Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, cerebellum, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9ORF72 -linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 31 genotypically diverse ALS cases with or without FTD, including four cases with UBQLN2 mutations (resulting in p.P497H, p.P506S, and two cases with p.T487I). Using double-, triple-, and five-label fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=19), unknown familial (n=3), SOD1 -linked (n=1), C9ORF72 -linked (n=4), and UBQLN2 -linked (n=4) cases. We differentiate between i) ubiquilin 2 aggregation together with, or driven by, pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation driven by UBQLN2 gene mutations. Together we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene mutations and to understand the mechanisms of UBQLN2 -linked disease. ### Competing Interest Statement The authors have declared no competing interest. * List of abbreviations : ALS : Amyotrophic lateral sclerosis bvFTD : Behavioural variant FTD C9ORF72 : Chromosome 9 open reading frame 72 DPR : Dipeptide repeat FFPE : Formalin-fixed paraffin-embedded FTD : Frontotemporal dementia IBD : Identity by descent LLPS : Liquid-liquid phase separation NCI : Neuronal cytoplasmic inclusions PBS : Phosphate-buffered saline PolyGA : Poly(glycine-arginine) PolyGP Poly(glycine-proline) pTDP-43 : Phosphorylated TDP-43 SOD1 Superoxide dismutase STED : Stimulated emission depletion SQSTM1 : Sequestosome 1 TDP-43 : Transactive response DNA binding protein 43 kDa