HEK293 cells but not mouse L cells cleave htt to produce a cp-A/1 like fragment.

<p>HEK293 and mouse L cells [thymidine kinase negative (TK⁻)] were transiently transfected with httN171-18Q constructs. After 48 hours, the cells were harvested and analyzed by immunoblot with the htt antibody 2B4. Only the HEK293 cells produce a cleavage product. The image shown is representative of at least 3 repetitions.</p

Analysis of spinal and muscle pathology in transgenic mice overexpressing wild-type and ALS-linked mutant MATR3

Abstract Mutations in MATR3 have been associated with amyotrophic lateral sclerosis (ALS) as well as a form of distal myopathy termed vocal cord pharyngeal distal myopathy (VCPDM). To begin to understand how mutations in MATR3 may cause disease, here we provide initial characterization of transgenic (Tg) mice expressing human wild-type (WT) MATR3 (MATR3WT) and ALS-mutant F115C MATR3 (MATR3F115C) proteins under the control of the mouse prion promoter (MoPrP). For each construct, we established multiple independent lines of mice that stably transmitted the transgene. Unexpectedly, for all stably-transmitting lines examined, MATR3 transgenic mRNA expression was more robust in muscle, with minimal expression in spinal cord. The levels of transgenic mRNA in muscle did not differ between mice from our lead MATR3F115C line and lead MATR3WT line, but mice from the lead MATR3F115C line had significantly higher levels of MATR3 protein in muscle over the lead MATR3WT line. Mice from the three independent, established lines of MATR3F115C mice developed weakness in both fore- and hind-limbs as early as  20 months were not overtly distinguishable from non-transgenic (NT) littermates based on basic motor phenotype. Muscle of both MATR3WT and MATR3F115C mice showed vacuoles by 2 months of age which worsened by ~ 10 months, but vacuolation was noticeably more severe in MATR3F115C mice. Overall, our results indicate that increasing the levels of MATR3 in muscle can cause pathologic changes associated with myopathy, with MATR3F115C expression causing overt muscle atrophy and a profound motor phenotype. The findings suggest that analysis of muscle pathology in individuals harboring ALS-linked MATR3 mutations should be routinely considered

Analysis of Proteolytic Processes and Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments in an HEK293 Cell Model

<div><h3>Background</h3><p>N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90–115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.</p> <h3>Results</h3><p>Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400–600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115–124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.</p> <h3>Conclusions</h3><p>Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.</p> </div

Gamma-secretase inhibitors fail to block htt proteolysis.

<p>A, HEK293 cells were transfected with N171-18Q, treated with 3-MA, and either untreated, or treated with DAPT or LY411,575 followed by immunoblot analysis for htt (lane 4 is non-transfected). In all treatment scenarios, no change was detected in htt proteolysis; cp-A/1 (arrowhead on left panel) was detected at equivalent levels regardless of treatment. B, The efficacy of the γ-secretase inhibitor was confirmed by immunoblotting for APP C-terminal fragments (10 kDa CTF; arrow on right panel). The images shown are representative of at least 3 repetitions of the experiment.</p

Autophagy activators or inhibitors do not block htt proteolysis.

<p>Top: inhibitors (3-methyladenine, 3-MA and Chloroquine, Chlrq) or activators (Rapamycin, Rapa) of autophagy failed to block proteolysis, as detected by immunoblot with 2B4 antibody (1∶1000). Treatment with 3-MA caused an increase in cp-B/2 and cp-A/1. Bottom: Detection of LC3-I and LC3-II. LC3 antibody was used at 1∶1000. The images shown are representative of at least 3 repetitions of the experiment.</p

Polyglutamine length does not affect cleavage efficiency.

<p>A, HEK293 cells were non-transfected (NTf) or transfected with cDNA encoding htt N171-18Q. Cell lysates were analyzed by immunoblots incubated with antibodies 2B4 or 1H6. B, HEK293 cells were transfected with htt cDNAs shown at the top of the figure, followed by lysis and SDS-PAGE. Arrowheads identify substrates or cleavage products that are generated at approximately equally levels for short and expanded polyglutamine lengths. The lack of an effect of polyglutamine length on cleavage efficiency was observed for both N171 and N586 based constructs. Immunoblots were probed with the htt81-90 antibody (1∶3000). The images shown are representative of at least 3 repetitions of the experiment. EH = endogenous htt.</p

Htt cleavage occurs C-terminal to cysteine 105.

<p>A, The sequence of htt is shown through residue 171, the substrate used in these experiments. The first cysteine in htt is at residue 105 (bold) and represents the most N-terminal cysteine that could be labeled with sulfhydrylated biotin (Biotin-SH). This cysteine must be present for any labeling to occur. Subsequent immunoprecipitation (IP) followed by detection with streptavidin or htt antibodies was used to visualize the biotinylation. B, Detection using streptavidin-HRP of transfected cell lysate (Tfx N171-18Q, +) reacted with biotinylation reagent (Biotin, +) shows cp-B/2 and cp-A/1-sized products (arrows). Asterisks (*) signify possible multimers of htt. C, Detection of htt with the antibody htt3-16 confirms the identities of cp-B/2 and cp-A/1 (bottom two arrows). IgG from the immunoprecipitation is observed in all lanes (top arrow). The images shown are representative of at least 3 repetitions of the experiment.</p

Knock-down of calpain-1 does not block htt cleavage.

<p>HEK293 cells which stably express a shRNA against calpain-1 (shRNAi +) do not block cleavage of htt following transfection with cDNA encoding N171-18Q (Tfx +). Knock-down of capn1 was confirmed by immunoblot with an antibody to activated calpain-1. Htt cp-A/1 was detected with the htt81-90 (1∶3000) antibody. The images shown are representative of at least 3 repetitions of the experiment.</p

Retraction Note: Transgenic mice overexpressing the ALS-linked protein Matrin 3 develop a profound muscle phenotype

Abstract The authors are retracting this article. The article describes mice expressing wild-type human MATR3. However, since publication the authors have become aware that all of the lines of mice described express human MATR3 containing the F115C mutation. Transgenic mice expressing wild-type and mutant Matrin were created simultaneously in their laboratory and, at a crucial stage of generating the DNA for embryo injection, as confirmed by an investigation by the University of Florida, the DNA preparations were accidentally mislabelled. All of the founders created were mosaic, requiring extensive breeding to isolate stable lines. Mice mislabelled as expressing wild-type MATR3 were the first to produce lines that stably transmitted the transgene and thus were the first to be characterized. However, as lines of mice that were mislabelled as expressing the mutant (F115C) MATR3 were ultimately established, the data began to suggest that an error had been made. Sequence analysis of amplified tail DNA from mice descended from the lines reported in the article have revealed that they express the F115C variant. The data described are therefore an accurate description of the pathology of mice that express the F115C variant of MATR3, but not of mice expressing wild-type MATR3. The authors are preparing a new manuscript reporting data from both mice expressing the F115C variant of MATR3 and mice expressing wild-type MATR3

Microglia-specific targeting by novel capsid-modified AAV6 vectors

Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies