A Novel Protocol for Directed Differentiation of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease, therapeutic drug screening, and transplant-based regenerative medicine. However, methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here, we present a novel, small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3beta (GSK3beta) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC), sporadic FTD, and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid, efficient, and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE: Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient, novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases

A Multiancestral Genome-Wide Exome Array Study of Alzheimer Disease, Frontotemporal Dementia, and Progressive Supranuclear Palsy

Importance Previous studies have indicated a heritable component of the etiology of neurodegenerative diseases such as Alzheimer disease (AD), frontotemporal dementia (FTD), and progressive supranuclear palsy (PSP). However, few have examined the contribution of low-frequency coding variants on a genome-wide level. Objective To identify low-frequency coding variants that affect susceptibility to AD, FTD, and PSP. Design, Setting, and Participants We used the Illumina HumanExome BeadChip array to genotype a large number of variants (most of which are low-frequency coding variants) in a cohort of patients with neurodegenerative disease (224 with AD, 168 with FTD, and 48 with PSP) and in 224 control individuals without dementia enrolled between 2005-2012 from multiple centers participating in the Genetic Investigation in Frontotemporal Dementia and Alzheimer’s Disease (GIFT) Study. An additional multiancestral replication cohort of 240 patients with AD and 240 controls without dementia was used to validate suggestive findings. Variant-level association testing and gene-based testing were performed. Main Outcomes and Measures Statistical association of genetic variants with clinical diagnosis of AD, FTD, and PSP. Results Genetic variants typed by the exome array explained 44%, 53%, and 57% of the total phenotypic variance of AD, FTD, and PSP, respectively. An association with the known AD gene ABCA7 was replicated in several ancestries (discovery P = .0049, European P = .041, African American P = .043, and Asian P = .027), suggesting that exonic variants within this gene modify AD susceptibility. In addition, 2 suggestive candidate genes, DYSF (P = 5.53 × 10−5) and PAXIP1 (P = 2.26 × 10−4), were highlighted in patients with AD and differentially expressed in AD brain. Corroborating evidence from other exome array studies and gene expression data points toward potential involvement of these genes in the pathogenesis of AD. Conclusions and Relevance Low-frequency coding variants with intermediate effect size may account for a significant fraction of the genetic susceptibility to AD and FTD. Furthermore, we found evidence that coding variants in the known susceptibility gene ABCA7, as well as candidate genes DYSF and PAXIP1, confer risk for AD

Lack of Association Between the CCR5-delta32 Polymorphism and Neurodegenerative Disorders.

ObjectiveRecent studies have suggested that diminished Ccr5 functioning has an effect on synaptic plasticity and hippocampal memory in mouse models. CCR5-delta32, a 32-bp frameshift deletion in human CCR5 encoding a nonfunctional receptor, has been reported to have a protective effect against human immunodeficiency virus infection but its role as a modifier of neurodegenerative disease has been minimally explored. We investigated whether the CCR5-delta32 polymorphism could have an effect in the context of human neurodegenerative diseases.MethodsWe examined the frequency of the CCR5-delta32 polymorphism in a large and well-characterized cohort including 1425 patients with neurodegenerative dementias and 2032 controls.ResultsWe did not observe a significant association between the CCR5-delta32 polymorphism and any of the neurodegenerative diseases screened in this study. However, we observed an earlier age of onset among neurodegenerative disease patients carrying the CCR5-delta32 allele.ConclusionsAlthough our findings were inconclusive, the earlier age of onset observed among neurodegenerative disease patients carrying the CCR5-delta32 allele suggests that the deletion may have a detrimental effect in the context of neurodegeneration

ApoE &egr;4 Is Associated With Cognition, Brain Integrity, and Atrophy in HIV Over Age 60

