The Cure VCP Scientific Conference 2021: Molecular and clinical insights into neurodegeneration and myopathy linked to multisystem proteinopathy-1 (MSP-1).

The 2021 VCP Scientific Conference took place virtually from September 9-10, 2021. This conference, planned and organized by the nonprofit patient advocacy group Cure VCP Disease, Inc. (https://www.curevcp.org), was the first VCP focused meeting since the 215th ENMC International Workshop VCP-related multi-system proteinopathy in 2016 (Evangelista et al., 2016). Mutations in VCP cause a complex and heterogenous disease termed inclusion body myopathy (IBM) with Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) (IBMPFD), or multisystem proteinopathy 1 (MSP-1) Kimonis (n.d.), Kovach et al. (2001), Kimonis et al. (2000). In addition, VCP mutations also cause other age-related neurodegenerative disorders including amyptrophic lateral sclerosis (ALS), Parkinsonism, Charcot-Marie type II-B, vacuolar tauopathy among others (Korb et al., 2022). The objectives of this conference were as follows: (1) to provide a forum that facilitates sharing of published and unpublished information on physiological roles of p97/VCP, and on how mutations of VCP lead to diseases; (2) to bolster understanding of mechanisms involved in p97/VCP-relevant diseases and to enable identification of therapeutics to treat these conditions; (3) to identify gaps and barriers of further discoveries and translational research in the p97/VCP field; (4) to set a concrete basic and translational research agenda for future studies including crucial discussions on biomarker discoveries and patient longitudinal studies to facilitate near-term clinical trials; (5) to accelerate cross-disciplinary research collaborations among p97/VCP researchers; (6) to enable attendees to learn about new tools and reagents with the potential to facilitate p97/VCP research; (7) to assist trainees in propelling their research and to foster mentorship from leaders in the field; and (8) to promote diversity and inclusion of under-represented minorities in p97/VCP research as diversity is critically important for strong scientific research. Given the range of topics, the VCP Scientific Conference brought together over one hundred and forty individuals representing a diverse group of research scientists, trainees, medical practitioners, industry representatives, and patient advocates. Twenty-five institutions with individuals from thirteen countries attended this virtual meeting. In this report, we summarize the major topics presented at this conference by a range of experts.

Isoform- and cell type-specific structure of apolipoprotein E lipoparticles as revealed by a novel Forster resonance energy transfer assay.

Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.

ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons.

Mutations in the AAA+ ATPase p97 cause multisystem proteinopathy 1, which includes amyotrophic lateral sclerosis; however, the pathogenic mechanisms that contribute to motor neuron loss remain obscure. Here, we use two induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using quantitative proteomics, we find that motor neurons harboring the p97 R155H mutation have deficits in the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to clear damaged lysosomes and have reduced viability. Lysosomes in mutant motor neurons have increased pH compared with wild-type cells. The clearance of damaged lysosomes involves UBXD1-p97 interaction, which is disrupted in mutant motor neurons. Finally, inhibition of the ATPase activity of p97 using the inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

Racial and Gender Differences in Medical Student Burnout: A 2021 National Survey.

OBJECTIVE: To measure racial and gender differences in medical student burnout and identify possible contributing factors. PATIENTS AND METHODS: Electronic surveys were distributed to medical students at 9 US medical schools from December 27, 2020, through January 17, 2021. Questions covered demographic characteristics, stressors contributing to burnout, and the 2-item Maslach Burnout Inventory. RESULTS: Of 5500 invited students, 1178 (21%) responded (mean age, 25.3 years; 61% identified as female). Fifty-seven percent of respondents identified as White, 26% as Asian, and 5% as Black. Overall, 75.6% of students met the criteria for burnout. Women reported more burnout (78% vs 72%; P=.049). There were no differences in burnout prevalence by race. Students commonly reported that lack of sleep (42%), decreased engagement in hobbies or self-care (41%), stress about grades (37%), feeling socially disconnected (36%), and lack of exercise (35%) contributed to burnout. Compared with students of other races, Black students reported that their feelings of burnout were affected significantly more by lack of sleep and poor diet, and Asian students more by stress about grades, residency, and publishing pressure (all P<.05). Female students were more affected than male students by stress about grades, poor diet, and feelings of social disconnectedness and inadequacy (all P<.05). CONCLUSION: Burnout (75.6%) was higher than historical norms, and female students reported higher burnout than male students. There was no difference in burnout prevalence by race. There were racial and gender differences in self-identified contributors of burnout. Additional research is needed to confirm whether stressors were contributors to or consequences of burnout, as well as how to address them.

Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9.

The capacity of certain adeno-associated virus (AAV) vectors to cross the blood-brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9) expressing the green fluorescent protein (GFP) either under an astrocyte (glial fibrillary acidic (GFA) protein) or neuronal (Synapsin (Syn)) promoter, after intravenous injection of adult mice (2 × 1013 vg/kg). ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11%) and neurons (17%), respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter), significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive) and resting astrocytes (GFAP-negative) express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS) gene therapy approaches in which peripheral expression of transgene is a concern.

Social stress and escalated drug self-administration in mice I. Alcohol and corticosterone.

RATIONALE: Stress experiences have been shown to be a risk factor for alcohol abuse in humans; however, a reliable mouse model using episodic social stress has yet to be developed. OBJECTIVES: The current studies investigated the effects of mild and moderate social defeat protocols on plasma corticosterone, voluntary alcohol drinking, and motivation to drink alcohol. METHODS: Outbred Carworth Farms Webster (CFW) mice were socially defeated for 10 days during which the intruder mouse underwent mild (15 bites: mean = 1.5 min) or moderate (30 bites: mean = 3.8 min) stress. Plasma corticosterone was measured on days 1 and 10 of the defeat. Ethanol drinking during continuous access to alcohol was measured 10 days following the defeat or 10 days prior to, during, and 20 days after the defeat. Motivation to drink was determined using a progressive ratio (PR) operant conditioning schedule during intermittent access to alcohol. RESULTS: Plasma corticosterone was elevated in both stress groups on days 1 and 10. Ethanol consumption and preference following moderate stress were higher (13.3 g/kg/day intake) than both the mild stress group (8.0 g/kg/day) and controls (7.4 g/kg/day). Mice with previously acquired ethanol drinking showed decreased alcohol consumption during the moderate stress followed by an increase 20 days post-defeat. Moderately stressed mice also showed escalated ethanol intake and self-administration during a schedule of intermittent access to alcohol. CONCLUSION: Social defeat experiences of moderate intensity and duration led to increased ethanol drinking and preference in CFW mice. Ongoing work investigates the interaction between glucocorticoids and dopaminergic systems as neural mechanisms for stress-escalated alcohol consumption.