analysis in post-mitotic neurons. In order to examine their cellular pathogenesis, we analyzed their effects in the endo-lysosomal pathway in post-mitotic neurons. Interestingly, of the missense mutant proteins, CHMP2B(T104N) mostly accumulated in the Rab5- and Rab7-positive endosomes and caused delayed degradation of EGFR as compared to CHMP2B(WT). Furthermore, CHMP2B(T104N) showed less association with Vps4 ATPase and was avidly associated with Snf7-2, a core component of ESCRT-III, suggesting that it may cause defects in the process of dissociation from ESCRT. Of the missense variants, CHMP2B(T104N) caused prominent accumulation of autophagosomes. However, neuronal cell survival was not dramatically affected by expression of CHMP2B(T104N). These findings suggested that, from among the various missense mutants, CHMP2B(T104N) was associated with relatively mild cellular pathogenesis in post-mitotic neurons. This study provided a better understanding of the cellular pathogenesis of neurodegenerative diseases associated with various missense mutations of CHMP2B as well as endocytic defects.