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Abstract Background Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1−/−) murine primary hippocampal neuron model to investigate whether PGRN’s neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN’s neurotrophic effects. Results As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn−/− neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1−/− cultures. Conclusion We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.

Background: Obstructive sleep apnea (OSA) is increasingly recognized as a common condition in the general population and causes significant OSA-associated morbidities including cardiovascular and cerebrovascular events such as cerebral small vessel disease (CSVD) and stroke., Methods: In this study, using sensitive ELISA immunoassays, we measured subset of endothelial/vascular and inflammatory biomarkers as well as neurofilament light chain (NfL), a sensitive marker for neuroaxonal injury, using plasma from OSA patients post-stroke (Acute Cerebral Infarction (ACI), N = 26) to determine their usefulness as potential prognostic markers in disease progression., Results: Our results showed significantly increased plasma TNFα and NfL concentrations and decreased concentrations of platelet derived growth factor (PDGF-AA) in post-stroke OSA patients with more severe white matter hyperintensities (WMHs). And after separating the patients based on sex, compared to females, male post-stroke OSA patients with severe WMHs have increased circulating levels of inflammatory chemokine CXCL10 and cytokine Interleukin-10 (IL-10) and significantly decreased levels of Angiopoietin-1 (Ang-1) an important protein responsible for endothelial/vascular integrity functions. Importantly, in a subset of newly diagnosed OSA patients (without prior history of stroke), significantly increased plasma CXCL10 levels and decreased plasma Ang-1 levels were also readily observed when compared to healthy controls, indicating possible altered endothelial integrity and ongoing vascular inflammation in these newly diagnosed OSA patients., Conclusions: In summary, our study has identified a novel set of plasma biomarkers including PDGF-AA, CXCL10 and Ang-1 for their potential prognostic value for disease outcomes pre- and post-stroke in OSA patients and use as surrogate markers to measure efficacy of treatment modalities., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genetic variation at the transmembrane protein 106B gene ( TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

C9orf72 repeat expansions are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly(GR) proteins are toxic to neurons by forming cytoplasmic inclusions that sequester RNA-binding proteins including stress granule (SG) proteins. However, little is known of the factors governing poly(GR) inclusion formation. Here, we show that poly(GR) infiltrates a finely tuned network of protein-RNA interactions underpinning SG formation. It interacts with G3BP1, the key driver of SG assembly and a protein we found is critical for poly(GR) inclusion formation. Moreover, we discovered that N 6 -methyladenosine (m6A)-modified mRNAs and m6A-binding YTHDF proteins not only co-localize with poly(GR) inclusions in brains of c9FTD/ALS mouse models and patients with c9FTD, they promote poly(GR) inclusion formation via the incorporation of RNA into the inclusions. Our findings thus suggest that interrupting interactions between poly(GR) and G3BP1 or YTHDF1 proteins or decreasing poly(GR) altogether represent promising therapeutic strategies to combat c9FTD/ALS pathogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Inclusions of TAR DNA-binding protein 43 kDa (TDP-43) has been designated limbic-predominant, age-related TDP-43 encephalopathy (LATE), with or without co-occurrence of Alzheimer's disease (AD). Approximately, 30-70% AD cases present TDP-43 proteinopathy (AD-TDP), and a greater disease severity compared to AD patients without TDP-43 pathology. However, it remains unclear to what extent TDP-43 dysfunction is involved in AD pathogenesis., Methods: To investigate whether TDP-43 dysfunction is a prominent feature in AD-TDP cases, we evaluated whether non-conserved cryptic exons, which serve as a marker of TDP-43 dysfunction in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), accumulate in AD-TDP brains. We assessed a cohort of 192 post-mortem brains from three different brain regions: amygdala, hippocampus, and frontal cortex. Following RNA and protein extraction, qRT-PCR and immunoassays were performed to quantify the accumulation of cryptic RNA targets and phosphorylated TDP-43 pathology, respectively., Results: We detected the accumulation of misspliced cryptic or skiptic RNAs of STMN2, KCNQ2, UNC13A, CAMK2B, and SYT7 in the amygdala and hippocampus of AD-TDP cases. The topographic distribution of cryptic RNA accumulation mimicked that of phosphorylated TDP-43, regardless of TDP-43 subtype classification. Further, cryptic RNAs efficiently discriminated AD-TDP cases from controls., Conclusions: Overall, our results indicate that cryptic RNAs may represent an intriguing new therapeutic and diagnostic target in AD, and that methods aimed at detecting and measuring these species in patient biofluids could be used as a reliable tool to assess TDP-43 pathology in AD. Our work also raises the possibility that TDP-43 dysfunction and related changes in cryptic splicing could represent a common molecular mechanism shared between AD-TDP and FTLD-TDP., (© 2023. Editorial Group and BioMed Central Ltd., part of Springer Nature.)

