HaloTag as a substrate-based macroautophagy reporter.

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

Advice to People with Parkinson's in My Clinic: Cannabis.

Cannabis (in all the varied methods of delivery) continues to garner significant attention as a potential therapeutic intervention for neurodegenerative disorders, including Parkinson's disease (PD). The recent legalization of personal use of cannabis products in some parts of the world has increased this interest and with it, potential availability to many more people. However, such access has led to more questions than answers for both patients and health care professionals. These include what symptom(s) of PD will cannabis products treat; what dose; what type of cannabis product to use and what are the side effects?

Multiple system atrophy with amyloid-ß-predominant Alzheimer's disease neuropathologic change.

Multiple system atrophy is a neurodegenerative disease with α-synuclein pathology predominating in the striatonigral and olivopontocerebellar systems. Mixed pathologies are considered to be of low frequency and mostly comprise primary age-related tauopathy or low levels of Alzheimer's disease-related neuropathologic change. Therefore, the concomitant presence of different misfolded proteins in the same brain region is less likely in multiple system atrophy. During the neuropathological evaluation of 21 consecutive multiple system atrophy cases, we identified four cases exhibiting an unusual discrepancy between high Thal amyloid-ß phase and low transentorhinal Braak neurofibrillary tangle stage. We mapped α-synuclein pathology, measured the size and number of glial cytoplasmic inclusions and compared the amyloid-ß peptides between multiple system atrophy and Alzheimer's disease. In addition, we performed α-synuclein seeding assay from the affected putamen samples. We performed genetic testing for APOE, MAPT, PSEN1, PSEN2 and APP. We refer to the four multiple system atrophy cases with discrepancy between amyloid-ß and tau pathology as 'amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy' to distinguish these from multiple system atrophy with primary age-related tauopathy or multiple system atrophy with typical Alzheimer's disease neuropathologic change. As most multiple system atrophy cases with mixed pathologies reported in the literature, these cases did not show a peculiar clinical or MRI profile. Three amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy cases were available for genetic testing, and all carried the APOE ɛ4 allele. The extent and severity of neuronal loss and α-synuclein pathology were not different compared with typical multiple system atrophy cases. Analysis of amyloid-ß peptides revealed more premature amyloid-ß plaques in amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy compared with Alzheimer's disease. α-Synuclein seeding amplification assay showed differences in the kinetics in two cases. This study highlights a rare mixed pathology variant of multiple system atrophy in which there is an anatomical meeting point of amyloid-ß and α-synuclein, i.e. the striatum or cerebellum. Since biomarkers are entering clinical practice, these cases will be recognized, and the clinicians have to be informed that the prognosis is not necessarily different than in pure multiple system atrophy cases but that the effect of potential α-synuclein-based therapies might be influenced by the co-presence of amyloid-ß in regions where α-synuclein also aggregates. We propose that mixed pathologies should be interpreted not only based on differences in the clinical phenotype but also on whether protein depositions regionally overlap, potentially leading to a different response to α-synuclein-targeted therapies.

Twelve Years of Drug Prioritization to Help Accelerate Disease Modification Trials in Parkinson's Disease: The International Linked Clinical Trials Initiative.

In 2011, the UK medical research charity Cure Parkinson's set up the international Linked Clinical Trials (iLCT) committee to help expedite the clinical testing of potentially disease modifying therapies for Parkinson's disease (PD). The first committee meeting was held at the Van Andel Institute in Grand Rapids, Michigan in 2012. This group of PD experts has subsequently met annually to assess and prioritize agents that may slow the progression of this neurodegenerative condition, using a systematic approach based on preclinical, epidemiological and, where possible, clinical data. Over the last 12 years, 171 unique agents have been evaluated by the iLCT committee, and there have been 21 completed clinical studies and 20 ongoing trials associated with the initiative. In this review, we briefly outline the iLCT process as well as the clinical development and outcomes of some of the top prioritized agents. We also discuss a few of the lessons that have been learnt, and we conclude with a perspective on what the next decade may bring, including the introduction of multi-arm, multi-stage clinical trial platforms and the possibility of combination therapies for PD.

Evaluating the Effect of Alzheimer's Disease-Related Biomarker Change in Corticobasal Syndrome and Progressive Supranuclear Palsy.

OBJECTIVES: To evaluate the effect of Alzheimer's disease (AD) -related biomarker change on clinical features, brain atrophy and functional connectivity of patients with corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP). METHODS: Data from patients with a clinical diagnosis of CBS, PSP, and AD and healthy controls were obtained from the 4-R-Tauopathy Neuroimaging Initiative 1 and 2, the Alzheimer's Disease Neuroimaging Initiative, and a local cohort from the Toronto Western Hospital. Patients with CBS and PSP were divided into AD-positive (CBS/PSP-AD) and AD-negative (CBS/PSP-noAD) groups based on fluid biomarkers and amyloid PET scans. Cognitive, motor, and depression scores; AD fluid biomarkers (cerebrospinal p-tau, t-tau, and amyloid-beta, and plasma ptau-217); and neuroimaging data (amyloid PET, MRI and fMRI) were collected. Clinical features, whole-brain gray matter volume and functional networks connectivity were compared across groups. RESULTS: Data were analyzed from 87 CBS/PSP-noAD and 23 CBS/PSP-AD, 18 AD, and 30 healthy controls. CBS/PSP-noAD showed worse performance in comparison to CBS/PSP-AD in the PSPRS [mean(SD): 34.8(15.8) vs 23.3(11.6)] and the UPDRS scores [mean(SD): 34.2(17.0) vs 21.8(13.3)]. CBS/PSP-AD demonstrated atrophy in AD signature areas and brainstem, while CBS/PSP-noAD patients displayed atrophy in frontal and temporal areas, globus pallidus, and brainstem compared to healthy controls. The default mode network showed greatest disconnection in CBS/PSP-AD compared with CBS/PSP-no AD and controls. The thalamic network connectivity was most affected in CBS/PSP-noAD. INTERPRETATION: AD biomarker positivity may modulate the clinical presentation of CBS/PSP, with evidence of distinctive structural and functional brain changes associated with the AD pathology/co-pathology. ANN NEUROL 2024;96:99-109.

Ethnic background and distribution of clinical phenotypes in patients with probable progressive supranuclear palsy.

BACKGROUND: Progressive Supranuclear Palsy (PSP) is a sporadic neurodegenerative disease without a clear geographic prevalence. Cohorts studied in the UK and India showed no higher prevalence of atypical parkinsonism in South Asian patients. We describe the ethnic and racial background of PSP patients in the Greater Toronto Area (GTA), Canada. METHODS: A prospective observational study of patients with clinically probable PSP evaluated at the dedicated Rossy PSP program. Demographic and clinical data were collected at baseline including PSP phenotype. Results were compared with the latest demographic information from the greater Toronto area. RESULTS: Of the 197 patients screened, 135 had probable PSP and resided within the GTA. The mean age at visit was 71.1 years, disease duration 4.4 years, and disease severity moderate. Compared to our catchment area, there was a higher proportion of patients with a South Asian origin and a lower proportion of patients from East and Southeastern Asia and Africa. A secondary analysis using population census data limited to individuals greater than 65 confirmed the significantly higher representation of South Asians in our clinic but found no differences for other racial and ethnic origins. CONCLUSION: Evaluation of this Toronto cohort found a greater than expected proportion of affected individuals with South Asian ethnic and racial origin. Despite limitations, our results suggest the possibility of a racial and ethnic predisposition to PSP. Further studies are needed to confirm and to address potential associated risk factors, and genome-environmental interactions.

Using artificial intelligence to identify drugs for repurposing to treat l-DOPA-induced dyskinesia.

Repurposing regulatory agency-approved molecules, with proven safety in humans, is an attractive option for developing new treatments for disease. We identified and assessed the efficacy of 3 drugs predicted by an in silico screen as having the potential to treat l-DOPA-induced dyskinesia (LID) in Parkinson's disease. We analysed ∼1.3 million Medline abstracts using natural language processing and ranked 3539 existing drugs based on predicted ability to reduce LID. 3 drugs from the top 5% of the 3539 candidates; lorcaserin, acamprosate and ganaxolone, were prioritized for preclinical testing based on i) having a novel mechanism of action, ii) having not been previously validated for the treatment of LID, iii) being blood-brain-barrier penetrant and orally bioavailable and iv) being clinical trial ready. We assessed the efficacy of acamprosate, ganaxolone and lorcaserin in a rodent model of l-DOPA-induced hyperactivity, with lorcaserin affording a 58% reduction in rotational asymmetry (P < 0.05) compared to vehicle. Acamprosate and ganaxolone failed to demonstrate efficacy. Lorcaserin, a 5HT2C agonist, was then further tested in MPTP lesioned dyskinetic macaques where it afforded an 82% reduction in LID (P < 0.05), unfortunately accompanied by a significant increase in parkinsonian disability. In conclusion, although our data do not support the repurposing of lorcaserin, acamprosate or ganaxolone per se for LID, we demonstrate value of an in silico approach to identify candidate molecules which, in combination with an in vivo screen, can facilitate clinical development decisions. The present study adds to a growing literature in support of this paradigm shifting approach in the repurposing pipeline.

Food Allergy Reaction Severity and Management in a Diverse Population.

BACKGROUND: Definitive treatment for food allergy reactions including anaphylaxis varies widely by reaction severity and socioeconomic status, but little data exist to characterize the relationship between severity, management, and race and ethnicity. OBJECTIVE: To analyze the differences in reaction severity, epinephrine use, and emergency room (ER) use by race and ethnicity in a large, diverse, food-allergic cohort. METHODS: We analyzed intake data from participants in the Food Allergy Outcomes Related to White and African-American Racial Differences cohort on the history of food allergy reactions, severity of the reactions, and management associated with each reaction. We used descriptive statistics as well as mixed-effects logistic and Poisson models to describe the differences in reaction severity, ER visits, and total lifetime epinephrine use by race and ethnicity. RESULTS: A total of 784 children were included in the analysis: 425 (54.2%) were non-Hispanic White, 282 (36.0%) were non-Hispanic Black, and 77 (9.8%) were Hispanic/Latino. Non-Hispanic Black children had increased odds of more severe reactions (odds ratio, 1.7; 95% CI, 1.2-2.3) and higher odds of going to the ER (odds ratio, 2.8; 95% CI, 1.4-5.4). Both non-Hispanic Black (incidence rate ratio, 0.4; 95% CI, 0.3-0.5) and Hispanic/Latino (incidence rate ratio, 0.3; 95% CI, 0.2-0.5) children had lower rates of total lifetime epinephrine use. CONCLUSIONS: There are significant disparities in the severity and treatment of food allergy reactions by race and ethnicity, resulting in increased ER use and decreased total lifetime epinephrine use. Equipping parents with resources and tools on management of food allergy reactions may result in decreased disparity in access to definitive care.

Medical, surgical, and physical treatments for Parkinson's disease.

Although dopamine replacement therapy remains a core component of Parkinson's disease treatment, the onset of motor fluctuations and dyskinetic movements might require a range of medical and surgical approaches from a multidisciplinary team, and important new approaches in the delivery of dopamine replacement are becoming available. The more challenging, wide range of non-motor symptoms can also have a major impact on the quality of life of a patient with Parkinson's disease, and requires careful multidisciplinary management using evidence-based knowledge, as well as appropriately tailored strategies according to the individual patient's needs. Disease-modifying therapies are urgently needed to prevent the development of the most disabling refractory symptoms, including gait and balance difficulties, cognitive impairment and dementia, and speech and swallowing impairments. In the third paper in this Series, we present the latest evidence supporting the optimal treatment of Parkinson's disease, and describe an expert approach to many aspects of treatment choice where an evidence base is insufficient.

Target Identification of a Class of Pyrazolone Protein Aggregation Inhibitor Therapeutics for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure, and current treatment options are very limited. Previously, we performed a high-throughput screen to identify small molecules that inhibit protein aggregation caused by a mutation in the gene that encodes superoxide dismutase 1 (SOD1), which is responsible for about 25% of familial ALS. This resulted in three hit series of compounds that were optimized over several years to give three compounds that were highly active in a mutant SOD1 ALS model. Here we identify the target of two of the active compounds (6 and 7) with the use of photoaffinity labeling, chemical biology reporters, affinity purification, proteomic analysis, and fluorescent/cellular thermal shift assays. Evidence is provided to demonstrate that these two pyrazolone compounds directly interact with 14-3-3-E and 14-3-3-Q isoforms, which have chaperone activity and are known to interact with mutant SOD1G93A aggregates and become insoluble in the subcellular JUNQ compartment, leading to apoptosis. Because protein aggregation is the hallmark of all neurodegenerative diseases, knowledge of the target compounds that inhibit protein aggregation allows for the design of more effective molecules for the treatment of ALS and possibly other neurodegenerative diseases.