BackgroundThere are contradicting reports on the associations between Apolipoprotein E4 (ApoE ε4) and brain outcomes in HIV with some evidence that relationships may be greatest in older age groups.MethodsWe assessed cognition in 76 clinically stable HIV-infected participants over age 60 and genotyped ApoE. Sixty-one of these subjects underwent structural brain magnetic resonance imaging and diffusion tensor imaging.ResultsThe median age of the participants was 64 years (range: 60-84) and the median estimated duration of HIV infection was 22 years. Apo ε4 carriers (n = 19) were similar to noncarriers (n = 57) in sex (95% vs. 96% male), and education (16.0 vs. 16.2 years) ApoE ε4 carriers demonstrated greater deficits in cognitive performance in the executive domain (P = 0.045) and had reduced fractional anisotropy and increased mean diffusivity throughout large white matter tracts within the brain compared with noncarriers. Tensor-based morphometry analyses revealed ventricular expansion and atrophy in the posterior corpus callosum, thalamus, and brainstem among HIV-infected ApoE ε4 carriers compared with ε4 noncarriers.ConclusionsIn this sample of older HIV-infected individuals, having at least 1 ApoE ε4 allele was associated with decreased cognitive performance in the executive functioning domain, reduced brain white matter integrity, and brain atrophy. Brain atrophy was most prominent in the posterior corpus callosum, thalamus, and brainstem. This pattern of cognitive deficit, atrophy, and damage to white matter integrity was similar to that described in HIV, suggesting an exacerbation of HIV-related pathology; although emergence of other age-associated neurodegenerative disorders cannot be excluded

Epigenetic clock and methylation studies in vervet monkeys.

DNA methylation-based biomarkers of aging have been developed for many mammals but not yet for the vervet monkey (Chlorocebus sabaeus), which is a valuable non-human primate model for biomedical studies. We generated novel DNA methylation data from vervet cerebral cortex, blood, and liver using highly conserved mammalian CpGs represented on a custom array (HorvathMammalMethylChip40). We present six DNA methylation-based estimators of age: vervet multi-tissue epigenetic clock and tissue-specific clocks for brain cortex, blood, and liver. In addition, we developed two dual species clocks (human-vervet clocks) for measuring chronological age and relative age, respectively. Relative age was defined as ratio of chronological age to maximum lifespan to address the species differences in maximum lifespan. The high accuracy of the human-vervet clocks demonstrates that epigenetic aging processes are evolutionary conserved in primates. When applying these vervet clocks to tissue samples from another primate species, rhesus macaque, we observed high age correlations but strong offsets. We characterized CpGs that correlate significantly with age in the vervet. CpG probes that gain methylation with age across tissues were located near the targets of Polycomb proteins SUZ12 and EED and genes possessing the trimethylated H3K27 mark in their promoters. The epigenetic clocks are expected to be useful for anti-aging studies in vervets

A novel temporal-predominant neuro-astroglial tauopathy associated with TMEM106B gene polymorphism in FTLD/ALS-TDP.

Polymorphisms in TMEM106B, a gene on chromosome 7p21.3 involved in lysosomal trafficking, correlates to worse neuropathological, and clinical outcomes in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) with TDP-43 inclusions. In a small cohort of C9orf72 expansion carriers, we previously found an atypical, neuroglial tauopathy in cases harboring a TMEM106B rs1990622 A/A genotype. To test whether TMEM106B genotype affects the risk of developing atypical tauopathy under a recessive genotype model (presence versus absence of two major alleles: A/A vs. A/G and G/G). We characterized the atypical tauopathy neuropathologically and determined its frequency by TMEM106B rs1990622 genotypes in 90 postmortem cases with a primary diagnosis of FTLD/ALS-TDP [mean age at death 65.5 years (±8.1), 40% female]. We investigated the effect of this new atypical tauopathy on demographics and clinical and neuropsychological metrics. We also genotyped TMEM106B in an independent series with phenotypically similar cases. Sixteen cases (16/90, 17.7 %) showed the temporal-predominant neuro-astroglial tauopathy, and 93.7% of them carried an A/A genotype (vs. ~35% in a population cohort). The odds ratio of FTLD/ALS-TDP individuals with the A/A genotype showing neuro-astroglial tauopathy was 13.9. Individuals with this tauopathy were older at onset (p = 0.01). The validation cohort had a similarly high proportion of rs1990622 A/A genotype. TDP-43 and tau changes co-occur in a subset of neurons. Our data add to the growing body of evidence that TMEM106B polymorphisms may modulate neurodegeneration. A distinctive medial temporal predominant, 4-repeat, neuro-astroglial tauopathy strongly correlates to TMEM106B A/A genotype in FTLD/ALS-TDP cases

