Human post-mortem brain and spinal cord samples

In accordance with the applicable laws in Belgium and upon written informed consent (UZ Leuven), brain and spinal cord tissue was collected. Ethical approval for the study was given by the Ethical Committee of UZ Leuven ({"type":"entrez-nucleotide","attrs":{"text":"S65097","term_id":"410617","term_text":"S65097"}}S65097, {"type":"entrez-protein","attrs":{"text":"S59292","term_id":"1363684","term_text":"pir||S59292"}}S59292, {"type":"entrez-protein","attrs":{"text":"S60803","term_id":"2126655","term_text":"pir||S60803"}}S60803, Leuven, Belgium). Brain samples of 17 ALS cases (nine C9orf72 and eight sporadic) and seven non-neurodegenerative controls and spinal cord samples of (16 ALS cases (eight C9orf72 and eight sporadic) and eight non-neurodegenerative controls were included in this study (Additional file 3: Table S2). The diagnosis of ALS or FTD was based on clinical assessment according to the consensus criteria for ALS [93–95] and FTD [96, 97]. The post-mortem diagnosis of ALS and FTLD-TDP was confirmed through pathological evaluation of pTDP-43 pathology in both brain and spinal cord. After the autopsy, the right hemisphere was dissected in coronal planes of approx. 2 cm and frozen at -80°C. 50 mg of brain or spinal cord tissue was weighed and mechanically homogenized using a micropestle in 0.5 mL 2% SDS in TBS with Nuclease (Pierce™ Universal Nuclease, Thermo Fisher Scientific) and a cocktail of protease/phosphatase inhibitors (Halt, Thermo Fisher Scientific). The samples were then sonicated, followed by centrifugation at 14 000 g for 30 min. The resulting supernatant was used for further analysis, and protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific).