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Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) that exist on a spectrum of neurodegenerative disease. A hallmark of pathology is cytoplasmic TDP-43 aggregates within neurons, observed in 97% of ALS cases and ~ 50% of FTLD cases. This mislocalisation from the nucleus into the cytoplasm and TDP-43 cleavage are associated with pathology, however, the drivers of these changes are unknown. p62 is invariably also present within these aggregates. We show that p62 overexpression causes TDP-43 mislocalisation into cytoplasmic aggregates, and aberrant TDP-43 cleavage that was dependent on both the PB1 and ubiquitin-associated (UBA) domains of p62. We further show that p62 overexpression induces neuron death. We found that stressors (proteasome inhibition and arsenic) increased p62 expression and that this shifted the nuclear:cytoplasmic TDP-43 ratio. Overall, our study suggests that environmental factors that increase p62 may thereby contribute to TDP-43 pathology in ALS and FTLD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCFCyclin F). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCFCyclin F substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS.