CHIP mutations affect the heat shock response differently in human fibroblasts and iPSC-derived neurons

Abstract

C-terminus of HSC70-interacting protein (CHIP) encoded by the gene STUB1 is a co-chaperone and E3 ligase that acts as a key regulator of cellular protein homeostasis. Mutations in STUB1 cause autosomal recessive spinocerebellar ataxia type 16 (SCAR16) with widespread neurodegeneration manifesting as spastic-ataxic gait disorder, dementia and epilepsy. CHIP−/− mice display severe cerebellar atrophy, show high perinatal lethality and impaired heat stress tolerance. To decipher the pathomechanism underlying SCAR16, we investigated the heat shock response (HSR) in primary fibroblasts of three SCAR16 patients. We found impaired HSR induction and recovery compared to healthy controls. HSPA1A/B transcript levels (coding for HSP70) were reduced upon heat shock but HSP70 remained higher upon recovery in patient- compared to control-fibroblasts. As SCAR16 primarily affects the central nervous system we next investigated the HSR in cortical neurons (CNs) derived from induced pluripotent stem cells of SCAR16 patients. We found CNs of patients and controls to be surprisingly resistant to heat stress with high basal levels of HSP70 compared to fibroblasts. Although heat stress resulted in strong transcript level increases of many HSPs, this did not translate into higher HSP70 protein levels upon heat shock, independent of STUB1 mutations. Furthermore, STUB1(−/−) neurons generated by CRISPR/Cas9-mediated genome editing from an isogenic healthy control line showed a similar HSR to patients. Proteomic analysis of CNs showed dysfunctional protein (re)folding and higher basal oxidative stress levels in patients. Our results question the role of impaired HSR in SCAR16 neuropathology and highlight the need for careful selection of proper cell types for modeling human diseases.

CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity

“…These were largely unchanged in P/KO, WT/KO, and P/WT SWATH-MS analyses, arguing against the activation of compensatory mechanisms due to the absence of CHIP expression. This is consistent with data from CHIP −/- mouse cells ( Morishima et al., 2008 ) and with a study, published during preparation of this manuscript, showing that CHIP KO neurons (at day 36) do not have an impaired heat shock response ( Schuster et al, 2020 ). Notably, the only detected HSP that met the criterion for significant fold-change was HSP27 ( HSPB1 ).…”

“…This view was supported by a recent study comparing iPSC-derived neurons from SCAR16 patients (with various STUB1 mutations) to those from healthy individuals. The proteomic changes identified in the Schuster et al, 2020 study were primarily associated with oxidative and proteotoxic stress. In the current study we were able to uncouple the role of CHIP in oxidative stress and protein quality control (QC) from its non-canonical activities by using isogenic cells in an early stage disease background.…”

“…A recent study demonstrated some degree of cell-type variability in the response to loss of CHIP function. Whilst fibroblasts from patients with ataxia (SCAR16), expressing mutant CHIP, had an impaired heat shock response, the response in induced pluripotent stem cell (iPSC)-derived neurons from the same patients was relatively unaffected ( Schuster et al, 2020 ).…”

A de novo STUB1 variant associated with an early adult-onset multisystemic ataxia phenotype

“…The generation of iPSCs from fibroblasts was performed according to a previously published protocol [13]. In brief, human dermal fibroblasts were nucleofected with 1 µg of each episomal plasmid [pCXLE-hUL, pCXLE-hSK and pCXLE-hOCT4 (Addgene numbers 27080, 27078 and 27076, respectively)] with Nucleofector 2b (Lonza, Basel, Switzerland).…”

“…iPSC-derived neurons were generated according to a published protocol [13]. In brief, iPSCs were seeded at a density of 3 × 10 5 /cm 2 in E8 medium supplemented with 10 µM Y-27632.…”