Protocol for the collection and analysis of the different immune cell subsets in the murine intestinal lamina propria.

The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP.

Upregulation of miR-25 and miR-181 Family Members Correlates with Reduced Expression of ATXN3 in Lymphocytes from SCA3 Patients.

BACKGROUND: Spinocerebellar ataxia type 3 (SCA3), the most common spinocerebellar ataxia, is caused by a polyglutamine (polyQ) expansion in the protein ataxin-3 (ATXN3). Silencing the expression of polyQ-expanded ATXN3 rescues the cellular disease phenotype. OBJECTIVE: This study investigated the differential expression of microRNAs (miRNAs), small noncoding RNAs targeting gene expression, in lymphoblastoid cells (LCs) from SCA3 patients and the capability of identified deregulated miRNAs to target and alter ATXN3 expression. METHODS: MiRNA profiling was performed by microarray hybridization of total RNA from control and SCA3-LCs. The capability of the identified miRNAs and their target sites to suppress ATXN3 expression was analyzed using mutagenesis, reverse transcription PCR, immunoblotting, luciferase reporter assays, mimics and precursors of the identified miRNAs. RESULTS: SCA3-LCs showed significantly decreased expression levels of ATXN3 and a significant upregulation of the ATXN3-3'UTR targeting miRNAs, miR-32 and miR-181c and closely related members of the miR-25 and miR-181 family, respectively. MiR-32 and miR-181c effectively targeted the 3'UTR of ATXN3 and suppressed the expression of ATXN3. CONCLUSIONS: The simultaneous upregulation of closely related miRNAs targeting the 3'UTR of ATXN3 and the significantly reduced ATXN3 expression levels in SCA3-LCs suggests that miR-25 and miR-181 family members cooperatively bind to the 3'UTR to suppress the expression of ATXN3. The findings further suggest that the upregulation of miR-25 and miR-181 family members in SCA3- LCs reflects a cell type-specific, protective mechanism to diminish polyQ-mediated cytotoxic effects. Thus, miRNA mimics of miR-25 and miR-181 family members may prove useful for the treatment of SCA3.