Mutant huntingtin confers cell-autonomous phenotypes on Huntington's disease iPSC-derived microglia.

Huntington's disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way.

Human iPSC-derived glia models for the study of neuroinflammation.

Neuroinflammation is a complex biological process that plays a significant role in various brain disorders. Microglia and astrocytes are the key cell types involved in inflammatory responses in the central nervous system. Neuroinflammation results in increased levels of secreted inflammatory factors, such as cytokines, chemokines, and reactive oxygen species. To model neuroinflammation in vitro, various human induced pluripotent stem cell (iPSC)-based models have been utilized, including monocultures, transfer of conditioned media between cell types, co-culturing multiple cell types, neural organoids, and xenotransplantation of cells into the mouse brain. To induce neuroinflammatory responses in vitro, several stimuli have been established that can induce responses in either microglia, astrocytes, or both. Here, we describe and critically evaluate the different types of iPSC models that can be used to study neuroinflammation and highlight how neuroinflammation has been induced and measured in these cultures.

Evaluation of a Community Suicide Prevention Project (Roots of Hope): Protocol for an Implementation Science Study.

BACKGROUND: Roots of Hope (RoH) is a multisite Canadian community-based suicide prevention initiative developed by the Mental Health Commission of Canada (MHCC), which is based on evidence for intervention effectiveness and World Health Organization recommendations. Seven communities developed local activities in the following 5 pillars: specialized supports, training and networks, public awareness, means safety, and evaluation research. OBJECTIVE: We aim to use an implementation research approach to understand the RoH model for reducing suicidal behaviors and their impacts in communities, and the lessons learned for the equitable development and implementation of RoH in different contexts. Moreover, we want to understand how the program is implemented in relation to the context, the causal pathways, and the factors influencing successful implementation. The evaluation includes assessments of short-term and intermediate effects at each site and overall. METHODS: The principal investigator (PI) developed a consensus among local research coordinators on common approaches and indicators through ongoing participation in an online community of practice, and regular virtual and in-person meetings. At the completion of the pilot phase, the PI will summarize evaluation results across sites and conduct pooled analyses. The RoH theory of change and evaluation model shows how evaluation activities from the planning phase through the implementation of activities in each of the pillars can help clarify the viability of the RoH model and identify factors that facilitate and inhibit effective and equitable implementation in different contexts. Beginning with a situational analysis to identify resources in each community and local specificities, we will examine the implementation characteristics of conformity, dosage, coverage, quality, utility, equity, appreciation, facilitators, and impediments. Evaluation of short-term effects will focus on changes in knowledge, attitudes, behaviors, help-seeking, service use, stigma, media reports, empowerment, and care experiences. Intermediate effects, long-term effects, and impact will include assessments of the changes in suicides, suicide attempt rates, and suicide risk indicators. A variety of data sources, both quantitative and qualitative, will be used. RESULTS: The quantitative and qualitative data from all sites will be summarized by the PI in March 2023 to draw conclusions to help the MHCC in its improvements to the RoH model, and to inform communities about how to better implement RoH. Since the COVID-19 pandemic occurred at the beginning of program implementation, its impact and influence will be documented. The validity of RoH in contributing to the prevention of suicides and suicidal behaviors will be clarified in a variety of contexts. The final evaluation report will be available in September 2023. CONCLUSIONS: The evaluation results, including the identification of factors that facilitate and inhibit the implementation of RoH and the adaptations to challenges, will be useful to the MHCC, current RoH communities, and those considering adopting the RoH model. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/39978.

Assaying Microglia Functions In Vitro.

