Recent insights from human induced pluripotent stem cell models into the role of microglia in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, primarily leading to the degeneration of motor neurons. The traditional focus on motor neuron-centric mechanisms has recently shifted towards understanding the contribution of non-neuronal cells, such as microglia, in ALS pathophysiology. Advances in induced pluripotent stem cell (iPSC) technology have enabled the generation of iPSC-derived microglia monocultures and co-cultures to investigate their role in ALS pathogenesis. Here, we briefly review the insights gained from these studies into the role of microglia in ALS. While iPSC-derived microglia monocultures have revealed intrinsic cellular dysfunction due to ALS-associated mutations, microglia-motor neuron co-culture studies have demonstrated neurotoxic effects of mutant microglia on motor neurons. Based on these findings, we briefly discuss currently unresolved questions and how they could be addressed in future studies. iPSC models hold promise for uncovering disease-relevant pathways in ALS and identifying potential therapeutic targets

Factors associated with foreign body infection in methicillin-resistant Staphylococcus aureus bacteremia

BackgroundWe aimed to compare patient characteristics, MRSA sequence types, and biofilm production of MRSA strains that did and did not cause a foreign body infection in patients with MRSA bloodstream infections (BSI)MethodsAll adult patients with MRSA BSI hospitalized in two hospitals were identified by clinical microbiology laboratory surveillance. Only patients who had at least one implanted foreign body during the episode of BSI were included.ResultsIn July 2018 - March 2022, of 423 patients identified with MRSA BSI, 118 (28%) had ≥1 foreign body. Among them, 51 (43%) had one or more foreign body infections. In multivariable analysis, factors associated with foreign body infection were history of MRSA infection in the last year (OR=4.7 [1.4-15.5], p=0.012) community-associated BSI (OR=68.1 [4.2-1114.3], p=0.003); surgical site infection as source of infection (OR=11.8 [2-70.4], p=0.007); presence of more than one foreign body (OR=3.4 [1.1-10.7], p=0.033); interval between foreign body implantation and infection <18 months (OR=3.3 [1.1-10], p=0.031); and positive blood culture ≥48h (OR=16.7 [4.3-65.7], p<0.001). The most prevalent sequence type was ST8 (39%), followed by ST5 (29%), and ST105 (20%) with no significant difference between patients with or without foreign body infection. Only 39% of MRSA isolates formed a moderate/strong biofilm. No significant difference was observed between patients with foreign body infection and those without foreign body infection. In multivariable analysis, subjects infected with a MRSA isolate producing moderate/strong in vitro biofilm were more likely to have a history of MRSA infection in the last year (OR=3.41 [1.23-9.43]), interval between foreign body implantation and MRSA BSI <18 months (OR=3.1 [1.05-9.2]) and ST8 (OR=10.64 [2-57.3]).ConclusionMost factors associated with foreign body infection in MRSA BSI were also characteristic of persistent infections. Biofilm-forming isolates were not associated with a higher risk of foreign-body infection but appeared to be associated with MRSA genetic lineage, especially ST8

Oligogenic genetic variation of neurodegenerative disease genes in 980 postmortem human brains.

BACKGROUND: Several studies suggest that multiple rare genetic variants in genes causing monogenic forms of neurodegenerative disorders interact synergistically to increase disease risk or reduce the age of onset, but these studies have not been validated in large sporadic case series. METHODS: We analysed 980 neuropathologically characterised human brains with Alzheimer's disease (AD), Parkinson's disease-dementia with Lewy bodies (PD-DLB), frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) and age-matched controls. Genetic variants were assessed using the American College of Medical Genetics criteria for pathogenicity. Individuals with two or more variants within a relevant disease gene panel were defined as 'oligogenic'. RESULTS: The majority of oligogenic variant combinations consisted of a highly penetrant allele or known risk factor in combination with another rare but likely benign allele. The presence of oligogenic variants did not influence the age of onset or disease severity. After controlling for the single known major risk allele, the frequency of oligogenic variants was no different between cases and controls. CONCLUSIONS: A priori, individuals with AD, PD-DLB and FTD-ALS are more likely to harbour a known genetic risk factor, and it is the burden of these variants in combination with rare benign alleles that is likely to be responsible for some oligogenic associations. Controlling for this bias is essential in studies investigating a potential role for oligogenic variation in neurodegenerative diseases

Frequency and signature of somatic variants in 1461 human brain exomes.