Treatments for neurodegenerative disease, including Frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS), remain rather limited, underscoring the need for greater mechanistic insight and disease-relevant models. Our ability to develop novel disease models of genetic risk factors, disease modifiers, and other FTD/ALS-relevant targets is impeded by the significant amount of time and capital required to develop conventional knockout and transgenic mice. To overcome these limitations, we have generated a novel CRISPRi interference (CRISPRi) knockin mouse. CRISPRi uses a catalytically dead form of Cas9, fused to a transcriptional repressor to knockdown protein expression, following the introduction of single guide RNA against the gene of interest. To validate the utility of this model we have selected the TAR DNA binding protein (TDP-43) splicing target, stathmin-2 ( STMN2 ). STMN2 RNA is downregulated in FTD/ALS due to loss of TDP-43 activity and STMN2 loss is suggested to play a role in ALS pathogenesis. The involvement of STMN2 loss of function in FTD has yet to be determined. We find that STMN2 protein levels in familial FTD cases are significantly reduced compared to controls, supporting that STMN2 depletion may be involved in the pathogenesis of FTD. Here, we provide proof-of-concept that we can simultaneously knock down Stmn2 and express the expanded repeat in the Chromosome 9 open reading frame 72 ( C9ORF72 ) gene, successfully replicating features of C9-associated pathology. Of interest, depletion of Stmn2 had no effect on expression or deposition of dipeptide repeat proteins (DPRs), but significantly decreased the number of phosphorylated Tdp-43 (pTdp-43) inclusions. We submit that our novel CRISPRi mouse provides a versatile and rapid method to silence gene expression in vivo and propose this model will be useful to understand gene function in isolation or in the context of other neurodegenerative disease models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pickles, Zanetti Alepuz, Koike, Yue, Tong, Liu, Zhou, Jansen-West, Daughrity, Song, DeTure, Oskarsson, Graff-Radford, Boeve, Petersen, Josephs, Dickson, Ward, Dong, Prudencio, Cook and Petrucelli.)

Introduction: Perry syndrome (PS) is a hereditary neurodegenerative disorder caused by mutations in the DCTN1 gene and characterized by TDP-43 pathology. As the diagnosis is usually made at the advanced stages of the disease, there are no studies on the asymptomatic mutation carriers and their conversion to overt disease., Methods: We personally examined 27 members of the large kindred of 104 individuals with familial parkinsonism. We evaluated each case with clinical (neurological examination; motor and non-motor scales), genetic testing (whole-exome or Sanger sequencing), and laboratory (neurofilament light, NFL; glial fibrillary acidic protein, GFAP) measures. Autopsy study was done on two individuals., Results: The mean age at evaluation was 49 years. Comorbidities were present in 20 cases, including sleep problems (n = 15 total, sleep apnea in 7), dysautonomia (n = 10), weight loss (n = 8), and anxiety/depression (n = 8). Neurological abnormalities were present in 18, including parkinsonism (n = 7), isolated tremor (n = 2), and varied isolated signs in individual cases. Cognition and smell were preserved. Genetic testing revealed a novel c.200G > T (Gly67Val) mutation in the DCTN1 gene in 10 individuals. The mutation, segregated with the PS phenotype (n = 4), was absent in gnomAD, and in silico predictions indicated it was pathogenic. Three young mutation carriers were monosymptomatic (prodromal), and three were asymptomatic. Plasma NFL and GFAP values were similar among the cases. Autopsy studies showed typical PS neuropathological findings., Conclusions: We identified a novel pathogenic Gly67Val DCTN1 mutation. We report prodromal disease of PS in some mutation carriers; however, more investigation is necessary to confirm this observation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1, and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter cell lines and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation - suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders., Competing Interests: CONFLICT OF INTEREST The authors declare no competing interests.

A major function of TAR DNA-binding protein-43 (TDP-43) is to repress the inclusion of cryptic exons during RNA splicing. One of these cryptic exons is in UNC13A, a genetic risk factor for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of cryptic UNC13A in disease is heightened by the presence of a risk haplotype located within the cryptic exon itself. Here, we revealed that TDP-43 extreme N-terminus is important to repress UNC13A cryptic exon inclusion. Further, we found hnRNP L, hnRNP A1, and hnRNP A2B1 bind UNC13A RNA and repress cryptic exon inclusion, independently of TDP-43. Finally, higher levels of hnRNP L protein associate with lower burden of UNC13A cryptic RNA in ALS/FTD brains. Our findings suggest that while TDP-43 is the main repressor of UNC13A cryptic exon inclusion, other hnRNPs contribute to its regulation and may potentially function as disease modifiers., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LP serves as a Consultant for Expansion Therapeutics., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Empathic function is essential for the well-being of social species. Empathy loss is associated with various brain disorders and represents arguably the most distressing feature of frontotemporal dementia (FTD), a leading form of presenile dementia. The neural mechanisms are unknown. We established an FTD mouse model deficient in empathy and observed that aged somatic transgenic mice expressing GGGGCC repeat expansions in C9orf72, a common genetic cause of FTD, exhibited blunted affect sharing and failed to console distressed conspecifics by affiliative contact. Distress-induced consoling behavior activated the dorsomedial prefrontal cortex (dmPFC), which developed profound pyramidal neuron hypoexcitability in aged mutant mice. Optogenetic dmPFC inhibition attenuated affect sharing and other-directed consolation in wild-type mice, whereas chemogenetically enhancing dmPFC excitability rescued empathy deficits in mutant mice, even at advanced ages when substantial cortical atrophy had occurred. These results establish cortical hypoexcitability as a pathophysiological basis of empathy loss in FTD and suggest a therapeutic strategy., Competing Interests: Declaration of interests F.-B.G. has an active research agreement with and receives funding from Stealth BioTherapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 ( TBK1 ) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg 228 →His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72 , TBK1 , and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited cerebellar ataxia caused by the expansion of a polyglutamine (polyQ) repeat in the gene encoding ATXN3. The polyQ expansion induces protein inclusion formation in the neurons of patients and results in neuronal degeneration in the cerebellum and other brain regions. We used adeno-associated virus (AAV) technology to develop a new mouse model of SCA3 that recapitulates several features of the human disease, including locomotor defects, cerebellar-specific neuronal loss, polyQ-expanded ATXN3 inclusions, and TDP-43 pathology. We also found that neurofilament light is elevated in the cerebrospinal fluid (CSF) of the SCA3 animals, and the expanded polyQ-ATXN3 protein can be detected in the plasma. Interestingly, the levels of polyQ-ATXN3 in plasma correlated with measures of cerebellar degeneration and locomotor deficits in 6-month-old SCA3 mice, supporting the hypothesis that this factor could act as a biomarker for SCA3., Competing Interests: ZKW serves as PI or Co-PI on Biohaven Pharmaceuticals, Inc (BHV4157-206 and BHV3241-301), Neuraly, Inc (NLY01-PD-1), and Vigil Neuroscience, Inc (VGL101 -01.001) grants. ZKW serves as Co-PI of the Mayo Clinic APDA Center for Advanced Research and as an external advisory board member for the Vigil Neuroscience, Inc. LP and Mayo Clinic have licensed technology involving C9orf72 Repeat Expansion Constructs and Virus and AAV-C9orf72-149 Repeat Expansion Mouse Model. This technology was not associated with or part of the current study. LP is a consultant for Expansion Therapeutics. MP serves as a consultant for Target ALS. The other authors declare that they have no competing interests., (Copyright © 2022 Jansen-West, Todd, Daughrity, Yue, Tong, Carlomagno, Del Rosso, Kurti, Jones, Dunmore, Castanedes-Casey, Dickson, Wszolek, Fryer, Petrucelli and Prudencio.)

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord 1 . A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing 2-4 . Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies 5,6 , but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function., (© 2022. The Author(s).)

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 ( C9orf72 ), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS., Competing Interests: Authors KL and FR were employed by Ionis Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 del Rosso, Carlomagno, Todd, Jones, Prudencio, Daughrity, Yue, Jansen-West, Tong, Shao, Wu, Castanedes-Casey, Tabassian, Oskarsson, Ling, Rigo, Dickson, Yao, Petrucelli, Cook and Zhang.)

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G 4 C 2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G 4 C 2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR) 200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G 4 C 2 repeat-expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G 4 C 2 -targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A G 4 C 2 hexanucleotide repeat expansion in an intron of C9orf72 is the most common cause of frontal temporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). A remarkably similar intronic TG 3 C 2 repeat expansion is associated with spinocerebellar ataxia 36 (SCA36). Both expansions are widely expressed, form RNA foci, and can undergo repeat-associated non-ATG (RAN) translation to form similar dipeptide repeat proteins (DPRs). Yet, these diseases result in the degeneration of distinct subsets of neurons. We show that the expression of these repeat expansions in mice is sufficient to recapitulate the unique features of each disease, including this selective neuronal vulnerability. Furthermore, only the G 4 C 2 repeat induces the formation of aberrant stress granules and pTDP-43 inclusions. Overall, our results demonstrate that the pathomechanisms responsible for each disease are intrinsic to the individual repeat sequence, highlighting the importance of sequence-specific RNA-mediated toxicity in each disorder., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The human genome contains "dark" gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions with few mappable reads that we call dark by depth, and others that have ambiguous alignment, called camouflaged. We assess how well long-read or linked-read technologies resolve these regions., Results: Based on standard whole-genome Illumina sequencing data, we identify 36,794 dark regions in 6054 gene bodies from pathways important to human health, development, and reproduction. Of these gene bodies, 8.7% are completely dark and 35.2% are ≥ 5% dark. We identify dark regions that are present in protein-coding exons across 748 genes. Linked-read or long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduce dark protein-coding regions to approximately 50.5%, 35.6%, and 9.6%, respectively. We present an algorithm to resolve most camouflaged regions and apply it to the Alzheimer's Disease Sequencing Project. We rescue a rare ten-nucleotide frameshift deletion in CR1, a top Alzheimer's disease gene, found in disease cases but not in controls., Conclusions: While we could not formally assess the association of the CR1 frameshift mutation with Alzheimer's disease due to insufficient sample-size, we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

Tauopathies are neurodegenerative disorders characterized by aggregation of microtubule associated tau protein in neurons and glia. They are clinically and pathologically heterogeneous depending on the isoform of tau protein that accumulates (three or four 31-to-32-amino-acid repeats [3R or 4R] in the microtubule binding domain), as well as the cellular and neuroanatomical distribution of tau pathology. Growing evidence suggests that distinct tau conformers may contribute to the characteristic features of various tauopathies. Globular glial tauopathy (GGT) is a rare 4R tauopathy with globular cytoplasmic inclusions within neurons and glial cells. Given the unique cellular distribution and morphology of tau pathology in GGT, we sought to determine if tau species in GGT had distinctive biological properties. To address this question, we performed seeding analyses with postmortem brain tissues using a commercial tau biosensor cell line. We found that brain lysates from GGT cases had significantly higher seeding competency than other tauopathies, including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease (AD). The robust seeding activity of GGT brain lysates was independent of phosphorylated tau burden and diminished upon removal of tau from samples, suggesting that seeding properties were indeed mediated by tau in the lysates. In addition, cellular inclusions in the tau biosensor cell line induced by GGT had a distinct, globular morphology that was markedly different from inclusions induced by other tauopathies, further highlighting the unique nature of tau species in GGT. Characterization of different tau species in GGT showed that detergent-insoluble, fibril-like tau contained the highest seeding activity, as reflected in its ability to increase tau aggregation in primary glial cultures. Taken together, our data suggest that unique seeding properties differentiate GGT-tau from other tauopathies, which provides new insight into pathogenic heterogeneity of primary neurodegenerative tauopathies.