Sex-Related Differences in the Relationship Between β-Amyloid and Cognitive Trajectories in Older Adults

Objective: We aimed to test the hypothesis that elevated neocortical β-amyloid (Aβ), a hallmark feature of Alzheimer's disease (AD), predicts sex-specific cognitive trajectories in clinically normal older adults, with women showing greater risk of decline than men. Method: Florbetapir Aβ positron emission tomography (PET) was acquired in 149 clinically normal older adults (52% female, Mage = 74). Participants underwent cognitive testing at baseline and during annual follow-up visits over a timespan of up to 5.14 years. Mixed-effects regression models evaluated whether relations between baseline neocortical Standardized Uptake Value Ratio (SUVR) and composite scores of episodic memory, executive functioning, and processing speed were moderated by sex (male/female) and apolipoprotein E (APOE) status (ε4 carrier/noncarrier). Results: Higher baseline SUVR was associated with longitudinal decline in episodic memory in women (b = -1.32, p < .001) but not men (b = -0.30, p = .28). Female APOE ε4 carriers with elevated SUVR showed particularly precipitous declines in episodic memory (b = -4.33, p < .001) whereas other cognitive domains were spared. SUVR did not predict changes in executive functioning or processing speed, regardless of sex (ps >.63), though there was a main effect of SUVR on processing speed (b = 2.50, p = .003). Conclusions: Clinically normal women with elevated Aβ are more vulnerable to episodic memory decline than men. Understanding sex-related differences in AD, particularly in preclinical stages, is crucial for guiding precision medicine approaches to early detection and intervention. (PsycInfo Database Record (c) 2020 APA, all rights reserved)

Amyloid in dementia associated with familial FTLD: not an innocent bystander.

Patients with frontotemporal lobar degeneration (FTLD) can show superimposed amyloid pathology, though the impact of amyloid on the clinical presentation of FTLD is not well characterized. This cross-sectional case-control study compared clinical features, fluorodeoxyglucose-positron emission tomography metabolism and gray matter volume loss in 30 patients with familial FTLD in whom amyloid status was confirmed with autopsy or Pittsburgh compound B-PET. Compared to the amyloid-negative patients, the amyloid-positive patients performed significantly worse on several cognitive tests and showed hypometabolism and volume loss in more temporoparietal regions. Our results suggest that in FTLD amyloid positivity is associated with a more Alzheimer's disease-like pattern of neurodegeneration

Amyloid in dementia associated with familial FTLD: not an innocent bystander

<div><p>Patients with frontotemporal lobar degeneration (FTLD) can show superimposed amyloid pathology, though the impact of amyloid on the clinical presentation of FTLD is not well characterized. This cross-sectional case–control study compared clinical features, fluorodeoxyglucose-positron emission tomography metabolism and gray matter volume loss in 30 patients with familial FTLD in whom amyloid status was confirmed with autopsy or Pittsburgh compound B-PET. Compared to the amyloid-negative patients, the amyloid-positive patients performed significantly worse on several cognitive tests and showed hypometabolism and volume loss in more temporoparietal regions. Our results suggest that in FTLD amyloid positivity is associated with a more Alzheimer’s disease-like pattern of neurodegeneration.</p></div