Analysis of Chaperone mRNA Expression in the Adult Mouse Brain by Meta Analysis of the Allen Brain Atlas

The pathology of many neurodegenerative diseases is characterized by the accumulation of misfolded and aggregated proteins in various cell types and regional substructures throughout the central and peripheral nervous systems. The accumulation of these aggregated proteins signals dysfunction of cellular protein homeostatic mechanisms such as the ubiquitin/proteasome system, autophagy, and the chaperone network. Although there are several published studies in which transcriptional profiling has been used to examine gene expression in various tissues, including tissues of neurodegenerative disease models, there has not been a report that focuses exclusively on expression of the chaperone network. In the present study, we used the Allen Brain Atlas online database to analyze chaperone expression levels. This database utilizes a quantitative in situ hybridization approach and provides data on 270 chaperone genes within many substructures of the adult mouse brain. We determined that 256 of these chaperone genes are expressed at some level. Surprisingly, relatively few genes, only 30, showed significant variations in levels of mRNA across different substructures of the brain. The greatest degree of variability was exhibited by genes of the DnaJ co-chaperone, Tetratricopeptide repeat, and the HSPH families. Our analysis provides a valuable resource towards determining how variations in chaperone gene expression may modulate the vulnerability of specific neuronal populations of mammalian brain

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

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

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

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

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

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

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