George T. Kannarkat

Alpha–Synuclein (α–syn) aggregation is central to Parkinson′s disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn–IMAGR, a single–molecule imaging platform with sub–femtomolar sensitivity, we show that purified α–syn aggregates undergo dilution–induced disassembly, revealing a concentration–dependent equilibrium. Applied to postmortem brain lysates, Syn–IMAGR distinguishes physiological α–syn multimers, which are dimmer and readily dissociate upon dilution, from PD–associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that α–syn assemblies occupy distinct stability regimes, with PD–associated aggregates representing a more persistent and less dilution–sensitive structural state. Syn–IMAGR thus provides a quantitative framework for resolving α–syn species and for probing their concentration–dependent equilibrium.

The development of biomarkers capable of reliably detecting pathologic forms of α-synuclein (aSyn) in vivo marks a significant step forward in the field of neurodegeneration. Over the recent years, CSF aSyn seed amplification assays (aSyn-SAA) and skin biopsy phospho-aSyn immunofluorescence (skin aSyn-IF) testing were developed that detect pathologic aSyn seeds and phosphorylated aSyn aggregates, respectively, in patients with synucleinopathies, which include Parkinson disease, dementia with Lewy bodies, and also multiple system atrophy. High rates of positivity have also been documented in research participants at a risk of developing future aSyn-related disease (e.g., hyposmia and REM sleep behavior disorder), as well as other contexts. These assays have numerous potential applications in research settings and clinical care. Here, we review the currently published evidence supporting CSF aSyn-SAA and skin aSyn-IF testing and discuss their potential research applications in clinical trials. As CSF aSyn-SAA and skin aSyn-IF testing is now commercially available to clinicians in nonresearch settings, we also explore their current utility and limitations as diagnostic tools and call for the development of a formal clinical use guidelines. We also highlight where critical knowledge gaps remain and explore emerging developments related to these assays.

: Spread and aggregation of misfolded α-synuclein (aSyn) within the brain is the pathologic hallmark of Lewy body diseases (LBD), including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). While evidence exists for multiple aSyn protein conformations, often termed “strains” for their distinct biological properties, it is unclear whether PD and DLB result from aSyn strain differences, and biomarkers that differentiate PD and DLB are lacking. Moreover, while pathological forms of aSyn have been detected outside the brain ( e.g., in skin, gut, blood), the functional significance of these peripheral aSyn species is unclear. Here, we developed assays using monoclonal antibodies selective for two different aSyn species generated in vitro – termed Strain A and Strain B – and used them to evaluate human brain tissue, cerebrospinal fluid (CSF), and plasma, through immunohistochemistry, enzyme-linked immunoassay, and immunoblotting. Surprisingly, we found that plasma aSyn species detected by these antibodies differentiated individuals with PD vs. DLB in a discovery cohort (UPenn, n=235, AUC 0.83) and a multi-site replication cohort (Parkinson’s Disease Biomarker Program, or PDBP, n=200, AUC 0.72). aSyn plasma species detected by the Strain A antibody also predicted rate of cognitive decline in PD. We found no evidence for aSyn strains in CSF, and ability to template aSyn fibrillization differed for species isolated from plasma vs. brain, and in PD vs. DLB. Taken together, our findings suggest that aSyn conformational differences may impact clinical presentation and cortical spread of pathological aSyn. Moreover, the enrichment of these aSyn strains in plasma implicates a non-central nervous system source.

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. As EVs represent a way of capturing molecular information about the proteins inside of cells, EVs from biofluids could be used to better understand and diagnose disease from difficult to access organs such as the brain. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs isolated from the blood. However, the measurement of cargo proteins in EVs has been challenging because EVs are present at low levels and EV isolation methods are imperfect at separating EVs from free proteins. Thus, it is difficult to know whether a protein measured after EV isolation is truly inside EVs. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size exclusion chromatography (SEC) protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital ELISA assays for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze key proteins involved in neurodegenerative diseases: α-synuclein, Tau, Aβ40, and Aβ42. We found that α-synuclein and Tau are present in plasma EVs at a small fraction of the levels in total plasma, whereas Aβ40 and Aβ42 are undetectable in plasma EVs. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Parkinson disease (PD) is the second-most common neurodegenerative disease worldwide. The gold standard diagnosis is the presence of bradykinesia plus tremor or muscular rigidity along with symptomatic improvement when taking levodopa, a precursor to the neurotransmitter dopamine. The classic, yet not universal, pathologic hallmark of PD is the loss of dopamine-producing neurons in the midbrain associated with Lewy bodies, intraneuronal aggregates of misfolded alpha-synuclein (aSyn). While advanced imaging techniques to measure dopamine transporter binding can support the diagnosis of PD, these tests are nonspecific and can be abnormal in other disorders of dopamine dysfunction unrelated to neuronal aSyn pathology. Despite recognition that clinical symptoms, like hyposmia and rapid eye movement sleep behavior disorder (RBD), and certain gene variants, including GBA or LRRK2, predict increased risk of PD development, aSyn-specific biomarkers measurable during life have remained elusive until recently. Adapted from the diagnosis of prion diseases, seed amplification assays (SAA), including real time quaking-induced conversion based on serial shaking or protein misfolding cyclic amplification based on serial sonication, show promise as tests for an in vivo aSyn biomarker ( 1).

The last decade has seen development of numerous novel antipsychotic drugs with unique mechanisms including long-acting formulations for clinical use. A comparative assessment of these new drugs with each other and previous antipsychotics have not been performed with regards to risk for drug-induced movement disorders (DIMD). Medline was searched from January 2010 to February 2022 for primary research articles and review articles in English using the search terms “extrapyramidal” and “tardive” with individual drug names of novel antipsychotics. We identified articles describing the risk of DIMD with 6 novel antipsychotics, 4 novel formulations, and 3 experimental antipsychotics. Both short- and long-term data generally showed comparable to lower risk of DIMD with novel antipsychotics and recent long-acting formulations compared to previously marketed antipsychotics. Several novel antipsychotics, particularly lumateperone and pimavanserin, show promise in being able to treat psychosis while reducing the risk of DIMD. Long-acting paliperidone may reduce risk of DIMD while other long-acting injectable formulations of SGA have similar risk of DIMD compared to oral formulations. New drug targets for treating psychosis without dopamine blockade also show promise.

Background: Exercise and physical therapy (PT) can improve motor function and quality of life in individuals with Parkinson's disease (PD), but their role in hospitalization avoidance is not well‐studied.Objectives: To determine the longitudinal and temporal association of exercise and PT use with hospital encounter.Methods: Longitudinal regression and χ² analyses were performed on Parkinson's Foundation Parkinson's Outcome Project exercise and PT use data from 4674 and 9259 persons with PD, respectively.Results: Greater exercise duration and intensity were associated with reduced odds of hospital encounter, whereas both PT and occupational therapy use were associated with increased odds. In the 2 years before a hospital encounter, there was an increased frequency of PT use, but not reductions in exercise.Conclusions: Consistent exercise may reduce hospitalization risk whereas PT referral may identify at‐risk individuals without preventing this outcome. Further work to incentivize consistent exercise in PD may reduce healthcare use.