Aggregation of α-synuclein is a hallmark for several neurodegenerative diseases including Dementia with Lewy bodies (DLB), Parkinson's Disease (PD), and multiple systems atrophy. Here we report a family with a history of synucleinopathy. The index case (61 y/o, APOE3/4) developed DLB by the age of 59, while his father (APOE3/3) died of DLB at the age of 75 (age of onset at 70). Two controls (paternal aunts of the proband, both APOE3/3) remain cognitively healthy at ages 77 and 73, respectively. Given the extensive familial history, we undertook a search for putative, pathogenic variants driving the underlying synucleinopathy. Visual hallucinations and cognitive decline with prominent parkinsonism were documented for the index case at the Stanford Center for Memory Disorders. One of the aunts was confirmed to be a healthy control following a research-based clinical and neuropsychological evaluations. The other aunt underwent a phone interview with a clinical dementia rating of 0. DNA of living subjects were collected from saliva samples, while the father's DNA was brain derived. Whole exome sequencing (WES) was performed on the Illumina HiSeq2000 platform. Raw data were analyzed with BWA (read mapping) and GATK (variant calling) software packages. Variants present in father-son pair and not carried by the two healthy aunts were selected for further investigation. These filtered variants were screened against 4449 controls aged 80 and up (1802 males, 2647 females) from the Alzheimer's Disease Sequencing Project. Variants not found in any controls were annotated using the Ensembl Variant Effect Predictor toolkit. Two variants remained after the filtering process—a missense variant on ZNF679 (NM_153363.2, 4:p.Ser253Leu) and a missense variant on CTD-3214H19.16 (ENSP00000469811.1:p.Ala128Thr). The father-son pair are heterozygous for each variant. Presence of these two variants in the father-son pair, combined with their absence in the two family controls and nearly 4,500 controls suggest that they are candidate pathogenic variants underlying the synucleinopathy in this family. Additional analysis of this family is underway, with WES being performed on a third paternal aunt (age 75) to further validate our findings . Pedigree of the family with an extensive history of synucleinopathy. Subjects with known APOE status have whole exome sequencing data available and were included in the analysis. Two cases of PD and three cases of dementia were reported by family members for generation I. DLB subjects are heterozygous for both c.380G>A CTD-3214H19.16 and c.758C>T ZNF679. Filtering scheme used for the discovery of possible putative, pathogenic variants.
Abstract Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin’s tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.
The most common and severe disease causing allele of Alpha 1-Antitrypsin Deficiency (1ATD) is Z-1AT. This protein aggregates in the endoplasmic reticulum, which is the main cause of liver disease in childhood. Based on recent evidences and on the frequency of liver disease occurrence in Z-1AT patients, it seems that liver disease progression is linked to still unknown genetic factors.We used an innovative approach combining yeast genetic screens with next generation exome sequencing to identify and functionally characterize the genes involved in 1ATD associated liver disease.Using yeast genetic screens, we identified HRD1, an Endoplasmic Reticulum Associated Degradation (ERAD) associated protein, as an inducer of Z-mediated toxicity. Whole exome sequencing of 1ATD patients resulted in the identification of two variants associated with liver damages in Z-1AT homozygous cases: HFE H63D and HERPUD1 R50H. Functional characterization in Z-1AT model cell lines demonstrated that impairment of the ERAD machinery combined with the HFE H63D variant expression decreased both cell proliferation and cell viability, while Unfolded Protein Response (UPR)-mediated cell death was hyperstimulated.This powerful experimental pipeline allowed us to identify and functionally validate two genes involved in Z-1AT-mediated severe liver toxicity. This pilot study moves forward our understanding on genetic modifiers involved in 1ATD and highlights the UPR pathway as a target for the treatment of liver diseases associated with 1ATD. Finally, these findings support a larger scale screening for HERPUD1 R50H and HFE H63D variants in the sub-group of 1ATD patients developing significant chronic hepatic injuries (hepatomegaly, chronic cholestasis, elevated liver enzymes) and at risk developing liver cirrhosis.
Endoplasmic-reticulum-associated protein degradation
Abstract Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from model systems. We also uncovered new potent modifiers of DPR toxicity whose gene products function in the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. Since regulators of ER stress emerged prominently from the screens, we further investigated one such modifier, TMX2 , which we identified as a modulator of the ER-stress signature elicited by C9orf72 DPRs in neurons. Together, this work identifies novel suppressors of DPR toxicity that represent potential therapeutic targets and demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases. One Sentence Summary Genome-wide CRISPR-Cas9 screens in human cells reveal mechanisms and targets for ALS-associated C9orf72 dipeptide repeat protein toxicity.
Summary Antisense oligonucleotide therapy targeting ATXN2 —a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis—has entered clinical trials in humans. Additional methods to lower ataxin-2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin-2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v-ATPase) as regulators of ataxin-2 levels. We demonstrate that Etidronate—a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v-ATPase—lowers ataxin-2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.
We describe the clinical and neuropathologic features of patients with Lewy body spectrum disorder (LBSD) carrying a nonsense variant, c.604C>T; p.R202X, in the glucocerebrosidase 1 (GBA) gene. While this GBA variant is causative for Gaucher's disease, the pathogenic role of this mutation in LBSD is unclear. Detailed neuropathologic evaluation was performed for one index case and a structured literature review of other GBA p.R202X carriers was conducted. Through the systematic literature search, we identified three additional reported subjects carrying the same GBA mutation, including one Parkinson's disease (PD) patient with early disease onset, one case with neuropathologically-verified LBSD, and one unaffected relative of a Gaucher's disease patient. Among the affected subjects carrying the GBA p.R202X, all males were diagnosed with Lewy body dementia, while the two females presented as PD. The clinical penetrance of GBA p.R202X in LBSD patients and families argues strongly for a pathogenic role for this variant, although presenting with a striking phenotypic heterogeneity of clinical and pathological features.
Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15 , in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila , with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.
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