How hexanucleotide GGGGCC (G 4 C 2 ) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G 4 C 2 repeats. The expression of green fluorescent protein-conjugated (PR) 50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72 -associated FTD and ALS., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: A G 4 C 2 hexanucleotide repeat expansion in the noncoding region of C9orf72 is the major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). Putative disease mechanisms underlying c9FTD/ALS include toxicity from sense G 4 C 2 and antisense G 2 C 4 repeat-containing RNA, and from dipeptide repeat (DPR) proteins unconventionally translated from these RNA products., Methods: Intracerebroventricular injections with adeno-associated virus (AAV) encoding 2 or 149 G 4 C 2 repeats were performed on postnatal day 0, followed by assessment of behavioral and neuropathological phenotypes., Results: Relative to control mice, gliosis and neurodegeneration accompanied by cognitive and motor deficits were observed in (G 4 C 2 ) 149 mice by 6 months of age. Recapitulating key pathological hallmarks, we also demonstrate that sense and antisense RNA foci, inclusions of poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA) DPR proteins, and inclusions of endogenous phosphorylated TDP-43 (pTDP-43) developed in (G 4 C 2 ) 149 mice but not control (G 4 C 2 ) 2 mice. Notably, proteins that play a role in the regulation of stress granules - RNA-protein assemblies that form in response to translational inhibition and that have been implicated in c9FTD/ALS pathogenesis - were mislocalized in (G 4 C 2 ) 149 mice as early as 3 months of age. Specifically, we observed the abnormal deposition of stress granule components within inclusions immunopositive for poly(GR) and pTDP-43, as well as evidence of nucleocytoplasmic transport defects., Conclusions: Our in vivo model of c9FTD/ALS is the first to robustly recapitulate hallmark features derived from both sense and antisense C9orf72 repeat-associated transcripts complete with neurodegeneration and behavioral impairments. More importantly, the early appearance of persistent pathological stress granules prior to significant pTDP-43 deposition implicates an aberrant stress granule response as a key disease mechanism driving TDP-43 proteinopathy in c9FTD/ALS.

Pathogenic mutations in the tau gene (microtubule associated protein tau, MAPT) are linked to the onset of tauopathy, but the A152T variant is unique in acting as a risk factor for a range of disorders including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB). In order to provide insight into the mechanism by which A152T modulates disease risk, we developed a novel mouse model utilizing somatic brain transgenesis with adeno-associated virus (AAV) to drive tau expression in vivo, and validated the model by confirming the distinct biochemical features of A152T tau in postmortem brain tissue from human carriers. Specifically, Tau A152T -AAV mice exhibited increased tau phosphorylation that unlike animals expressing the pathogenic P301L mutation remained localized to the soluble fraction. To investigate the possibility that the A152T variant might alter the phosphorylation state of tau on T152 or the neighboring T153 residue, we generated a novel antibody that revealed significant accumulation of soluble tau species that were hyperphosphorylated on T153 (pT153) in Tau A152T -AAV mice, which were absent the soluble fraction of Tau P301L -AAV mice. Providing new insight into the role of A152T in modifying risk of tauopathy, as well as validating the Tau A152T -AAV model, we demonstrate that the presence of soluble pT153-positive tau species in human postmortem brain tissue differentiates A152T carriers from noncarriers, independent of disease classification. These results implicate both phosphorylation of T153 and an altered solubility profile in the mechanism by which A152T modulates disease risk.

Background: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 'GGGGCC' (G 4 C 2 ) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences' (PacBio) and Oxford Nanopore Technologies' (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G 4 C 2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G 4 C 2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9., Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual's repeat region was > 99% G 4 C 2 content, though we cannot rule out small interruptions., Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G 4 C 2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 G 4 C 2 repeat expansion 1,2 . Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP-(GR) 100 in the brain. GFP-(GR) 100 mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP-(GR) 100 mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP-(GR) 100 mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.

Expansion of G 4 C 2 repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G 4 C 2 translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNA Met i . Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G 4 C 2 sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.

We previously found C9orf72-associated (c9ALS) and sporadic amyotrophic lateral sclerosis (sALS) brain transcriptomes comprise thousands of defects, among which, some are likely key contributors to ALS pathogenesis. We have now generated complementary methylome data and combine these two data sets to perform a comprehensive "multi-omic" analysis to clarify the molecular mechanisms initiating RNA misregulation in ALS. We found that c9ALS and sALS patients have generally distinct but overlapping methylome profiles, and that the c9ALS- and sALS-affected genes and pathways have similar biological functions, indicating conserved pathobiology in disease. Our results strongly implicate SERPINA1 in both C9orf72 repeat expansion carriers and non-carriers, where expression levels are greatly increased in both patient groups across the frontal cortex and cerebellum. SERPINA1 expression is particularly pronounced in C9orf72 repeat expansion carriers for both brain regions, where SERPINA1 levels are strictly down regulated across most human tissues, including the brain, except liver and blood, and are not measurable in E18 mouse brain. The altered biological networks we identified contain critical molecular players known to contribute to ALS pathology, which also interact with SERPINA1. Our comprehensive combined methylation and transcription study identifies new genes and highlights that direct genetic and epigenetic changes contribute to c9ALS and sALS pathogenesis.

