The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals

Impaired haematopoietic stem cell differentiation and enhanced skewing towards myeloid progenitors in aged caspase-2-deficient mice

The apoptotic cysteine protease caspase-2 has been shown to suppress tumourigenesis in mice and its reduced expression correlates with poor prognosis in some human malignancies. Caspase-2-deficient mice develop normally but show ageing-related traits and, when challenged by oncogenic stimuli or certain stress, show enhanced tumour development, often accompanied by extensive aneuploidy. As stem cells are susceptible to acquiring age-related functional defects because of their self-renewal and proliferative capacity, we examined whether loss of caspase-2 promotes such defects with age. Using young and aged Casp2−/− mice, we demonstrate that deficiency of caspase-2 results in enhanced aneuploidy and DNA damage in bone marrow (BM) cells with ageing. Furthermore, we demonstrate for the first time that caspase-2 loss results in significant increase in immunophenotypically defined short-term haematopoietic stem cells (HSCs) and multipotent progenitors fractions in BM with a skewed differentiation towards myeloid progenitors with ageing. Caspase-2 deficiency leads to enhanced granulocyte macrophage and erythroid progenitors in aged mice. Colony-forming assays and long-term culture-initiating assay further recapitulated these results. Our results provide the first evidence of caspase-2 in regulating HSC and progenitor differentiation, as well as aneuploidy, in vivo.Swati Dawar, Nur Hezrin Shahrin, Nikolina Sladojevic, Richard J D, Andrea, Loretta Dorstyn, Devendra K Hiwase and Sharad Kuma

Caspase-2 deficiency enhances whole-body carbohydrate utilisation and prevents high-fat diet-induced obesity

Published online 26 October 2017Caspase-2 has been shown to be involved in metabolic homeostasis. Here, we show that caspase-2 deficiency alters basal energy metabolism by shifting the balance in fuel choice from fatty acid to carbohydrate usage. At 4 weeks of age, whole-body carbohydrate utilisation was increased in Casp2-/- mice and was maintained into adulthood. By 17 weeks of age, Casp2-/- mice had reduced white adipose mass, smaller white adipocytes decreased fasting blood glucose and plasma triglycerides but maintained normal insulin levels. When placed on a 12-week high-fat diet (HFD), Casp2-/- mice resisted the development of obesity, fatty liver, hyperinsulinemia and insulin resistance. In addition, HFD-fed Casp2-/- mice had reduced white adipocyte hypertrophy, apoptosis and expansion of both subcutaneous and visceral adipose depots. Increased expression of UCP1 and the maintenance of adiponectin levels in white adipose tissue of HFD-fed Casp2-/- mice indicated increased browning and adipocyte hyperplasia. We found that while the preference for whole-body carbohydrate utilisation was maintained, HFD-fed Casp2-/- mice were not impaired in their ability to switch to utilising fats as a fuel source. Our findings suggest that caspase-2 impacts basal energy metabolism by regulating adipocyte biology and fat expansion, most likely via a non-apoptotic function. Furthermore, we show that caspase-2 deficiency shifts the balance in fuel choice towards increased carbohydrate utilisation and propose that this is due to mild energy stress. As a consequence, Casp2-/- mice show an adaptive remodelling of adipose tissue that protects from HFD-induced obesity and improves glucose homeostasis while paradoxically increasing their susceptibility to oxidative stress induced damage and premature ageing.Claire H Wilson, Andrej Nikolic, Stephen J Kentish, Marianne Keller, George Hatzinikolas, Loretta Dorstyn, Amanda J Page and Sharad Kuma

Caspase-2-mediated cell death is required for deleting aneuploid cells

Caspase-2, one of the most evolutionarily conserved of the caspase family, has been implicated in maintenance of chromosomal stability and tumour suppression. Caspase-2 deficient (Casp2-/-) mice develop normally but show premature ageing-related traits and when challenged by certain stressors, succumb to enhanced tumour development and aneuploidy. To test how caspase-2 protects against chromosomal instability, we utilized an ex vivo system for aneuploidy where primary splenocytes from Casp2-/- mice were exposed to anti-mitotic drugs and followed up by live cell imaging. Our data show that caspase-2 is required for deleting mitotically aberrant cells. Acute silencing of caspase-2 in cultured human cells recapitulated these results. We further generated Casp2C320S mutant mice to demonstrate that caspase-2 catalytic activity is essential for its function in limiting aneuploidy. Our results provide direct evidence that the apoptotic activity of caspase-2 is necessary for deleting cells with mitotic aberrations to limit aneuploidy.S Dawar, Y Lim, J Puccini, M White, P Thomas, L Bouchier-Hayes, D R Green, L Dorstyn and S Kuma

The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c

The prevalence of insomnia in multiple sclerosis: A meta-analysis.

Insomnia is a common complaint for adults with multiple sclerosis and can severely impact health-related quality of life. Point prevalence estimates of insomnia are, however, difficult to determine in this population due to the use of different measurement tools as well as the highly variable clinical presentation of multiple sclerosis. This review consolidates the current evidence base to provide a global estimate of insomnia disorders and symptoms in multiple sclerosis, with consideration of both measurement and sample issues. A comprehensive review of the PUBMED, EMBASE, PsycINFO and CINAHL databases from database inception until January 31st, 2023 identified 1649 records, of which 34 (7636 participants total) were eligible for inclusion. Findings were meta-analysed using a random-effects model. Estimates based on self-reported symptoms (52%, CI: 44%-59%) were significantly higher than those obtained by diagnostic tools (22%, CI: 16%-29%). Gender was identified as a potential moderator, with women more likely to report insomnia than men. One in two adults with multiple sclerosis endorse symptoms of poor sleep quality and daytime sleepiness, with 1 in 5 diagnosed with an insomnia disorder. Future research is needed to enhance understanding of these comorbid conditions, including the trajectory of insomnia with disease progression. PROSPERO registration number CRD42021281524

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus

The PIDDosome (PIDD–RAIDD–caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2–dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function

Online Resource to Promote Vocational Interests Among Job Seekers With Multiple Sclerosis: A Randomized Controlled Trial in Australia

© 2017 American Congress of Rehabilitation Medicine Objective: To provide a preliminary evaluation of the effectiveness of an online resource for job seekers with multiple sclerosis (MS). Design: Randomized controlled design. Setting: Community-dwelling cohort. Participants: Adults (N = 95) with relapsing-remitting or progressive MS were randomly assigned to one of two groups. Forty-five accessed an email delivered, 7 module resource, Work and MS, over a 4 week period. Waitlist control participants (n=50) were offered the opportunity to access Work and MS 4 weeks postenrollment. Main Outcome Measures: Primary outcomes focused on vocational interests (My Vocational Situation Scale) and self-efficacy in job-seeking activities (Job-Procurement Self Efficacy Scale). Secondary outcomes focused on perceived workplace difficulties (Multiple Sclerosis Work Difficulties Questionnaire [MSWDQ]), optimism (Life Orientation Test – Revised), and mood (Patient Health Questionnaire-9). Results: Intention-to-treat analyses revealed pre-post gains: participants who accessed Work and MS reported improved confidence in their career goals (My Vocational Situation Scale g=.55; 95% confidence interval [CI],.14–.96; P=.008) and positively reappraised potential workplace difficulties (MSWDQ g range,.42–.47; P range,.023–.042). The effect on job self-efficacy was not significant, but changed in the expected direction (g=.17; 95% CI, –.23 to.57; P=.409). Completer data revealed larger, significant effect estimates (g range,.52–.64; P range,.009–.035). Conclusions: Findings provide preliminary support for the utility of a job information resource, Work and MS, to augment existing employment services. The results also suggest the need to test employment-ready interventions in a larger study population. This might include the addition of online peer support to increase intervention compliance

Belonging in the Online World: Older Adults' Use of Internet for Community

Objective: To explore older Australians’ experiences of using computermediated communication (CMC) to engage with their social networks and communities. Background: Use of CMC among older adults has been associated with favourable social outcomes. How older adults engage with others to foster these outcomes is less well known. Understanding this may be useful when developing programs to encourage older adults’ use of CMC for social purposes. Methods: In-depth semi-structured interviews with 12 adults (five women, seven men; aged 69 to 81) were conducted. Interview questions focused on individuals’ use of CMC to engage with online communities. Data were transcribed and thematically analysed. Results: Two overarching themes relating to a sense of Belonging and Support emerged. Belonging was most heavily emphasised, and included subthemes on how participants experienced their close social networks online, as well as their broader engagement with building interests and identity. Support arose to a lesser extent, and included subthemes relating to how CMC was used not only for the provision and receipt of such, but also to signal availability or need for support. Throughout, participants consistently weighed the benefits of CMC against the disadvantages. Conclusion: The findings highlight the importance of social networks and online communities for older adults and, in particular, how CMC facilitates feelings of belongingness and provides opportunities for reciprocal instrumental, emotional, and informational support. Future research needs to consider the importance of having a sense of belonging when describing the social functioning of digitally literate older adults.Belinda Grace Fuss, Diana Dorstyn, Lynn War

The p53-caspase-2 axis in the cell cycle and DNA damage response

Caspase-2 was discovered almost three decades ago. It was one of the first two mammalian homologs of CED-3, the other being interleukin 1β-converting enzyme (ICE/caspase-1). Despite high similarity with CED-3 and its fly and mammalian counterparts (DRONC and caspase-9, respectively), the function of caspase-2 in apoptosis has remained enigmatic. A number of recent studies suggest that caspase-2 plays an important role in the regulation of p53 in response to cellular stress and DNA damage to prevent the proliferation and accumulation of damaged or aberrant cells. Here, we review these recent observations and their implications in caspase-2-mediated cellular death, senescence, and tumor suppression.Yoon Lim, Loretta Dorstyn and Sharad Kuma