Integration of population-level data sources into an individual-level clinical prediction model for dengue virus test positivity.

The differentiation of dengue virus (DENV) infection, a major cause of acute febrile illness in tropical regions, from other etiologies, may help prioritize laboratory testing and limit the inappropriate use of antibiotics. While traditional clinical prediction models focus on individual patient-level parameters, we hypothesize that for infectious diseases, population-level data sources may improve predictive ability. To create a clinical prediction model that integrates patient-extrinsic data for identifying DENV among febrile patients presenting to a hospital in Thailand, we fit random forest classifiers combining clinical data with climate and population-level epidemiologic data. In cross-validation, compared to a parsimonious model with the top clinical predictors, a model with the addition of climate data, reconstructed susceptibility estimates, force of infection estimates, and a recent case clustering metric significantly improved model performance.

Epigallocatechin Gallate Modulates Microglia Phenotype to Suppress Pro-inflammatory Signalling Cues and Inhibit Phagocytosis.

Microglia are crucial players in the pathogenesis of late-onset Alzheimer's disease (AD), with evidence for both deleterious and beneficial effects. Identifying interventions to modulate microglial responsiveness, promote amyloid ß (Aß) clearance, disrupt plaque formation, or dampen excessive inflammation has therapeutic potential. Bioavailable flavonoids, such as the flavan 3-ols, are of interest due to their antioxidant, metal chelating, signalling, and anti-inflammatory potential. Primary microglia were treated with a series of structurally related flavanol 3-ols to assess effects on phagocytosis, cytokine release, and transcriptional responses by RNA sequencing. Data indicated that the extent of hydroxylation and the presence of the galloyl moiety were strong determinants of flavan 3-ol activity. Epigallocatechin gallate (EGCG) was the most effective flavan-3-ol tested and strongly inhibited phagocytosis of Aß independent of any metal chelating properties, suggesting a more direct modulation of microglia responsiveness. EGCG was broadly anti-inflammatory, reducing cytokine release and downregulating transcription, particularly of components of the microglia extracellular matrix such as MMP3 and SerpinB2. Collectively, this brings new insight into the actions of flavonoids on microglial responsiveness with potential implications for the therapeutic use of EGCG and structurally related flavanol-3-ols in AD.

Demographic, gambling, and psychological characteristics of sports bettors in a stratified sample of adults who gamble regularly.

OBJECTIVE: Sports betting accounts for the greatest proportion of online gambling behavior globally and has been linked to various harms. Few studies have examined the average sports bettor profile using stratified samples of adults who gamble regularly. The present study builds upon existing research on the demographic variables of sports bettors and provides an in-depth examination of the clinical and gambling-related factors associated with sports betting. METHOD: Participants (N = 10,039; 53.6% female) consisted of a stratified sample of Canadian adults who endorsed participating in gambling at least once per month in the past year. Participants completed standardized measures assessing demographics, gambling behavior, problem gambling severity, gambling-related harms, gambling motives, and psychological characteristics (e.g., substance use, mental health). RESULTS: About 1,816 participants (18.1%) reported engaging in sports betting in the past year. Sports bettors tended to be younger, male, and employed full-time compared to nonsports bettors. Sports bettors endorsed different patterns of clinical comorbidities and greater substance use. Sports betting was also associated with unique gambling motives and greater time and money spent on gambling. Among participants who endorsed problem gambling, sports betting was associated with greater impulsivity and likelihood of using illicit substances while gambling. CONCLUSIONS: The results highlight the characteristics of individuals who bet on sports, as well as the characteristics of sports bettors with problem gambling which may help to inform the development of targeted prevention and intervention efforts to mitigate the potential harms of sports betting. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Predictors of problem gambling remission in adults: A Canadian longitudinal study.

OBJECTIVE: Remission from problem gambling (PG) continues to be a priority of clinicians and researchers. Data from cross-sectional studies indicate that some correlates are more predictive of PG, and existing longitudinal studies have exclusively examined risk factors that predict emergence of PG. This study's objective is to fill in the remaining pieces of the puzzle by identifying factors that might facilitate remission from PG. METHOD: A stratified sample of 10,199 Canadian adult gamblers were recruited from an online panel. Respondents who screened positively for PG at baseline and completed a follow-up assessment 1 year later (n = 468) were assessed on a series of modifiable gambling, psychosocial, mental health, and substance use variables. A forward stepwise logistic regression was conducted to identify the strongest predictors of remission from PG at follow-up. A Least Absolute Shrinkage and Selection Operator regression was also conducted to confirm the most relevant predictors. RESULTS: Out of 75 candidate variables, 10 were retained by the regression model. Two were related to cessation of specific gambling activities, two were related to gambling motivations, two were psychosocial in nature, two were related to substance use while gambling, and one was related to remission from a mental health disorder. The final and strongest predictor was PG severity at baseline. CONCLUSIONS: Although PG remission predictors were mostly gambling-related, psychosocial aspects may also be targeted by stakeholders aiming to reduce PG. Ceasing to use tobacco while gambling and diversifying leisure activities may be promising targets. Other mental health and substance use predictors may still possibly be relevant, but only for a subset of people with PG. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Are There Riskier Types of Gambling?

Gambling-related harm is a public health issue requiring market regulation and efforts aimed at prevention and treatment. An important consideration for the regulation of gambling is whether certain types of gambling are intrinsically more harmful than others. The present study was a comprehensive investigation of this issue in a nationwide sample of 10,199 Canadian adult gamblers that included 1346 individuals with problem gambling. We investigated (a) the univariate cross-sectional association between individual types of gambling and problem gambling; (b) the cross-sectional association between individual gambling types and problem gambling when controlling for breadth of gambling involvement; (c) the prospective/lagged relationship between participation in different gambling types and future problem gambling; and (d) the self-reports of people with gambling problems concerning the types and modalities they consider to be most problematic. Our collective results indicate that breadth of gambling involvement is a stronger predictor of gambling problems than involvement in any particular type, but that involvement in certain types (electronic gambling machines in particular, and casino table games and online gambling to a lesser extent) does confer additional risk.

Cognitive Factors that Predict Gambling Fallacy Endorsement.

Gambling fallacies are a collection of error-stricken beliefs about gambling and how gambling works. Gambling fallacies, while common in the general public, appear to increase as a function of gambling severity. This being the case, many interventions have focused on reducing gambling fallacies as a means of treating problem-gambling. Less research, however, has investigated what factors contributes to gambling fallacy susceptibility in the first place. Available studies have identified associations between gambling fallacy susceptibility and isolated individual differences in, for example, statistical reasoning/understanding, intelligence, or cognitive ability. The current study aimed to assess these cognitive factors in conjunction, and their relative predictive potential for gambling fallacy susceptibility. In an Australian university student sample (n = 90) it was found that there were moderate to strong association between gambling fallacy endorsement and general intelligence, probabilistic reasoning ability, rational cognitive style and the ability to suppress intuitive thought, however, only probabilistic reasoning, rational cognitive style and the ability to suppress intuitive thinking contributed to the prediction of fallacy endorsement. Importantly, each of these factors are malleable. Interventions for the correction of gambling-specific fallacious beliefs should focus on these factors.

Problem gambling severity, gambling behavior, substance use, and mental health in gamblers who do and do not use cannabis: Evidence from a Canadian national sample.

INTRODUCTION: Cannabis use frequently co-occurs with gambling, and evidence indicates that both acute and chronic cannabis use may influence gambling behavior. The primary aim of the present study was to further contribute to the literature on this relationship by examining data collected from a Canadian national study of gambling. METHODS: Respondents consisted of 10,054 Canadian gamblers recruited from Leger Opinion's (LEO) online panel. In this study, gamblers who used cannabis were compared with non-users across a number of gambling as well as demographic and mental health variables. RESULTS: Of the total sample, 25.4 % reported past 12-month cannabis use. Among the 2,553 cannabis-users, 21.3 % reported daily use, and 69.9 % reported using once a month or more. A total of 56.2 % indicated they had used cannabis while gambling in the past 12 months. Bivariate analysis found significant differences between cannabis use and non-use on numerous demographic, mental health, and gambling-related variables. Individuals with greater problem gambling severity scores, more hours gambling, and a larger range of gambling activities were more likely to endorse using cannabis. Hierarchical logistic regression revealed that tobacco use, and having experienced significant child abuse were predictors of cannabis use. Non-use of cannabis was associated with older age, less engagement in online gambling, and being less likely to consume alcohol. CONCLUSION: The present findings both corroborate previous studies and expand upon the relationship between cannabis and gambling.

Etiology of problem gambling in Canada.

OBJECTIVE: To conduct a large-scale national cohort study to identify the current etiological risk factors for problem gambling in Canada. METHOD: A cohort of 10,119 Canadian gamblers completed a comprehensive self-administered online questionnaire in 2018 and were reassessed in 2019. At baseline, the sample contained 1,388 at-risk gamblers, 1,346 problem gamblers, and 2,710 with a major DSM-5 mental health disorder. A total of 108 independent variables (IVs) were available for analysis, as well as the self-report of perceived causes of gambling-related problems for 1,261 individuals. RESULTS: The strongest multivariate predictors of current and future problem gambling were "gambling-related" variables (i.e., current and past problem gambling, intensive gambling involvement, playing electronic gambling machines (EGMs), gambling fallacies, socializing with other people having gambling-related problems, and family history of having gambling-related problems). Beyond gambling-related variables, greater impulsivity and lower household income were robustly predictive. Thirteen additional variables were either concurrently or prospectively predictive, but not both. In contrast to the many different quantitative predictors, self-reported causes tended to be singular and psychologically oriented (i.e., desire to win money, boredom, stress, poor self-control). CONCLUSIONS: The predictors of problematic gambling in the present study are very similar to the predictors identified in prior international longitudinal and cross-sectional research. This implies core cross-cultural risk factors, with gambling-related variables and impulsivity being most important, and comorbidities and demographic variables having more modest contributions. The additional value of the present results is that they comprehensively identify the relative importance of all known etiologically relevant variables within a current Canadian context. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Individual differences and predictors of general awareness in problem gambling.

In Canada, up to 3% of individuals have or are at risk of gambling disorder. Among these individuals, a lack of awareness of their problem gambling is a common barrier to treatment and negatively affects treatment adherence. A secondary analysis was conducted on data from 1346 individuals (mean age = 43.4, SD = 14.4; 54.3 % male) with problem gambling who did and did not perceive having a problem with their gambling as assessed by the fifth item of the Problem Gambling Severity Index, "In the past twelve months, how often have you felt that you might have a problem with gambling?" Additionally, we investigated predictors of increased general awareness at 12-month follow-up. At baseline, individuals who perceived a problem with their gambling experienced more gambling-related harms (OR = 1.714), had greater total gambling losses (OR = 1.067), were more likely to have a family history of problem gambling (OR = 2.143), experienced a greater loss of control (OR = 1.313) and more often gambled alone than with others (OR = 0.879), accounting for 26.6 % of the variance in general awareness. Baseline problem awareness was positively associated with attempts to cut down or control gambling at follow-up (χ2=11.350,p<.001), but negatively associated with remission (χ2=18.392,p<.001). Increases in awareness were related to an increase in the number of gambling-related harms, gambling involvement, impaired control, and lower educational attainment, explaining 35.5% of the variance in increased general awareness. The results indicate that experiencing more gambling-related harms increases the salience and awareness of problem gambling, and that awareness is also associated with an individual's gambling context, their loss of control, and their level of gambling involvement. The findings highlight the importance of gambling harms as they pertain to general awareness and suggest that improving the recognition of gambling harms could be beneficial for the prevention and intervention of gambling disorder.

Harm Minimization Training, Knowledge, and Behaviour of Canadian Casino Employees.

Casino employees regularly interact with problem and at-risk gamblers and thus have considerable potential to both prevent and reduce gambling-related harm. While harm minimization (HM) and responsible gambling (RG) are routinely espoused by the casino industry, the actual level of employee HM/RG training, knowledge, and behaviour is unknown. The present study investigated this issue in the Canadian context by examining employee surveys collected by the RG Check accreditation program (8,262 surveys from 78 Canadian casinos/racinos collected between 2011 and 2020). These surveys revealed that almost all casino employees receive HM/RG training, but the amount of training tends to be quite limited (one hour) except for supervisors, managers, and security personnel. Basic HM/RG knowledge among all employees appears adequate, although their understanding of probability is incomplete. The most important consideration is whether this training and knowledge translates into meaningful HM/RG behaviour towards patrons. The large majority of employees (83.1%) report engaging in at least one HM/RG interaction with a patron at some point during the course of their employment (median length of 4 to 9 years), with security personnel reporting the highest rates. However, the frequency, nature, and impact of these interactions is unknown.

Anionic lipid vesicles have differential effects on the aggregation of early onset-associated α-synuclein missense mutants.

α-synuclein (αS) is the key component of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-ß amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity. Furthermore, lipids are known to be important for native αS function, where they play a key role in the regulation of synaptic vesicle docking to presynaptic membranes and dopamine transmission. However, the role of lipids in the function of mutant αS is unclear. Here, we studied αS aggregation properties of WT αS and five of the most predominant single-point missense mutants associated with early onset PD in the presence of anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine lipid vesicles. Our results highlight significant differences between aggregation rates, the number of aggregates produced, and overall fibril morphologies of WT αS and the A30P, E46K, H50Q, G51D, and A53T missense mutants in the presence of lipid vesicles. These findings have important implications regarding the interplay between the lipids required for αS function and the individual point mutations known to accelerate PD and related diseases.

A comprehensive analysis of all known fishes from Sydney Harbour.

Fishes represent an important natural resource and yet their diversity and function in dynamic estuaries with relatively high levels of human pressure such as Sydney Harbour have rarely been quantified. Further, Eastern Australia supports the survival and persistence of an increasing number of tropical species found within temperate estuaries owing to increasing average ocean temperatures. A re-valuation of the number of fish species known from Sydney Harbour is therefore needed. In this study, we generated an up-to-date and annotated checklist of fishes recorded from Sydney Harbour based on verified natural history records as well as newly available citizen science records based on opportunistic observations and structured surveys. We explored the spatial and temporal distribution of these records. In addition, we quantified the function, conservation status, and commercial importance of the identified fishes. The number of fish species recorded from Sydney Harbour now stands at 675, an increase of 89 species (15 %) when compared to the most recent evaluation in 2013. We attribute this increase in fish diversity over a relatively short time to the contribution of newer citizen science programs as well as the influx and survival of fishes in the Harbour with preferences for warmer waters. Some fish families were also overrepresented in the more urbanized and polluted sections of the Harbour. In forecasting further environmental impacts on the fishes of Sydney Harbour, we recommend increased integration of collaborative citizen science programs and natural history collections as a means to track these changes.

Making Change: Attempts to Reduce or Stop Gambling in a General Population Sample of People Who Gamble.

Objective: This study examined past year attempts to reduce or quit gambling among people who gamble generally and those with gambling problems specifically. Methods: Regular gamblers recruited from an online panel (N = 10,054) completed a survey of gambling, mental health and substance use comorbidity and attempts to reduce or quit gambling. The sample was weighted to match the gambling and demographic profile for the same subsample (i.e., past month gamblers) in a recent Canadian national survey. Results: 5.7% reported that they tried to cutback or stop gambling in the past year. As predicted, individuals making a change attempt had greater levels of problem gambling severity and were more likely to have a gambling problem. Of individuals with problem gambling, 59.8% made a change attempt. Of those, 90.2% indicated that they did this primarily on their own, and 7.7% accessed formal or informal treatment. Most people attempting self- change indicated that this was a personal preference (55%) but about a third reported feeling too ashamed to seek help. Over a third (31%) reported that their attempt was successful. Of the small group of people accessing treatment, 39% described it as helpful. Conclusions: Whereas gambling treatment-seeking rates are low, rates of self-change attempts are high. The public health challenge is to promote self-change efforts among people beginning to experience gambling problems, facilitate success at self-change by providing accessible support for use of successful strategies, and provide seamless bridges to a range of other treatments when desired or required.

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

Library-Derived Peptide Aggregation Modulators of Parkinson's Disease Early-Onset α-Synuclein Variants.

Parkinson's Disease (PD) is characterized by the accumulation of Lewy bodies in dopaminergic neurons. The main protein component of Lewy bodies, α-synuclein (αS), is also firmly linked to PD through the identification of a number of single point mutations that are autosomal dominant for early-onset disease. Consequently, the misfolding and subsequent aggregation of αS is thought to be a key stage in the development and progression of PD. Therefore, modulating the aggregation pathway of αS is an attractive therapeutic target. Owing to the fact that all but one of the familial mutations is located in the preNAC 45-54 region of αS, we previously designed a semi-rational library using this sequence as a design scaffold. The 45-54 peptide library was screened using a protein-fragment complementation assay approach, leading to the identification of the 4554W peptide. The peptide was subsequently found to be effective in inhibiting primary nucleation of αS, the earliest stage of the aggregation pathway. Here, we build upon this previous work by screening the same 45-54 library against five of the known αS single-point mutants that are associated with early-onset PD (A30P, E46K, H50Q, G51D, and A53T). These point mutations lead to a rapid acceleration of PD pathology by altering either the rate or type of aggregates formed. All ultimately lead to earlier disease onset and were therefore used to enforce increased assay stringency during the library screening process. The ultimate aim was to identify a peptide that is effective against not only the familial αS variant from which it has been selected but that is also effective against WT αS. Screening resulted in five peptides that shared common residues at some positions, while deviating at others. All reduced aggregation of the respective target, with several also identified to be effective at reducing aggregation when incubated with other variants. In addition, our results demonstrate that a previously optimized peptide, 4554W(N6A), is highly effective against not only WT αS but also several of the single-point mutant forms and hence is a suitable baseline for further work toward a PD therapeutic.

Gambling in Canada During the COVID Lockdown: Prospective National Survey.

The current study investigated the impact of the COVID pandemic lockdown on gambling and problem gambling in Canada. The AGRI National Project's online panel participants (N = 3449) provided baseline gambling data 6 months prior to the pandemic. Re-surveying this sample during the lockdown provided an opportunity to make quantitative comparisons of the changes. Nearly one-third of gamblers reported ceasing gambling altogether during the lockdown. For the continuing gamblers, quantitative data indicated significant decreases in gambling frequency, time spent in gambling sessions, money spent, and the number of game types played. Qualitative perceptions of changes in gambling were examined and the accuracy of these reports were not closely aligned with actual changes in gambling. Gambling platform was the only gambling engagement metric where increases were found with ~ 17% of the gambling sample migrating to online gambling during the lockdown. Although problem gambling within the sample generally declined, consistent with previous literature, it was also found that gambling online-among other biopsychosocial factors-was a significant predictor for classification as a problem gambler during the lockdown. COVID-specific influences on health, employment, leisure time and social isolation were moderately associated with problem gambling scores but were not independent predictors of changes in gambling engagement during lockdown. Future studies are required to assess if the pandemic related changes in gambling evidenced in this study remain stable, or if engagement reverts to pre-pandemic levels when the pandemic response allows for the re-opening of land-based gambling venues.

Responsible Gambling in Canada: An Analysis of the RG Check Patron Surveys.

This study analyzed the Responsible Gambling Check patron survey data from Canadian casinos and racinos collected from 2011-2019 (18,580 patrons and 75 venues). The results indicated increasing awareness and use over time of harm minimization tools among more frequent patrons. Despite these promising trends, it is concerning that a substantial percentage of gamblers are still unaware of the harm minimization tools available. Further, the actual impact of this awareness on responsible gambling behaviour is largely unknown. We suggest greater efforts are needed nation-wide to promote the awareness, utilization, and evaluation of these harm minimization tools.

Indigenous Gambling and Problem Gambling in Canada.

The present study provides a profile of Canadian Indigenous gambling and problem gambling using the 2018 Canadian Community Health Survey (CCHS) (n = 23,952 adults; 1,324 Indigenous) and an online panel survey of 10,199 gamblers (n = 589 Indigenous). The relative popularity of different types of gambling was similar between Indigenous and non-Indigenous samples. However, there was higher Indigenous participation in electronic gambling machines (EGMs), bingo, instant lotteries, overall gambling and a higher rate of problem gambling (2.0% versus 0.5%). Variables predictive of Indigenous problem gambling were EGM participation, gambling fallacies, having a mental or substance use disorder, sports betting, and male gender. Compared to non-Indigenous problem gamblers, Indigenous problem gamblers had higher substance use and lower impulsivity. In general, variables predictive of Indigenous problem gambling were the same ones predictive of problem gambling in all populations, with elevated Indigenous problem gambling rates primarily being due to elevated rates of these generic risk factors. Many of these risk factors are modifiable. Particular consideration should be given to reducing the disproportionate concentration of EGMs in geographic areas having the highest concentration of Indigenous people and ameliorating the disadvantageous social conditions in this population that are conducive to mental health and substance use problems.

Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors for cancer treatment.

A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments will help inform treatment regimes. The nonreceptor tyrosine kinase SRC regulates many cellular signalling processes, and pharmacological inhibition has long been a target of cancer drug discovery projects. Here, we describe the in vitro and in vivo characterisation of the small-molecule SRC inhibitor AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-419 of SRC (IC50 ~ 100 nm) in many cancer cell lines; however, inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a subset of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using reverse-phase protein array (RPPA) analysis. We demonstrate that SRC is activated in response to treatment of KRAS-mutant colorectal cell lines with MEK inhibitors (trametinib or AZD6244) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 had reduced EGFR, FAK and SRC compensatory activation, and cell viability was synergistically inhibited. In vivo, trametinib treatment of mice-bearing HCT116 tumours increased phosphorylation of SRC on Tyr419, and, when combined with AZD0424, inhibition of tumour growth was greater than with trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however, inhibition of SRC remains an effective way to block invasion of trametinib-resistant tumour cells. These data imply that SRC inhibition may offer a useful addition to MEK inhibitor combination strategies.

A Downsized and Optimised Intracellular Library-Derived Peptide Prevents Alpha-Synuclein Primary Nucleation and Toxicity Without Impacting Upon Lipid Binding.

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson's disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45-54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson's disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function.