Microglia, the main immune modulators of the central nervous system, have key roles in both the developing and adult brain. These functions include shaping healthy neuronal networks, carrying out immune surveillance, mediating inflammatory responses, and disposing of unwanted material. A wide variety of pathological conditions present with microglia dysregulation, highlighting the importance of these cells in both normal brain function and disease. Studies into microglial function in the context of both health and disease thus have the potential to provide tremendous insight across a broad range of research areas. In vitro culture of microglia, using primary cells, cell lines, or induced pluripotent stem cell derived microglia, allows researchers to generate reproducible, robust, and quantifiable data regarding microglia function. A broad range of assays have been successfully developed and optimised for characterizing microglial morphology, mediation of inflammation, endocytosis, phagocytosis, chemotaxis and random motility, and mediation of immunometabolism. This review describes the main functions of microglia, compares existing protocols for measuring these functions in vitro, and highlights common pitfalls and future areas for development. We aim to provide a comprehensive methodological guide for researchers planning to characterise microglial functions within a range of contexts and in vitro models.

PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages.

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Scoping Review of Clinical Practice Guidelines for Fall Risk Screening and Assessment in Older Adults across the Care Continuum.

Given the rising numbers of older adults in Canada experiencing falls, evidence-based identification of fall risks and plans for prevention across the continuum of care is a significant priority for health care providers. A scoping review was conducted to synthesize published international clinical practice guidelines (CPGs) and recommendations for fall risk screening and assessment in older adults (defined as 65 years of age and older). Of the 22 CPGs, 6 pertained to multiple settings, 9 pertained to community-dwelling older adults only, 2 each pertained to acute care and long-term care settings only, and 3 did not specify setting. Two criteria, prior fall history and gait and balance abnormalities, were applied either independently or sequentially in 19 CPG fall risk screening algorithms. Fall risk assessment components were more varied across CPGs but commonly included: detailed fall history; detailed evaluation of gait, balance, and/or mobility; medication review; vision; and environmental hazards assessment. Despite these similarities, more work is needed to streamline assessment approaches for heterogeneous and complex older adult populations across the care continuum. Support is also needed for sustainable implementation of CPGs in order to improve health outcomes.

TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.

BACKGROUND: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. METHODS: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. RESULTS: R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVß3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFß1, as a candidate upstream regulator. TGFß1 failed to rescue vitronectin adhesion of pMac, although it improved αVß3 expression. CONCLUSIONS: The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.

CIHR Health System Impact Fellows: Reflections on "Driving Change" Within the Health System.

Learning health systems necessitate interdependence between health and academic sectors and are critical to address the present and future needs of our health systems. This concept is being supported through the new Canadian Institutes of Health Research (CIHR) Health System Impact (HSI) Fellowship, through which postdoctoral fellows are situated within a health system-related organization to help propel evidence-informed organizational transformation and change. A voluntary working group of fellows from the inaugural cohort representing diversity in geography, host setting and personal background, collectively organized a panel at the 2018 Canadian Association for Health Services and Policy Research Conference with the purpose of describing this shared scholarship experience. Here, we present a summary of this panel reflecting on our experiential learning in a practice environment and its ability for impact.

Microglial inflammation and phagocytosis in Alzheimer's disease: Potential therapeutic targets.

One of the largest unmet medical needs is a disease-modifying treatment for Alzheimer's disease (AD). Recently, the role of microglia in disease, particularly AD, has gained great interest, following the identification of several disease risk-associated genes that are highly expressed in microglia. Microglia play a critical homeostatic role in the brain, with neuroinflammatory and phagocytic mechanisms being of particular importance. Here, we review the role of NLRP3, the complement system, and the triggering receptor expressed in myeloid cells 2 (TREM2) in modulating microglial functions. We have reviewed the targets, their molecular pathways and the therapeutic interventions aimed at modulating these targets, in the hope of discovering a novel therapeutic approach for the treatment of AD. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Individual- and area-level socioeconomic inequalities in diabetes mellitus in Saskatchewan between 2007 and 2012: a cross-sectional analysis.

