Aditi Methi

Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. Moreover, pathological proteomic signatures are ...

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase, characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum. Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from endoplasmic reticulum-associated protein degradation in a catalytic-independent manner, promote autophagy, and address ...

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully ...

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have ...

Multiple sclerosis (MS) is an inflammatory neurological disease of the central nervous system with a subclinical phase preceding frank neuroinflammation. CD8+ T cells are abundant within MS lesions, but their potential role in disease pathology remains unclear. Using high-throughput single-cell RNA sequencing and single-cell T cell receptor analysis, we compared CD8+ T cell clones from the blood and cerebrospinal fluid (CSF) of monozygotic twin pairs in which the cotwin had either no or subclinical ...

The microglial proteome of homozygous Cln3Δex7/8/Δex7/8 was compared with those of littermate control heterozygous (Cln3Δex7/8/+) or wildtype (Cln3+/+) mice at 3 (Cln3Δex7/8/Δex7/8 n=5 vs controls n=7) and 12 months (Cln3Δex7/8/Δex7/8 n=4 vs controls n=5) of age. Microglia were acutely isolated using MACS with CD11b microbeads (Miltenyi Biotec) as previously described in Colombo et al. 36 Sample preparation and mass spectrometric measurements using data independent acquisition were performed as ...

Wild-type (WT) and ADAM17 knockout (KO) mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 1% L-Glutamine, 1% Penicilin and Streptomycin, 1% Sodium Pyruvate and 10% Fetal Bovine Serum (FBS) at 37 °C, 5% CO2 (all reagents were purchased from Euroclone, Pero, Italy). HTB94 were kindly provided by Prof Hideaki Nagase, and cultured in DMEM containing 1% L-Glutamine, 1% Penicillin and Streptomycin, 1% Sodium Pyruvate and 10% FBS at 37° C, 5% CO2. ...

HeLa WT and CTS DBLZ-deficient as well as SH-SY5Y WT and CTS DBL-deficient cell clones (each n=6) were seeded 24 h after cell pellet collection. Cells were washed in ice cold PBS and harvested with a cell scraper. A modified RIPA lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 50 mM TrisHCl pH 8) was used to lyse the cells one ice for 30 min. The supernatant was collected after centrifugation at 16.000 rpm for 10 min at 4°C. 10 μl were used to determine ...

CD8+ T cells from PBMCs of the MS twin cohort (n = 12; 6 healthy twins, 6 twins with SCNI, and 12 twins with MS; 193,771 cells) and the validation cohort (n = 17; 5 individuals with IIH and 12 individuals with MS; 89,859 cells) were isolated via FACS and processed using the 10X manufacturer's protocol to generate single-cell transcriptome and TCR libraries. The corresponding TCR sequences are included within the metadata of the uploaded objects. (Author states that fastq raw data files from humans ...

We treated male Oxt-ires-Cre;RiboTag mice with either vehicle or CCK, collected hypothalami 2h post injection, pooled 2-3 tissues per sample, and affinity purified conditionally HA-tagged ribosomes (incl translating mRNA) specifically from oxytocin neurons. We then performed gene expression profiling analysis using data obtained from RNA-seq of immunoprecipitates versus inputs (n=4 per group) from standard chow diet fed mice and inputs only from high-fat/high-sugar diet fed mice (n=4 per group). ...