C9orf72 poly-PR helps p53 escape from the ubiquitin-proteasome system and promotes its stability.

C9orf72-derived dipeptide repeats (DPRs) proteins have been regarded as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). As the most toxic DPRs in C9-ALS/FTD, poly-proline-arginine (poly-PR) is associated with the stability and accumulation of p53, which consequently induces neurodegeneration. However, the exact molecular mechanism via which C9orf72 poly-PR stabilizes p53 remains unclear. In this study, we showed that C9orf72 poly-PR induces not only neuronal damage but also p53 accumulation and p53 downstream gene activation in primary neurons. C9orf72 (PR)50 also slows down p53 protein turnover without affecting the p53 transcription level and thus promotes its stability in N2a cells. Interestingly, the ubiquitin-proteasome system but not the autophagy function was impaired in (PR)50 transfected N2a cells, resulting in defective p53 degradation. Moreover, we found that (PR)50 induces mdm2 mistranslocation from the nucleus to the cytoplasm and competitively binds to p53, reducing mdm2-p53 interactions in the nucleus in two different (PR)50 transfected cells. Our data strongly indicate that (PR)50 reduces mdm2-p53 interactions and causes p53 to escape from the ubiquitin-proteasome system, promoting its stability and accumulation. Inhibiting or at least downregulating (PR)50 binding with p53 may be therapeutically exploited for the treatment of C9-ALS/FTD.

Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity of CHCHD10-related diseases.

Following the involvement of CHCHD10 in FrontoTemporal-Dementia-Amyotrophic Lateral Sclerosis (FTD-ALS) clinical spectrum, a founder mutation (p.Gly66Val) in the same gene was identified in Finnish families with late-onset spinal motor neuronopathy (SMAJ). SMAJ is a slowly progressive form of spinal muscular atrophy with a life expectancy within normal range. In order to understand why the p.Ser59Leu mutation, responsible for severe FTD-ALS, and the p.Gly66Val mutation could lead to different levels of severity, we compared their effects in patient cells. Unlike affected individuals bearing the p.Ser59Leu mutation, patients presenting with SMAJ phenotype have neither mitochondrial myopathy nor mtDNA instability. The expression of CHCHD10S59L mutant allele leads to disassembly of mitochondrial contact site and cristae organizing system (MICOS) with mitochondrial dysfunction and loss of cristae in patient fibroblasts. We also show that G66V fibroblasts do not display the loss of MICOS complex integrity and mitochondrial damage found in S59L cells. However, S59L and G66V fibroblasts show comparable accumulation of phosphorylated mitochondrial TDP-43 suggesting that the severity of phenotype and mitochondrial damage do not depend on mitochondrial TDP-43 localization. The expression of the CHCHD10G66V allele is responsible for mitochondrial network fragmentation and decreased sensitivity towards apoptotic stimuli, but with a less severe effect than that found in cells expressing the CHCHD10S59L allele. Taken together, our data show that cellular phenotypes associated with p.Ser59Leu and p.Gly66Val mutations in CHCHD10 are different; loss of MICOS complex integrity and mitochondrial dysfunction, but not TDP-43 mitochondrial localization, being likely essential to develop a severe motor neuron disease.

Review: Induced pluripotent stem cell models of frontotemporal dementia.

The increasing prevalence of dementia in the ageing population combined with the lack of treatments and the burden on national health care systems globally make dementia a public health priority. Despite the plethora of important research findings published over the past two decades, the mechanisms underlying dementia are still poorly understood and the progress in pharmacological interventions is limited. Recent advances in cellular reprogramming and genome engineering technologies offer an unprecedented new paradigm in disease modeling. Induced pluripotent stem cells (iPSCs) have enabled the study of patient-derived neurons in vitro, a significant progress in the field of dementia research. The first studies using iPSCs to model dementia have recently emerged, holding promise for elucidating disease pathogenic mechanisms and accelerating drug discovery. In this review, we summarize the major findings of iPSC-based studies in frontotemporal dementia (FTD) and FTD overlapping with amyotrophic lateral sclerosis (FTD/ALS). We also discuss some of the main challenges in the use of iPSCs to model complex, late-onset neurodegenerative diseases such as dementias.

A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement.

Mitochondrial DNA instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare. We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mitochondrial DNA deletions found in skeletal muscle revealed a mitochondrial DNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. Using whole-exome sequencing we identified a missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven frontotemporal dementia-amyotrophic lateral sclerosis. We identified the same missense p.Ser59Leu mutation in one of these families. This work opens a novel field to explore the pathogenesis of the frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.

Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS.

GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C4G2 repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C4G2 RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by biallelic, non-reference pentameric AAGGG(CCCTT) repeat expansions within the second intron of replication factor complex subunit 1 (RFC1). To investigate how these repeats cause disease, we generated CANVAS patient induced pluripotent stem cell (iPSC) derived neurons (iNeurons) and utilized calcium imaging and transcriptomic analysis to define repeat-elicited gain-of-function and loss-of-function contributions to neuronal toxicity. AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway functions. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins that selectively accumulate in CANVAS patient brains. However, neither these proteins nor repeat RNA foci were detected in iNeurons, and overexpression of these repeats in isolation did not induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded allele. These phenotypic deficits were not replicated by knockdown of RFC1 in control neurons and were not rescued by ectopic expression of RFC1. These findings support a repeat-dependent but RFC1-independent cause of neuronal dysfunction in CANVAS, with important implications for therapeutic development in this currently untreatable condition.

Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs.

Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.

C9ORF72: What It Is, What It Does, and Why It Matters.

When the non-coding repeat expansion in the C9ORF72 gene was discovered to be the most frequent cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in 2011, this gene and its derived protein, C9ORF72, were completely unknown. The mutation appeared to produce both haploinsufficiency and gain-of-function effects in the form of aggregating expanded RNAs and dipeptide repeat proteins (DPRs). An unprecedented effort was then unleashed to decipher the pathogenic mechanisms and the functions of C9ORF72 in order to design therapies. A decade later, while the toxicity of accumulating gain-of-function products has been established and therapeutic strategies are being developed to target it, the contribution of the loss of function starts to appear more clearly. This article reviews the current knowledge about the C9ORF72 protein, how it is affected by the repeat expansion in models and patients, and what could be the contribution of its haploinsufficiency to the disease in light of the most recent findings. We suggest that these elements should be taken into consideration to refine future therapeutic strategies, compensating for the decrease of C9ORF72 or at least preventing a further reduction.

Frontal and Cerebellar Atrophy Supports FTSD-ALS Clinical Continuum.

BACKGROUND: Frontotemporal Spectrum Disorder (FTSD) and Amyotrophic Lateral Sclerosis (ALS) are neurodegenerative diseases often considered as a continuum from clinical, epidemiologic, and genetic perspectives. We used localized brain volume alterations to evaluate common and specific features of FTSD, FTSD-ALS, and ALS patients to further understand this clinical continuum. METHODS: We used voxel-based morphometry on structural magnetic resonance images to localize volume alterations in group comparisons: patients (20 FTSD, seven FTSD-ALS, and 18 ALS) versus healthy controls (39 CTR), and patient groups between themselves. We used mean whole-brain cortical thickness ( C T ¯ ) to assess whether its correlations with local brain volume could propose mechanistic explanations of the heterogeneous clinical presentations. We also assessed whether volume reduction can explain cognitive impairment, measured with frontal assessment battery, verbal fluency, and semantic fluency. RESULTS: Common (mainly frontal) and specific areas with reduced volume were detected between FTSD, FTSD-ALS, and ALS patients, confirming suggestions of a clinical continuum, while at the same time defining morphological specificities for each clinical group (e.g., a difference of cerebral and cerebellar involvement between FTSD and ALS). C T ¯ values suggested extensive network disruption in the pathological process, with indications of a correlation between cerebral and cerebellar volumes and C T ¯ in ALS. The analysis of the neuropsychological scores indeed pointed toward an important role for the cerebellum, along with fronto-temporal areas, in explaining impairment of executive, and linguistic functions. CONCLUSION: We identified common elements that explain the FTSD-ALS clinical continuum, while also identifying specificities of each group, partially explained by different cerebral and cerebellar involvement.

Diffusion tensor imaging evidence of corticospinal pathway involvement in frontotemporal lobar degeneration.

Motor neuron dysfunctions (MNDys) in Frontotemporal Lobar Degeneration (FTLD) have been consistently reported. Clinical and neurophysiological findings proved a variable range of pathological changes, also affecting the corticospinal tract (CST). This study aims to assess white-matter microstructural alterations in a sample of patients with FTLD, and to evaluate the relationship with MNDys. Fifty-four FTLD patients (21 bvFTD, 16 PPA, 17 CBS) and 36 healthy controls participated in a Diffusion Tensor Imaging (DTI) study. We analyzed distinctive and common microstructural alteration patterns across FTLD subtypes, including those affecting the CST, and performed an association analysis between CST integrity and the presence of clinical and/or neurophysiological signs of MNDys. The majority of FTLD patients showed microstructural changes in the motor pathway with a high prevalence of CST alterations also in patients not displaying clinical and/or neurophysiological signs of MNDys. Our results suggest that subtle CST alterations characterize FTLD patients regardless to the subtype. This may be due to the spread of the pathological process to the motor system, even without a clear clinical manifestation of MNDys.

Excitatory Dendritic Mitochondrial Calcium Toxicity: Implications for Parkinson's and Other Neurodegenerative Diseases.

Dysregulation of calcium homeostasis has been linked to multiple neurological diseases. In addition to excitotoxic neuronal cell death observed following stroke, a growing number of studies implicate excess excitatory neuronal activity in chronic neurodegenerative diseases. Mitochondria function to rapidly sequester large influxes of cytosolic calcium through the activity of the mitochondrial calcium uniporter (MCU) complex, followed by more gradual release via calcium antiporters, such as NCLX. Increased cytosolic calcium levels almost invariably result in increased mitochondrial calcium uptake. While this response may augment mitochondrial respiration, limiting classic excitotoxic injury in the short term, recent studies employing live calcium imaging and molecular manipulation of calcium transporter activities suggest that mitochondrial calcium overload plays a key role in Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and related dementias [PD with dementia (PDD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD)]. Herein, we review the literature on increased excitatory input, mitochondrial calcium dysregulation, and the transcriptional or post-translational regulation of mitochondrial calcium transport proteins, with an emphasis on the PD-linked kinases LRRK2 and PINK1. The impact on pathological dendrite remodeling and neuroprotective effects of manipulating MCU, NCLX, and LETM1 are reviewed. We propose that shortening and simplification of the dendritic arbor observed in neurodegenerative diseases occur through a process of excitatory mitochondrial toxicity (EMT), which triggers mitophagy and perisynaptic mitochondrial depletion, mechanisms that are distinct from classic excitotoxicity.

Unraveling the Role of RNA Mediated Toxicity of C9orf72 Repeats in C9-FTD/ALS.

The most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is intronic hexanucleotide (G4C2) repeat expansions (HRE) in the C9orf72 gene. The non-exclusive pathogenic mechanisms by which C9orf72 repeat expansions contribute to these neurological disorders include loss of C9orf72 function and gain-of-function determined by toxic RNA molecules and dipeptides repeats protein toxicity. The expanded repeats are transcribed bidirectionally and forms RNA foci in the central nervous system, and sequester key RNA-binding proteins (RBPs) leading to impairment in RNA processing events. Many studies report widespread transcriptome changes in ALS carrying a C9orf72 repeat expansion. Here we review the contribution of RNA foci interaction with RBPs as well as transcriptome changes involved in the pathogenesis of C9orf72- associated FTD/ALS. These informations are essential to elucidate the pathology and therapeutic intervention of ALS and/or FTD.

RNA biology of disease-associated microsatellite repeat expansions.

Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA in this review, which can mediate molecular disease pathology in multiple ways. This review focuses on the potential impact that simple tandem repeat expansions can have on the biology and metabolism of RNA that contain them and underscores important gaps in understanding. Merging the molecular biology of repeat expansion disorders with the current understanding of RNA biology, including splicing, transcription, transport, turnover and translation, will help clarify mechanisms of disease and improve therapeutic development.

Syntactic comprehension deficits across the FTD-ALS continuum.

To establish the frequency, severity, relationship to bulbar symptoms, and neural correlates of syntactic comprehension deficits across the frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) disease spectrum. In total, 85 participants were included in the study; 20 amyotrophic lateral sclerosis (ALS), 15 FTD-ALS, 27 progressive nonfluent aphasia (PNFA), and 23 controls. Syntactic comprehension was evaluated in ALS, FTD-ALS, PNFA, and controls using the Test for Reception of Grammar. Voxel-based morphometry examined neuroanatomical correlates of performance. Syntactic comprehension deficits were detected in 25% of ALS (p = 0.011), 92.9% of FTD-ALS (p < 0.001), and 81.5% of PNFA (p < 0.001) patients. FTD-ALS was disproportionately impaired compared to PNFA. Impaired Test for Reception of Grammar performance was frequent in ALS with early bulbar involvement but did not correlate with bulbar impairment overall. Left peri-insular atrophy correlated with syntactic comprehension deficits. Syntactic comprehension deficits are frequent in FTD-ALS, more severe than in PNFA, and related to left peri-insular atrophy. A significant minority of ALS patients are impaired, but the relationship between bulbar symptoms and syntactic impairment is not understood.

The role of CHMP2BIntron5 in autophagy and frontotemporal dementia.

Charged multivesicular body protein 2B (CHMP2B) - a component of the endosomal complex required for transport-III (ESCRT-III) - is responsible for the vital membrane deformation functions in autophagy and endolysosomal trafficking. A dominant mutation in CHMP2B (CHMP2BIntron5) is associated with a subset of heritable frontotemporal dementia - frontotemporal dementia linked to chromosome 3 (FTD-3). ESCRT-III recruits Vps4, an AAA-ATPase that abscises the membrane during various cellular processes including autophagy and intraluminal vesicle formation. CHMP2BIntron5 results in a C-terminus truncation removing an important Vps4 binding site as well as eliminating the normal autoinhibitory resting state of CHMP2B. CHMP2B is expressed in most cell types but seems to be especially vital for proper neuronal function. CHMP2BIntron5-mediated phenotypes include misregulation of transmembrane receptors, accumulation of multilamellar structures, abnormal lysosomal morphology, down regulation of a brain-specific micro RNA (miRNA-124), abnormal dendritic spine morphology, decrease in dendritic arborization, and cell death. Currently, transgenic-fly,-mouse, and -human cell lines are being used to better understand the diverse phenotypes and develop therapeutic approaches for the CHMP2BIntron5-induced FTD-3. This article is part of a Special Issue entitled SI:Autophagy.

Unstable repeat expansions in neurodegenerative diseases: nucleocytoplasmic transport emerges on the scene.

An astonishing number of neurological diseases result from expansion of unstable repetitive sequences causing alterations in key neuronal processes. Some are progressive late-onset conditions related to aging, such as the spinocerebellar ataxias. In several of these pathologies, the expanded repeat is transcribed, producing an expanded RNA repeat that causes neurodegeneration by a complex mechanism, comprising 3 main pathways. These include (1) accumulation in the nucleus of RNA foci, resulting from sequestration of RNA-binding proteins functioning in important neuronal cascades; (2) decrease in availability of RNA-binding proteins, such as splicing factors, causing alternative splicing misregulation with imbalance in the expression ratio of neuronal isoforms; and (3) generation of neurotoxic peptides, produced from repeat-associated non-ATG-initiated translation across the RNA repeat, in all reading frames. Recently, 2 pathologies characterized by impaired motor function, cognitive decline, or/and degeneration of motor neurons have been found that have broaden our understanding of these diseases. Moreover, the finding of compromised nucleocytoplasmic transport opens new avenues for research. This review will cover the amazing progress regarding these conditions.

Structural insight into C9orf72 hexanucleotide repeat expansions: Towards new therapeutic targets in FTD-ALS.

Hexanucleotide repeat expansions, (G4C2) in the C9orf72 gene are considered as the single most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). (G4C2), either as DNA or the transcribed RNA, can folds into unusual secondary structures, including G-quadruplex, R-loop, I-motif and hairpin. These structural polymorphism at both DNA and RNA levels were proposed to initiate molecular cascade leading to ALS/FTD. G-quadruplexes are composed of stacked G4 tetrads, held by hydrophobic bonds, and is highly stable secondary structure. Here, we covers the structural and functional features of G-quadruplexes with an emphasis on C9orf72-repeat-associated FTD and ALS (C9-FTD/ALS). We also highlighted tools and techniques used to study the G-quadruplexes. Current perspectives for molecules that target G-quadruplexes as potential therapeutic are discussed. Our extensive analysis of structural features of G-quadruplexes will be used for a better understanding of molecular mechanism of C9-FTD/ALS.

Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions.

TMEM106B was identified as a risk factor for frontotemporal lobar degeneration (FTD) with TAR DNA-binding protein 43 kDa inclusions. It has been reported that variants in this gene are genetic modifiers of the disease and that this association is stronger in patients carrying a GRN mutation or a pathogenic expansion in chromosome 9 open reading frame 72 (C9orf72) gene. Here, we investigated the contribution of TMEM106B polymorphisms in cohorts of FTD and FTD with amyotrophic lateral sclerosis patients from France and Italy. Patients carrying the C9orf72 expansion (n = 145) and patients with GRN mutations (n = 76) were compared with a group of FTD patients (n = 384) negative for mutations and to a group of healthy controls (n = 552). In our cohorts, the presence of the C9orf72 expansion did not correlate with TMEM106B genotypes but the association was very strong in individuals with pathogenic GRN mutations (p = 9.54 × 10(-6)). Our data suggest that TMEM106B genotypes differ in FTD patient cohorts and strengthen the protective role of TMEM106B in GRN carriers. Further studies are needed to determine whether TMEM106B polymorphisms are associated with other genetic causes for FTD, including C9orf72 repeat expansions.