Significant transcriptome alterations are detected in the brain of patients with amyotrophic lateral sclerosis (ALS), including carriers of the C9orf72 repeat expansion and C9orf72-negative sporadic cases. Recently, the expression of repetitive element transcripts has been associated with toxicity and, while increased repetitive element expression has been observed in several neurodegenerative diseases, little is known about their contribution to ALS. To assess whether aberrant expression of repetitive element sequences are observed in ALS, we analysed RNA sequencing data from C9orf72-positive and sporadic ALS cases, as well as healthy controls. Transcripts from multiple classes and subclasses of repetitive elements (LINEs, endogenous retroviruses, DNA transposons, simple repeats, etc.) were significantly increased in the frontal cortex of C9orf72 ALS patients. A large collection of patient samples, representing both C9orf72 positive and negative ALS, ALS/FTLD, and FTLD cases, was used to validate the levels of several repetitive element transcripts. These analyses confirmed that repetitive element expression was significantly increased in C9orf72-positive compared to C9orf72-negative or control cases. While previous studies suggest an important link between TDP-43 and repetitive element biology, our data indicate that TDP-43 pathology alone is insufficient to account for the observed changes in repetitive elements in ALS/FTLD. Instead, we found that repetitive element expression positively correlated with RNA polymerase II activity in postmortem brain, and pharmacologic modulation of RNA polymerase II activity altered repetitive element expression in vitro. We conclude that increased RNA polymerase II activity in ALS/FTLD may lead to increased repetitive element transcript expression, a novel pathological feature of ALS/FTLD., (© The Author 2017. Published by Oxford University Press.)

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G 4 C 2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G 4 C 2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72 -associated ALS (c9ALS). Therapeutics that target G 4 C 2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G 4 C 2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G 4 C 2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G 4 C 2 RNA and downstream G 4 C 2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G 4 C 2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention., (Copyright © 2017, American Association for the Advancement of Science.)

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease., Competing Interests: The authors declare no actual or potential conflict of interest., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately., (Copyright © 2016, American Association for the Advancement of Science.)

Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology., Competing Interests: The authors declare no competing financial interests.

The aggregation and mislocalization of RNA-binding proteins leads to the aberrant regulation of RNA metabolism and is a key feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. However, the pathological consequences of abnormal deposition of TDP-43 and other RNA-binding proteins remain unclear, as the specific molecular events that drive neurodegeneration have been difficult to identify and continue to be elusive. Here, we provide novel insight into the complexity of the RNA-binding protein network by demonstrating that the inclusion of exon 17b in the SORT1 mRNA, a pathologically relevant splicing event known to be regulated by TDP-43, is also considerably affected by additional RNA-binding proteins, such as hnRNP L, PTB/nPTB and hnRNP A1/A2. Most importantly, the expression of hnRNP A1/A2 and PTB/nPTB is significantly altered in patients with frontotemporal dementia with TDP-43-positive inclusions (FTLD-TDP), indicating that perturbations in RNA metabolism and processing in FTLD-TDP are not exclusively driven by a loss of TDP-43 function. These results also suggest that a comprehensive assessment of the RNA-binding protein network will dramatically advance our current understanding of the role of TDP-43 in disease pathogenesis, as well as enhance both diagnostic and therapeutic capabilities., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aberrant tau protein accumulation drives neurofibrillary tangle (NFT) formation in several neurodegenerative diseases. Currently, efforts to elucidate pathogenic mechanisms and assess the efficacy of therapeutic targets are limited by constraints of existing models of tauopathy. In order to generate a more versatile mouse model of tauopathy, somatic brain transgenesis was utilized to deliver adeno-associated virus serotype 1 (AAV1) encoding human mutant P301L-tau compared with GFP control. At 6 months of age, we observed widespread human tau expression with concomitant accumulation of hyperphosphorylated and abnormally folded proteinase K resistant tau. However, no overt neuronal loss was observed, though significant abnormalities were noted in the postsynaptic scaffolding protein PSD95. Neurofibrillary pathology was also detected with Gallyas silver stain and Thioflavin-S, and electron microscopy revealed the deposition of closely packed filaments. In addition to classic markers of tauopathy, significant neuroinflammation and extensive gliosis were detected in AAV1-Tau(P301L) mice. This model also recapitulates the behavioral phenotype characteristic of mouse models of tauopathy, including abnormalities in exploration, anxiety, and learning and memory. These findings indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved and are independent of cell death in this novel AAV-based model of tauopathy, which offers exceptional versatility and speed in comparison with existing transgenic models. Therefore, we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets., (© The Author 2015. Published by Oxford University Press.)

The major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 repeat expansion in C9ORF72. Efforts to combat neurodegeneration associated with "c9FTD/ALS" are hindered by a lack of animal models recapitulating disease features. We developed a mouse model to mimic both neuropathological and clinical c9FTD/ALS phenotypes. We expressed (G4C2)66 throughout the murine central nervous system by means of somatic brain transgenesis mediated by adeno-associated virus. Brains of 6-month-old mice contained nuclear RNA foci, inclusions of poly(Gly-Pro), poly(Gly-Ala), and poly(Gly-Arg) dipeptide repeat proteins, as well as TDP-43 pathology. These mouse brains also exhibited cortical neuron and cerebellar Purkinje cell loss, astrogliosis, and decreased weight. (G4C2)66 mice also developed behavioral abnormalities similar to clinical symptoms of c9FTD/ALS patients, including hyperactivity, anxiety, antisocial behavior, and motor deficits., (Copyright © 2015, American Association for the Advancement of Science.)

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the "c9RAN proteins" thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes "c9FTD/ALS" are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. Hexanucleotide (GGGGCC) repeat expansions in a noncoding region of C9ORF72 are the major genetic cause of FTD and ALS (c9FTD/ALS). The RNA structure of GGGGCC repeats renders these transcripts susceptible to an unconventional mechanism of translation-repeat-associated non-ATG (RAN) translation. Antibodies generated against putative GGGGCC repeat RAN-translated peptides (anti-C9RANT) detected high molecular weight, insoluble material in brain homogenates, and neuronal inclusions throughout the CNS of c9FTD/ALS cases. C9RANT immunoreactivity was not found in other neurodegenerative diseases, including CAG repeat disorders, or in peripheral tissues of c9FTD/ALS. The specificity of C9RANT for c9FTD/ALS is a potential biomarker for this most common cause of FTD and ALS. These findings have significant implications for treatment strategies directed at RAN-translated peptides and their aggregation and the RNA structures necessary for their production., (Copyright © 2013 Elsevier Inc. All rights reserved.)

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

We found that CNS-directed expression of interferon-γ (IFN-γ) resulted in basal ganglia calcification, reminiscent of human idiopathic basal ganglia calcification (IBGC), and nigrostriatal degeneration. Our results indicate that IFN-γ mediates age-progressive nigrostriatal degeneration in the absence of exogenous stressors. Further study of this model may provide insight into selective nigrostriatal degeneration in human IBGC and other Parkinson syndromes.

The use of recombinant adeno-associated viral (rAAV) vectors as a means of gene delivery to the central nervous system has emerged as a potentially viable method for the treatment of several types of degenerative brain diseases. However, a limitation of typical intracranial injections into the adult brain parenchyma is the relatively restricted distribution of the delivered gene to large brain regions such as the cortex, presumably due to confined dispersion of the injected particles. Optimizing the administration techniques to maximize gene distribution and gene expression is an important step in developing gene therapy studies. Here, we have found additive increases in distribution when 3 methods to increase brain distribution of rAAV were combined. The convection enhanced delivery (CED) method with the step-design cannula was used to deliver rAAV vector serotypes 5, 8 and 9 encoding GFP into the hippocampus of the mouse brain. While the CED method improved distribution of all 3 serotypes, the combination of rAAV9 and CED was particularly effective. Systemic mannitol administration, which reduces intracranial pressure, also further expanded distribution of GFP expression, in particular, increased expression on the contralateral hippocampi. These data suggest that combining advanced injection techniques with newer rAAV serotypes greatly improves viral vector distribution, which could have significant benefits for implementation of gene therapy strategies., (Copyright © 2010 Elsevier B.V. All rights reserved.)