Molecular Mechanisms of Synaptic Vesicle Degradation

Neurons rely on precise spatial and temporal control of neurotransmitter release to ensure proper communication. Neurotransmission occurs when synaptic vesicles in the presynaptic compartment fuse with the plasma membrane and release their contents into the synaptic cleft, where neurotransmitters bind to receptors on the postsynaptic neuron. Synaptic vesicle pools must maintain a functional repertoire of proteins in order to efficiently release neurotransmitter. Indeed, the accumulation of old or damaged proteins on synaptic vesicle membranes is linked to synaptic dysfunction and neurodegeneration. Despite the importance of synaptic vesicle protein turnover for neuronal health, the molecular mechanisms underlying this process are unknown. In this thesis, we present work that uncovers key components that regulate synaptic vesicle degradation. Specifically, we identify a pathway that mediates the activity-dependent turnover of a subset of synaptic vesicle membrane proteins in mammalian neurons. This pathway requires the synaptic vesicle-associated GTPase Rab35, the ESCRT machinery, and synaptic vesicle protein ubiquitination. We further demonstrate that neuronal activity stimulates synaptic vesicle protein turnover by inducing Rab35 activation and binding to the ESCRT-0 component Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to synaptic vesicle pools, thereby initiating synaptic vesicle protein degradation via the ESCRT pathway. Interestingly, we find that not all synaptic vesicle proteins are degraded by this mechanism, suggesting that synaptic vesicles are not degraded as units, but rather that SV proteins are degraded individually or in subsets. Moreover, we find that lysine-63 ubiquitination of VAMP2 is required for its degradation, and we identify an E3 ubiquitin ligase, RNF167, that is responsible for this activity. Our findings show that RNF167 and the Rab35/ESCRT pathway facilitate the removal of specific proteins from synaptic vesicle pools, thereby maintaining presynaptic protein homeostasis. Overall, our studies provide novel mechanistic insight into the coupling of neuronal activity with synaptic vesicle protein degradation, and implicate ubiquitination as a major regulator in maintaining functional synaptic vesicle pools. These findings will facilitate future studies determining the effects of perturbations to synaptic homeostasis in neuronal dysfunction and degeneration

Prescription Of analgesia in Emergency Medicine (POEM): a multicentre observational survey of pain relief in patients presenting with an isolated limb fracture and/or dislocation

Background: Acute pain is one of the most commonly cited reasons for attendance to the emergency department (ED), and the Royal College of Emergency Medicine (RCEM) Best Practice Guideline (2014) acknowledged that the current management of acute pain in UK EDs is inadequate and has a poor evidence base. Methods: The Prescription Of analgesia in Emergency Medicine (POEM) survey is a cross-sectional observational survey of consecutive patients presenting to 12 National Health Service (NHS) EDs with limb fracture and/or dislocation in England and Scotland and was carried out between 2015 and 2017. The primary outcome was to assess the adequacy of pain management in the ED against the recommendations in the RCEM Best Practice Guidelines. Results: In all, 8346 patients were identified as attending the ED with a limb fracture and/or dislocation but adherence to RCEM guidelines could only be evaluated for the 4160 (49.8%) patients with a recorded pain score. Of these, 2409/4160 (57.9%) patients received appropriate pain relief, but only 1347 patients were also assessed within 20 minutes of their arrival in the ED. Therefore, according to the RCEM guidelines, only 16.1% (1347/8346) of all patients were assessed and had satisfactory pain management in the ED. Conclusions: The POEM survey has identified that pain relief for patients with an isolated limb fracture remains inadequate when strictly compared to the RCEM Best Practice Guidelines. However, we have found that some patients receive analgesia despite having no pain score recorded, while other analgesic modalities are provided that are not currently encompassed by the Best Practice Guidelines. Future iterations of these guidelines may wish to encompass the breadth of available modalities of pain relief and the whole patient journey. In addition, more work is needed to improve timely and repeated assessment of pain and its recording, which has been better achieved in some EDs than others

Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice

Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones

Outcomes of population based language promotion for slow to talk toddlers at ages 2 and 3 years: Let’s Learn Language cluster randomised controlled trial

Objective To determine the benefits of a low intensity parent-toddler language promotion programme delivered to toddlers identified as slow to talk on screening in universal services

Inequity in exercise-based interventions for adults with rheumatoid arthritis : a systematic review

