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  • FTLD Patient-Derived Fibroblasts Show Defective Mitochondrial Function and Accumulation of p62.

FTLD Patient-Derived Fibroblasts Show Defective Mitochondrial Function and Accumulation of p62.

Molecular neurobiology (2021-07-31)
Stina Leskelä, Dorit Hoffmann, Hannah Rostalski, Nadine Huber, Rebekka Wittrahm, Päivi Hartikainen, Ville Korhonen, Ville Leinonen, Mikko Hiltunen, Eino Solje, Anne M Remes, Annakaisa Haapasalo
ABSTRACT

Frontotemporal lobar degeneration (FTLD) is a clinically, genetically, and neuropathologically heterogeneous group of neurodegenerative syndromes, leading to progressive cognitive dysfunction and frontal and temporal atrophy. C9orf72 hexanucleotide repeat expansion (C9-HRE) is the most common genetic cause of FTLD, but pathogenic mechanisms underlying FTLD are not fully understood. Here, we compared cellular features and functional properties, especially related to protein degradation pathways and mitochondrial function, of FTLD patient-derived skin fibroblasts from C9-HRE carriers and non-carriers and healthy donors. Fibroblasts from C9-HRE carriers were found to produce RNA foci, but no dipeptide repeat proteins, and they showed unchanged levels of C9orf72 mRNA transcripts. The main protein degradation pathways, the ubiquitin-proteasome system and autophagy, did not show alterations between the fibroblasts from C9-HRE-carrying and non-carrying FTLD patients and compared to healthy controls. An increase in the number and size of p62-positive puncta was evident in fibroblasts from both C9-HRE carriers and non-carriers. In addition, several parameters of mitochondrial function, namely, basal and maximal respiration and respiration linked to ATP production, were significantly reduced in the FTLD patient-derived fibroblasts from both C9-HRE carriers and non-carriers. Our findings suggest that FTLD patient-derived fibroblasts, regardless of whether they carry the C9-HRE expansion, show unchanged proteasomal and autophagic function, but significantly impaired mitochondrial function and increased accumulation of p62 when compared to control fibroblasts. These findings suggest the possibility of utilizing FTLD patient-derived fibroblasts as a platform for biomarker discovery and testing of drugs targeted to specific cellular functions, such as mitochondrial respiration.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, ≥98% (TLC), powder
Sigma-Aldrich
Anti-C9ORF72/C9RANT (poly-GA) Antibody, clone 5E9, clone 5E9, from mouse
Sigma-Aldrich
Anti-C9ORF72/C9RANT (poly-PA), serum, from rabbit
Sigma-Aldrich
Bafilomycin A1 from Streptomyces griseus, ≥90% (HPLC)
Sigma-Aldrich
Rotenone, ≥95%
Sigma-Aldrich
Antimycin A from Streptomyces sp.
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Anti-C9ORF72/C9RANT (poly-GR) Antibody, clone 5A2, clone 5A2, 1 mg/mL, from rat