The small heat shock protein B8 (HSPB8) efficiently removes aggregating species of dipeptides produced in C9ORF72-related neurodegenerative diseases

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not "classical" misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.

Keywords: HSPB8; Motor neuron diseases; Protein aggregation; Protein clearance; RAN translation.

Fig. 1

DPRs overexpression in NSC34 cells. a Confocal microscopy analysis of NSC34 cells shows DPRs localization (×63 magnification); scale bars 30 μm. b, c NSC34 cells were collected 48 h after transfection with FLAG-polyDPRs (GA, GP, GR, PR, PA). b WB shows DPR total levels. TUBA was used as loading control. c FRA shows PBS insoluble fraction of DPRs. Bar graph represents the FRA mean relative optical density computed over three independent biological samples for each condition (n = 3) ± SD (**p < 0.01, ***p < 0.001; one-way ANOVA followed by Tukey’s test)

Fig. 2

Effect of autophagy and proteasome inhibitors on the PBS insoluble levels of DPRs. NSC34 cells were collected 48 h after transfection with FLAG-polyDPRs (GA, GP, GR, PR, PA). a FRA shows PBS insoluble fraction of DPRs after 48 h of autophagy inhibition treatment with 10 mM 3-MA. Bar graph represents the FRA mean relative optical density computed over three independent biological samples for each condition (n = 3) ± SD (*p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test). b FRA shows PBS insoluble fraction of DPRs after 16 h of proteasome inhibition treatment with 10 μM MG-132. Bar graph represents the FRA mean relative optical density computed over three independent biological samples for each condition (n = 3) ± SD (***p < 0.001; Student’s t test)

Fig. 3

Effect of HSPB8 overexpression on DPRs distribution. NSC34 cells were fixed 48 h after transfection with FLAG-polyDPRs (GA, GP, GR, PR, PA) and pCI-HSPB8 or pcDNA3. IF shows DPRs distribution (×63 magnification); scale bars 30 μm

Fig. 4

Effect of HSPB8 overexpression on DPRs levels. NSC34 cells were collected 48 h after transfection with FLAG-polyDPRs (GA, GP, GR, PR, PA) and pCI-HSPB8 or pCDNA3. a FRA shows PBS insoluble fraction of DPRs. Bar graph represents the FRA mean relative optical density computed over three independent biological samples for each condition (n = 3) ± SD (**p < 0.01, ***p < 0.001; Student’s t test). b WB shows DPR total levels; TUBA was used as loading control

Fig. 5

Effect of HSPB8 silencing on DPRs levels. NSC34 cells were transfected with non-target or HspB8 siRNA and FLAG-polyDPRs (GA, GP, GR, PR, PA). a FRA shows PBS insoluble fraction of DPRs. Bar graph represents the FRA mean relative optical density computed over three independent biological samples for each condition (n = 3) ± SD (* = p < 0.05, *** = p < 0.001; Student’s t test). b WB shows DPRs total levels; TUBA was used as loading control. c WB shows HSPB8 levels; TUBA was used as loading control. Bar graph represents the HSPB8/TUBA ratio computed over three independent biological samples for each condition (n = 3) ± SD (** = p < 0.01; Student’s t test)