OBJECTIVES: This systematic review describes the extent to which PROGRESS-Plus equity factors were considered in the eligibility criteria of trials of exercise interventions for adults with RA. METHODS: Electronic databases were searched for published (Cinahl, Embase, Medline, Physiotherapy Evidence Database), unpublished (Opengrey) and registered ongoing (International Standard Randomized Controlled Trial Number registry) randomized controlled trials (RCTs) of exercise interventions for adults with RA. Two authors independently performed study selection and quality assessment (Cochrane risk of bias tool). RESULTS: A total of 9696 records were identified. After screening, 50 trials were included. All trials had either some concerns or high risk of bias and reported at least one PROGRESS-Plus equity factor within the eligibility criteria; this included place of residence, personal characteristics (age and disability), language, sex, social capital, time-dependent factors or features of relationship factors. Where reported, this equated to exclusion of 457 of 1337 potential participants (34%) based on equity factors. CONCLUSION: This review identified the exclusion of potential participants within exercise-based interventions for people with RA based on equity factors that might affect health-care opportunities and outcomes. This limits the generalizability of results, and yet this evidence is used to inform management and service design. Trials need to optimize participation, particularly for people with cardiovascular conditions, older adults and those with cognitive impairments. Reasons for exclusions need to be justified. Further research needs to address health inequalities to improve treatment accessibility and the generalizability of research findings. PROSPERO REGISTRATION: CRD42021260941

An occupational therapy intervention for residents with stroke related disabilities in UK care homes (OTCH): cluster randomised controlled trial

Objective To evaluate the clinical efficacy of an established programme of occupational therapy in maintaining functional activity and reducing further health risks from inactivity in care home residents living with stroke sequelae. Design Pragmatic, parallel group, cluster randomised controlled trial. Setting 228 care homes (>10 beds each), both with and without the provision of nursing care, local to 11 trial administrative centres across the United Kingdom. Participants 1042 care home residents with a history of stroke or transient ischaemic attack, including those with language and cognitive impairments, not receiving end of life care. 114 homes (n=568 residents, 64% from homes providing nursing care) were allocated to the intervention arm and 114 homes (n=474 residents, 65% from homes providing nursing care) to standard care (control arm). Participating care homes were randomised between May 2010 and March 2012. Intervention Targeted three month programme of occupational therapy, delivered by qualified occupational therapists and assistants, involving patient centred goal setting, education of care home staff, and adaptations to the environment. Main outcome measures Primary outcome at the participant level: scores on the Barthel index of activities of daily living at three months post-randomisation. Secondary outcome measures at the participant level: Barthel index scores at six and 12 months post-randomisation, and scores on the Rivermead mobility index, geriatric depression scale-15, and EuroQol EQ-5D-3L questionnaire, at all time points. Results 64% of the participants were women and 93% were white, with a mean age of 82.9 years. Baseline characteristics were similar between groups for all measures, personal characteristics, and diagnostic tests. Overall, 2538 occupational therapy visits were made to 498 participants in the intervention arm (mean 5.1 visits per participant). No adverse events attributable to the intervention were recorded. 162 (11%) died before the primary outcome time point, and 313 (30%) died over the 12 months of the trial. The primary outcome measure did not differ significantly between the treatment arms. The adjusted mean difference in Barthel index score at three months was 0.19 points higher in the intervention arm (95% confidence interval −0.33 to 0.70, P=0.48). Secondary outcome measures also showed no significant differences at all time points. Conclusions This large phase III study provided no evidence of benefit for the provision of a routine occupational therapy service, including staff training, for care home residents living with stroke related disabilities. The established three month individualised course of occupational therapy targeting stroke related disabilities did not have an impact on measures of functional activity, mobility, mood, or health related quality of life, at all observational time points. Providing and targeting ameliorative care in this clinically complex population requires alternative strategies

Arterial Elasticity in Ehlers-Danlos Syndromes

Ehlers-Danlos Syndromes (EDS) are a group of heritable disorders of connective tissue (HDCT) characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Orthostatic intolerance (OI) is highly prevalent in EDS however mechanisms linking OI to EDS remain poorly understood. We hypothesize that impaired blood pressure (BP) and heart rate control is associated with lower arterial stiffness in people with EDS. Orthostatic vital signs and arterial stiffness were assessed in a cohort of 60 people with EDS (49 female, 36 ± 16 years). Arterial elasticity was assessed by central and peripheral pulse wave velocity (PWV). Central PWV was lower in people with EDS compared to reference values in healthy subjects. In participants with EDS, central PWV was correlated to supine systolic BP (r = 0.387, p = 0.002), supine diastolic BP (r = 0.400, p = 0.002), and seated systolic BP (r = 0.399, p = 0.002). There were no significant correlations between PWV and changes in BP or heart rate with standing (p > 0.05). Between EDS types, there were no differences in supine hemodynamics or PWV measures (p > 0.05). These data demonstrate that increased arterial elasticity is associated with lower BP in people with EDS which may contribute to orthostatic symptoms and potentially provides a quantitative clinical measure for future genotype-phenotype investigations