PURPOSE: To systematically study somatic variants arising during development in the human brain across a spectrum of neurodegenerative disorders. METHODS: In this study we developed a pipeline to identify somatic variants from exome sequencing data in 1461 diseased and control human brains. Eighty-eight percent of the DNA samples were extracted from the cerebellum. Identified somatic variants were validated by targeted amplicon sequencing and/or PyroMark® Q24. RESULTS: We observed somatic coding variants present in >10% of sampled cells in at least 1% of brains. The mutational signature of the detected variants showed a predominance of C>T variants most consistent with arising from DNA mismatch repair, occurred frequently in genes that are highly expressed within the central nervous system, and with a minimum somatic mutation rate of 4.25 × 10-10 per base pair per individual. CONCLUSION: These findings provide proof-of-principle that deleterious somatic variants can affect sizeable brain regions in at least 1% of the population, and thus have the potential to contribute to the pathogenesis of common neurodegenerative diseases.Wellcome Trus

C9orf72-ALS human iPSC microglia are pro-inflammatory and toxic to co-cultured motor neurons via MMP9

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, with additional pathophysiological involvement of non-neuronal cells such as microglia. The commonest ALS-associated genetic variant is a hexanucleotide repeat expansion (HRE) mutation in C9orf72. Here, we study its consequences for microglial function using human iPSC-derived microglia. By RNA-sequencing, we identify enrichment of pathways associated with immune cell activation and cyto-/chemokines in C9orf72 HRE mutant microglia versus healthy controls, most prominently after LPS priming. Specifically, LPS-primed C9orf72 HRE mutant microglia show consistently increased expression and release of matrix metalloproteinase-9 (MMP9). LPS-primed C9orf72 HRE mutant microglia are toxic to co-cultured healthy motor neurons, which is ameliorated by concomitant application of an MMP9 inhibitor. Finally, we identify release of dipeptidyl peptidase-4 (DPP4) as a marker for MMP9-dependent microglial dysregulation in co-culture. These results demonstrate cellular dysfunction of C9orf72 HRE mutant microglia, and a non-cell-autonomous role in driving C9orf72-ALS pathophysiology in motor neurons through MMP9 signaling

Detection and Quantification of Novel C‐terminal TDP‐43 Fragments in ALS‐TDP

The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS‐PRM) can generate a high‐resolution map of pathological TDP‐43 peptide ratios to form the basis for quantitation of abnormal C‐terminal TDP‐43 fragment enrichment.Human cortex and spinal cord, microscopically staged for the presence of phosphoTDP‐43, p‐tau, alpha‐synuclein and beta‐amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full‐length and truncated (disease‐specific) TDP‐43 peptides. This informed synthesis of heavy isotope‐labelled peptides for absolute quantification of TDP‐43 by MS‐PRM across 16 ALS, 8 Parkinson’s and 8 Alzheimer’s disease and 8 aged control cases.We confirmed by immunoblot the previously described enrichment of pathological C‐terminal fragments in ALS‐TDP urea fractions. Subsequent MS analysis resolved specific TDP‐43 N‐ and C‐terminal peptides, including a novel N‐terminal truncation site‐specific peptide. Absolute quantification of peptides by MS‐PRM showed an increased C:N‐terminal TDP‐43 peptide ratio in ALS‐TDP brain compared to normal and disease controls. A C:N‐terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6‐100) and specificity of 100% (CI 68‐100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70‐100) and specificity of 100% (CI 68‐100). N‐terminal truncation site‐specific peptides were increased in ALS in line with C‐terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N‐terminal ratio but coexistent TDP‐43 pathology.In conclusion this is a novel, sensitive and specific method to quantify the enrichment of pathological TDP‐43 fragments in human brain, which could form the basis for an antibody‐free assay. Our methodology has the potential to help clarify if specific pathological TDP‐43 peptide signatures are associated with primary or secondary TDP‐43 proteinopathies

Author Correction:The Beta-adrenergic agonist, Ractopamine, increases skeletal muscle expression of Asparagine Synthetase as part of an integrated stress response gene program

© 2019, The Author(s). In the Supplementary Information file originally published with this Article, Table 2 was omitted. This error has been corrected in the Supplementary Information that now accompanies the Article

GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.</p

A proposal for new diagnostic criteria for ALS

© 2020 The Authors. Published by Elsevier B.V. on behalf of International Federation of Clinical Neurophysiology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Sclerosis (ALS) were initially published in 1994 and revised in 2000. Criteria were established because the ‘‘variety of clinical features which may be present early in the course of ALS makes absolute diagnosis difficult and compromises the certainty of diagnosis for clinical research purposes and therapeutic trials.” The original criteria described 4 categories of disease: Definite, Probable, Possible, and Suspected ALS. However, subsequent clinical experience made it clear that non-Definite categories included patients who would ultimately die of ALS with a high degree of clinical certainty.info:eu-repo/semantics/publishedVersio

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy