Han-Jou Chen

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

Summary The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

Familial amyotrophic lateral sclerosis is associated with a mutation in D-amino acid oxidase

We report a unique mutation in the D-amino acid oxidase gene (R199W DAO) associated with classical adult onset familial amyotrophic lateral sclerosis (FALS) in a three generational FALS kindred, after candidate gene screening in a 14.52 cM region on chromosome 12q22-23 linked to disease. Neuronal cell lines expressing R199W DAO showed decreased viability and increased ubiquitinated aggregates compared with cells expressing the wild-type protein. Similarly, lentiviral-mediated expression of R199W DAO in primary motor neuron cultures caused increased TUNEL labeling. This effect was also observed when motor neurons were cocultured on transduced astrocytes expressing R199W, indicating that the motor neuron cell death induced by this mutation is mediated by both cell autonomous and noncell autonomous processes. DAO controls the level of D-serine, which accumulates in the spinal cord in cases of sporadic ALS and in a mouse model of ALS, indicating that this abnormality may represent a fundamental component of ALS pathogenesis.

Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species

ABSTRACT TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.

TDP-43 Proteinopathy and ALS: Insights into Disease Mechanisms and Therapeutic Targets

Therapeutic options for patients with amyotrophic lateral sclerosis (ALS) are currently limited. However, recent studies show that almost all cases of ALS, as well as tau-negative frontotemporal dementia (FTD), share a common neuropathology characterized by the deposition of TAR-DNA binding protein (TDP)-43-positive protein inclusions, offering an attractive target for the design and testing of novel therapeutics. Here we demonstrate how diverse environmental stressors linked to stress granule formation, as well as mutations in genes encoding RNA processing proteins and protein degradation adaptors, initiate ALS pathogenesis via TDP-43. We review the progressive development of TDP-43 proteinopathy from cytoplasmic mislocalization and misfolding through to macroaggregation and the addition of phosphate and ubiquitin moieties. Drawing from cellular and animal studies, we explore the feasibility of therapeutics that act at each point in pathogenesis, from mitigating genetic risk using antisense oligonucleotides to modulating TDP-43 proteinopathy itself using small molecule activators of autophagy, the ubiquitin-proteasome system, or the chaperone network. We present the case that preventing the misfolding of TDP-43 and/or enhancing its clearance represents the most important target for effectively treating ALS and frontotemporal dementia.

The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis

Insoluble TDP-43 inclusions are the pathological hallmark of ALS and tau-negative frontotemporal lobar degeneration. Chen et al. show that the heat shock response (HSR), which regulates chaperone expression, is compromised in an ALS mouse model and in patients. Activation of the HSR clears insoluble TDP-43 and increases cell survival.

Allele-Specific Knockdown of ALS-Associated Mutant TDP-43 in Neural Stem Cells Derived from Induced Pluripotent Stem Cells

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43M337V mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43wt or GFP-TDP-43M337V or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43M337V allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

Expanded G4C2 repeats linked to C9ORF72 ALS and FTD form length-dependent RNA foci, sequester RNA binding proteins and are neurotoxic.

Background The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1,2]. The mechanism by which the G4C2 intronic repeats cause neurodegeneration is unknown. Decreased tissue levels of the C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [1].

Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.

Annexin A11 mutations, implicated in ALS, prevent binding to calcyclin and induce the formation of cytoplasmic inclusions. Annexing another protein in ALS pathogenesis Amyotrophic lateral sclerosis (ALS) causes progressive paralysis due to motor neuron degeneration. Smith et al. performed exome sequencing of 751 familial ALS cases and discovered six missense mutations in the ANXA11 gene in 13 individuals, which were absent or vanishingly rare in ~70,000 healthy controls. Abundant annexin 11 protein inclusions were detected in spinal motor neurons and hippocampal axons in a patient with the p.D40G mutation. Annexin 11 is known to play a role in vesicular trafficking between the Golgi and endoplasmic reticulum. Functional studies in transfected cells revealed abnormal binding of mutant annexin 11 to calcyclin, which implicates defective intracellular protein trafficking in ALS pathogenesis. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11–positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.

C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity

Abstract An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.

Erratum to: TDP-43 Proteinopathy and ALS: Insights into Disease Mechanisms and Therapeutic Targets

TDP-43 and TDP-43 Proteinopathy, Paragraph 1: BIn the nucleus, TDP-43 plays a critical role in regulating RNA splicing, as well as modulating microRNA biogenesis [8, 9].^ Reference 9 should be: Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes. Proc Natl Acad Sci U S A. 2012;109(9):3347-52. doi:10.1073/pnas.1112427109. TDP-43 and TDP-43 Proteinopathy, Paragraph 1: BIn addition to TDP-43 RNA, TDP-43 regulates the splicing and stability of a large number of other transcripts [10, 12–15], and thus influences diverse cellular processes.^ Reference 13 should be: Strong MJ, Volkening K, Hammond R, Yang W, Strong W, Leystra-Lantz C et al. TDP-43 is a human low molecular weight neurofilament (hNFL) mRNAbinding protein. Mol Cell Neurosci. 2007;35(2):320-327. Reference 14 should be: Buratti E, Dork T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J. 2001;20(7):1774-84. TDP-43 and TDP-43 Proteinopathy, Paragraph 2: BAlthough mostly nuclear, up to ~30 % of TDP-43 protein can be found in the cytoplasm [16], with nuclear efflux regulated by both activity and stress [17].^ Reference 17 should be: Wang IF, Wu LS, Chang HY, Shen CK. TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor. J Neurochem. 2008;105(3):797806. doi:10.1111/j.1471-4159.2007.05190.x. Protein degradation gene mutations, Paragraph 1: BTDP43 proteostasis is normally maintained by the coordinated action of the UPS and autophagy, which is particularly important for clearing TDP-43 oligomers and aggregates [81–87].^ This should be references 83-87 Protein degradation gene mutations, Paragraph 2: BNotably, VCP and p62 are required for the formation of Baggresomes^ [84, 88, 89], which are large perinuclear inclusions decorated with ubiquitin, ubiquilin, and p 62.^ Reference 84 should be: Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated prote in aggregates by autophagy. J Biol Chem. 2007;282(33):24131-45 (originally designated reference 79) Protein degradation gene mutations, Paragraph 2: BThe TDP43-positive aggregates that are the hallmark ALS pathology are likely aggresomes [76, 90].^ Reference 76 should be: Scotter EL, Vance C, Nishimura AL, et al. Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci 2014;127: 1263–1278. (originally designated reference 83) Protein degradation gene mutations, Paragraph 4: BCertainly, small molecule activators of the UPS or autophagy The online version of the original article can be found at http://dx.doi.org/ 10.1007/s13311-015-0338-x. E. L. Scotter :H.