BACKGROUND: Improving our understanding of social inequalities may improve prevention and treatment efforts for diabetes mellitus. We examined the association between individual- and area-level socioeconomic measures and physician-diagnosed diabetes in Saskatchewan over time. METHODS: In this cross-sectional study, we linked health administrative data with individual-level socioeconomic data from the Canadian Community Health Survey and area-level data from the 2006 Canadian census. We used general linear mixed-models regression to analyze the effect of each factor, controlling for geographic and demographic measures. RESULTS: Area-level deprivation was associated with medically diagnosed type 2 diabetes mellitus after adjustment for the individual-level factors of age, sex, household income and education. Individuals residing in areas ranked in the least deprived quintile had a lower likelihood of diabetes than those in the most deprived quintile (odds ratio 0.40, 95% confidence interval 0.18-0.88). However, this disparity existed only in urban areas. This result may reflect less pronounced health inequalities in rural areas, greater socioeconomic heterogeneity, larger geographic units or some combination of these factors. INTERPRETATION: Individual- and area-level socioeconomic factors were associated with the likelihood of medically diagnosed diabetes; however, the strength of this association varied between urban and rural communities. Acknowledgement of area-level deprivation as a modifiable risk factor related to the prevalence of diabetes is important in the development of effective interventions for urban, but not rural, areas.

Facilitators and barriers of sociodemographic data collection in Canadian health care settings: a multisite case study evaluation.

BACKGROUND: Despite growing awareness of the importance of social determinants of health, research remains limited about the implementation of sociodemographic data collection in Canadian health care settings. Little is known about the salient contextual factors that enable or hinder collection and use of social information to improve quality of care in clinical settings. This study examines the perceptions and experiences of managers and care providers to better understand how to support organizational efforts to collect and use sociodemographic data to provide equity-oriented care. METHODS: Case studies of three diverse urban health care settings employed semi-structured individual and group interviews with managers and care providers respectively to explore their experiences with implementation. Data was analyzed separately and in context for each site as part of an individual case study. A thematic analysis of interview transcripts was performed with an inductive approach to coding of segments of the text. Constructs of the Consolidated Framework for Implementation Research (CFIR) were used as an analytical framework to structure the data to support cross case comparisons of facilitators and barriers to implementation across settings. RESULTS: Several perceived facilitators and barriers to implementation were identified that clustered around three CFIR domains: intervention, inner setting and characteristics of individuals. Macro level (outer setting) factors were relatively unexplored. Sites were motivated by their recognition of need for social information to improve quality of care. Organizational readiness for implementation was demonstrated by priorities that reflected concern for equity in care, leadership support and commitment to an inclusive process for stakeholder engagement. Barriers included perceived relevance of only a subset of sociodemographic questions to service delivery, staff capacity and comfort with data collection as well as adequate resources (funding and time). CONCLUSION: Although system level mandates were underexplored, they may accelerate adoption and implementation of sociodemographic data collection in the presence of organizational readiness. Standardized tools integrated into information systems and workflows would support adequately trained personnel. More research is needed to understand important factors in rural health settings and with clinical application to inform care delivery pathways.

Knowledge, Attitudes and Barriers to Human Papillomavirus (HPV) Vaccine Uptake Among an Immigrant and Refugee Catch-Up Group in a Western Canadian Province.

Vaccination is a key strategy to prevent cervical cancer in developed countries. Lower uptake of human papillomavirus (HPV) vaccine among new immigrants and refugees has been documented, although exploration of underlying reasons remains an understudied area. Semi-structured interviews with eleven immigrant women (ages 18-26 years) were conducted to understand their knowledge, attitudes and barriers regarding HPV vaccination in a western Canadian province. Participants had limited knowledge about HPV and the vaccine. Most women perceived that their risk of HPV was low, however expressed willingness to receive the vaccine if it were recommended by their physician. Greater efforts are needed to increase knowledge about HPV among immigrant and refugee women and support for physicians to discuss and offer vaccination to this underserved population.

α-Synuclein increases ß-amyloid secretion by promoting ß-/γ-secretase processing of APP.

α-Synuclein misfolding and aggregation is often accompanied by ß-amyloid deposition in some neurodegenerative diseases. We hypothesised that α-synuclein promotes ß-amyloid production from APP. ß-Amyloid levels and APP amyloidogenic processing were investigated in neuronal cell lines stably overexpressing wildtype and mutant α-synuclein. γ-Secretase activity and ß-secretase expression were also measured. We show that α-synuclein expression induces ß-amyloid secretion and amyloidogenic processing of APP in neuronal cell lines. Certain mutations of α-synuclein potentiate APP amyloidogenic processing. γ-Secretase activity was not enhanced by wildtype α-synuclein expression, however ß-secretase protein levels were induced. Furthermore, a correlation between α-synuclein and ß-secretase protein was seen in rat brain striata. Iron chelation abolishes the effect of α-synuclein on neuronal cell ß-amyloid secretion, whereas overexpression of the ferrireductase enzyme Steap3 is robustly pro-amyloidogenic. We propose that α-synuclein promotes ß-amyloid formation by modulating ß-cleavage of APP, and that this is potentially mediated by the levels of reduced iron and oxidative stress.

Seeking a mechanism for the toxicity of oligomeric α-synuclein.

In a number of neurological diseases including Parkinson's disease (PD), α-synuclein is aberrantly folded, forming abnormal oligomers, and amyloid fibrils within nerve cells. Strong evidence exists for the toxicity of increased production and aggregation of α-synuclein in vivo. The toxicity of α-synuclein is popularly attributed to the formation of "toxic oligomers": a heterogenous and poorly characterized group of conformers that may share common molecular features. This review presents the available evidence on the properties of α-synuclein oligomers and the potential molecular mechanisms of their cellular disruption. Toxic α-synuclein oligomers may impact cells in a number of ways, including the disruption of membranes, mitochondrial depolarization, cytoskeleton changes, impairment of protein clearance pathways, and enhanced oxidative stress. We also examine the relationship between α-synuclein toxic oligomers and amyloid fibrils, in the light of recent studies that paint a more complex picture of α-synuclein toxicity. Finally, methods of studying and manipulating oligomers within cells are described.

Frequent HIV testing among participants of a routine HIV testing program.

Massachusetts developed a routine HIV testing program in four sites from January-September 2002. Of the 2,502 patients tested, 453 (18.1%) reported ≥2 HIV tests within the prior three years. In multivariate analyses, frequent HIV testing was associated with younger age (18-30 years, OR = 1.42), a history of injection drug use (OR = 6.35), and men who had sex with men (OR = 3.49). Participants who reported multiple sexual partners (OR = 2.17) and high risk sexual behavior (OR = 2.02) were significantly more likely to have had a prior HIV test. Patients whose HIV risk was unknown had the highest association with frequent testing (OR = 13.18). Because characteristics of frequent HIV testers may inform behavioral interventions, there is a need to understand the motivation for repeatedly accessing HIV testing services.

The cost-effectiveness of antiretroviral therapy for treating HIV disease in the Caribbean.

BACKGROUND: Antiretroviral therapy (ART) recently became available in the Organization of Eastern Caribbean States (OECS). Survival benefits and budgetary implications associated with universal access to ART have not been examined in the Caribbean. METHODS: Using a state-transition simulation model of HIV with regional data, we projected survival, cost, and cost-effectiveness of treating an HIV-infected cohort. We examined 1 or 2 ART regimens and cotrimoxazole. In sensitivity analysis, we varied HIV natural history and ART efficacy, cost, and switching criteria. RESULTS: Without treatment, mean survival was 2.30 years (mean baseline CD4 count = 288 cells/microL). One ART regimen with cotrimoxazole when the CD4 count was <350 cells/microL provided an additional 5.86 years of survival benefit compared with no treatment; the incremental cost-effectiveness ratio was $690 per year of life saved (YLS). A second regimen added 1.04 years of survival benefit; the incremental cost-effectiveness ratio was $10,960 per YLS compared with 1 regimen. Results were highly dependent on second-line ART costs. Per-person lifetime costs decreased from $17,020 to $9290 if second-line ART costs decreased to those available internationally, yielding approximately $8 million total savings. CONCLUSIONS: In the OECS, ART is cost-effective by international standards. Reducing second-line ART costs increases cost-effectiveness and affordability. Current funding supports implementing universal access regionally over the next year, but additional funding is required to sustain lifetime care for currently infected persons.