<i>In Vivo</i> Profiling Reveals a Competent Heat Shock Response in Adult Neurons: Implications for Neurodegenerative Disorders

<div><p>The heat shock response (HSR) is the main pathway used by cells to counteract proteotoxicity. The inability of differentiated neurons to induce an HSR has been documented in primary neuronal cultures and has been proposed to play a critical role in ageing and neurodegeneration. However, this accepted dogma has not been demonstrated <i>in vivo</i>. We used BAC transgenic mice generated by the Gene Expression Nervous System Atlas project to investigate the capacity of striatal medium sized spiny neurons to induce an HSR as compared to that of astrocytes and oligodendrocytes. We found that all cell populations were competent to induce an HSR upon HSP90 inhibition. We also show the presence and relative abundance of heat shock-related genes and proteins in these striatal cell populations. The identification of a competent HSR in adult neurons supports the development of therapeutics that target the HSR pathway as treatments for neurodegenerative disorders.</p></div

mRNA expression level of HS related genes in different striatal cell populations.

<p>Striatal cells were isolated from wild type and transgenic mice at 6 weeks of age 2 hours after treatment with HSP990 (12 mg/kg) or vehicle and sorted based on <i>Gfp</i> expression. RT-qPCR analysis of the expression levels of (A) <i>Hspa1a/b</i>, <i>Dnajb1</i> and <i>Hspb1</i> (B) <i>Hsf1</i> and <i>Sirt1</i> and (C) <i>Hsp90aa1</i> and <i>Hsp90ab1</i> in GFP⁺ cells isolated from mice treated with HSP990 as compared to those treated with vehicle.</p

Expression of the heat shock proteins and their regulators in the striatal cell populations Striatal cells were isolated from wild type and transgenic mice at 6 weeks of age 2 hours after treatment with HSP990 (12 mg/kg) or vehicle and sorted based on GFP expression.

<p>Western blot analysis of the expression levels of (A) SIRT1, HSP90, HSF1, HSP70 and HSP40 and (B) NEUN, GFAP and GFP in GFP⁺ cells isolated from mice treated with HSP990 as compared to those treated with vehicle. Loading controls were ATP5B and GAPDH.</p

Validation of the purity of the cell populations used for the mRNA analysis Striatal cells were isolated from wild type and transgenic mice at 6 weeks of age 2 hours after treatment with HSP990 (12 mg/kg) or vehicle and sorted based on <i>Gfp</i> expression.

<p>RT-qPCR analysis of the expression levels of (A) <i>Slc12a5</i>, <i>Drd1a</i>, <i>Drd2</i>, <i>Aldh1l1</i>, <i>Gfap</i> and <i>Mbp</i> and (B) <i>Gfp</i> in GFP⁺ cells isolated from mice treated with HSP990 or vehicle.</p

Native GFPu fluorescence in R6/2-GFPu mice.

<p>Native GFPu fluorescence is not notably increased in the hippocampus, cortex or striatum of R6/2; GFPu mice. Sections were stained with the nuclear-specific fluorescent dye TOPRO-3. Scale bars are 40 µM.</p

GFPu does not accumulate in the R6/2 brain.

<p>(A) Schematic showing the GFPu construct under control of the mouse prion promoter (PrP). GFPu protein is composed of GFP appended with a 16 amino acid C-terminal degradation signal, the CL-1 degron. (B) Western blot analysis and densitomeric quantification reveals no increase in steady-state levels of GFPu in 12 week R6/2 brains. α-tubulin was used as a loading control. (C) Expression of the GFPu transgene is unchanged in the 12-week-old R6/2 brain. Error bars represent the standard error of the mean.</p

Relationship between the UPS and inclusion formation.

<p>(A) Immunofluorescent double-staining of R6/2; GFPu brain sections shows that the presence of nuclear inclusion bodies does not correlate with the intensity of GFPu immunofluorescence in CA1 region of the hippocampus, the piriform cortex or the cortex (B). Quantification of GFPu immunofluorescence in nuclei with or without an inclusion body confirms that there is no relationship between inclusion body formation and impairment of the UPS in R6/2 mice. Sections were stained with the nuclear fluorescent dye TOPRO-3. Error bars represent the standard errors of the mean. Scale bars are 6 µM.</p

GFPu immunofluorescence and inclusion body formation in the hippocampus.

<p>GFPu immunofluorescence in the CA1 region of the hippocampus is comparable in 12-week-old GFPu and R6/2; GFPu brains. Staining with anti-htt antibody S830 shows widespread inclusion body formation in R6/2 mice. Sections were stained with the nuclear fluorescent dye TOPRO-3. Scale bars are 10 µM.</p

GFPu immunofluorescence in R6/2-GFPu mice.

<p>Immunofluorescent staining of brain sections with anti-GFP antibody followed by quantitation of fluorescent units reveals no difference in the levels of GFPu in R6/2 cortex (A), hippocampus (B) or striatum (C). Sections were counterstained with the nuclear fluorescent dye TOPRO-3. Error bars represent the standard error of the mean. Scale bars are 40 µM.</p

Hdac3 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.

<p>(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and <i>Hdac3</i> lysates were comparable to water. Aggregation levels augment with age but are not modified by <i>Hdac3</i> reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).</p