Peripheral artery disease (PAD) has a huge social and economic burden and is an important contributor to the global health burden. Sex differences in PAD are apparent, with recent data suggesting equal if not greater prevalence in women, and women having worse clinical outcomes. Why this occurs is not clear. To identify underlying reasons for gender inequalities in PAD, we executed a deeper exploration through a social constructive perspective. A scoping review was conducted using the World Health Organization model for analysis of gender-related needs in healthcare. Complex interacting factors, including biological, clinical, and societal variables, were reviewed to highlight gender-related inequities in the diagnosis, treatment, and management of PAD. Current gaps in knowledge were identified and insights into future directions aimed at improving these inequalities were discussed. Our findings highlight the multi-level complexities that need to be considered for strategies to improve gender-related needs in PAD healthcare.

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

Background Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders that share similar clinical and pathological hallmarks, as well as overlapping genetic aetiologies. Previously, we identified that mutations in CYLD can cause FTD‐ALS. CYLD is a lysine‐63 deubiquitinase, and the FTD‐ALS causal mutation CYLDM719V shows significantly increased deubiquitinase activity [Dobson‐Stone et al. 2020, Brain 143:783‐799]. CYLD is known to interact with the protein products of at least three established FTD and ALS genes. We sought to determine whether other FTD‐ALS proteins were influenced by modulation of CYLD. Method We performed mass spectrometry quantitative proteomic analysis of HEK293 lysates overexpressing wild‐type and mutant CYLD (CYLDWT & CYLDM719V), followed by western blots of total cell lysates from SH‐SY5Y and HEK293 cells overexpressing CYLDWT, CYLDM719V and the deubiquitinase‐inactive mutation CYLDD681G. Densitometry analysis was performed in Image Lab (Bio‐Rad, USA) and normalised to total protein. Result Mass spectrometry analysis indicated significant differences in expression of TIA1, MATR3, VCP and SFPQ when CYLD was overexpressed. Validation by western blot showed that overexpression of CYLD increased relative protein expression of SFPQ (2.7 fold increase for CYLDWT versus empty vector, p = 0.0048) in SH‐SY5Y cells. This is likely due to deubiquitinase activity, as CYLDD681G did not show such an increase. SFPQ is a nucleic acid binding protein that has been shown to mislocalise from the nucleus to the cytoplasm in FTD and ALS. We are currently examining whether CYLD affects subcellular localisation of SFPQ by western blot and immunofluorescent microscopy analyses. Conclusion Our study indicates that CYLD overexpression increases SFPQ protein expression via its deubiquitinase activity. However, it remains to be shown whether this mechanism is essential to the pathogenesis of FTD and ALS driven by CYLD.

Background Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1‐16q12.2 for a large European Australian family with FTD‐ALS [Dobson‐Stone et al 2013, Acta Neuropathol 125:523‐533]. We have recently identified the p.M719V substitution in CYLD as the causal mutation at this locus. CYLD is a lysine‐63 deubiquitinase and CYLDM719V shows significantly increased deubiquitinase activity [Dobson‐Stone et al, Brain, accepted Dec 2019]. We sought to determine what biological pathways are affected by the presence of this mutation. Method We performed label‐free mass spectrometry quantitative proteomic analysis of HEK293 lysates overexpressing wild‐type (CYLDwt), FTD‐ALS mutant CYLDM719V or deubiquitinase‐inactive mutant CYLDD681G. We used Ingenuity Pathway Analysis to predict dysregulated biological pathways. We examined necroptosis in L929 cells overexpressing CYLDwt and CYLDM719V, measuring cell viability (ATP production) and cell death (membrane permeabilisation). Result 276 proteins exhibited significantly altered expression when CYLDM719V was overexpressed, relative to CYLDwt. Pathway analysis predicted activation of apoptosis with CYLDM719V. This is likely related to deubiquitinase activity, as CYLDD681G was predicted to have the opposite effect. We are working to validate these findings by immunoblot and co‐immunoprecipitation analyses. CYLD has recently been identified as a mediator of another cell death pathway, necroptosis. We found a small but significant effect of CYLDM719V on cell viability after treatment with necroptosis inducer Z‐VAD‐FMK in L929 cells at 0.2 μM (8.8% decrease relative to control, Student’s t test p = 0.006), 2 μM (26.1% decrease, p = 0.0003) and 20 μM (38.6% decrease, p = 0.00005). CYLDwt showed significant effects only at higher doses (0.2 μM: 1.1% decrease, p = 0.396; 2 μM: 10.8% decrease, p = 0.049; 20 μM: 32.5% decrease, p = 0.0001). Conclusion Our study indicates that CYLDM719V may act in part by inappropriate activation of apoptosis and/or necroptosis, possibly via sensitising CYLD’s response to death stimuli. It remains to be seen which of these mechanisms is critical to CYLD’s effects on neurodegeneration in FTD and ALS, although our data lend support to the emerging research showing necroptosis as a plausible therapeutic target for ALS.