BackgroundParkinson’s disease (PD) is a neurodegenerative condition with progressive phosphorylated alpha-synuclein (P-SYN) deposition. Skin biopsies are a sensitive and specific source of tissue to detect intra-axonal P-SYN in patients with PD. Further, isolated REM sleep behavior disorder (iRBD) is a prodromal condition with a high risk of phenoconversion to PD. The objective of the Synuclein-Quantification (Syn-Q) study is to quantify cutaneous P-SYN across a range of PD severity and in iRBD and measure changes in P-SYN over time.MethodsAfter consent, participants with iRBD and PD across Hoehn and Yahr stages 1, 2, 3 will complete neurological and cognitive evaluation, motor and olfactory assessments, orthostatic vitals, and questionnaires. Skin biopsies (3 mm diameter and 3–4 mm depth) will be taken from distal leg 10 cm above the lateral malleolus, distal thigh 10 cm above the lateral knee, and posterior cervical region 3 cm lateral to the C-7 spinous process with quantitation of cutaneous intra-axonal P-SYN. Participants will return for follow-up visits to repeat study procedures at 6, 12, and 18 months following the baseline visit. This study is funded by the Michael J. Fox Foundation and is registered on Clinicaltrial.org (NCT06621602).Expected resultsWe anticipate that patients with more severe stages of PD will have higher levels of quantitative P-SYN. We also predict that increases in P-SYN over time will be associated with worsening disease severity.DiscussionIn patients with PD, the detection and quantification of P-SYN is a critical step to supporting clinical trials that seek to alter the natural history of disease. Skin biopsies offer a more accessible, repeatable and quantifiable approach to monitoring phosphorylated alpha-synuclein compared to cerebrospinal fluid. This study will define the longitudinal rates of P-SYN change in patients with PD from prodromal to advanced disease.Clinical Trial Registrationhttps://clinicaltrials.gov/study/NCT06621602

Multiple system atrophy is a progressive neurodegenerative disorder characterized by autonomic failure, parkinsonism, and cerebellar ataxia. Phosphorylated alpha-synuclein in skin nerves has emerged as a promising biomarker, but longitudinal studies remain limited. This study evaluated changes in cutaneous alpha-synuclein deposition over time, its association with autonomic dysfunction, and its concordance with seed amplification assay findings. Seventeen participants with probable multiple system atrophy were followed up for 12 months with clinical assessments, skin biopsies from posterior cervical and distal thigh regions, standardized orthostatic vital signs, and cerebrospinal fluid biomarker collection. Phosphorylated alpha-synuclein deposition was quantified using immunofluorescence microscopy and correlated with autonomic measures and seed amplification assay results. Cutaneous alpha-synuclein deposition increased from baseline to the12-month follow-up visit. Total deposition was associated with orthostatic systolic blood pressure drop (p = 0.80, p = 0.0001) and with higher scores on the Orthostatic Hypotension Questionnaire and Composite Autonomic Symptom Score. Posterior cervical deposition showed stronger autonomic associations than distal thigh deposition. No associations were found with global disease severity or neurofilament light chain levels. Two participants with negative seed amplification assay results showed phosphorylated alpha-synuclein on skin biopsy. In this cohort, cutaneous phosphorylated alpha-synuclein increased over time and correlated with objective orthostatic hypotension and autonomic symptom burden in early synucleinopathy. Skin biopsy may serve as a minimally invasive tool to support diagnosis, monitor progression, and stratify clinical trial participants.

IntroductionThe neurodegenerative disorders characterized by deposition of phosphorylated alpha-synuclein (P-SYN) include Parkinson’s disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Diagnosis of synucleinopathies in clinical practice has been enhanced by recent updates to diagnostic guidelines. Despite these advances, many patients still fall between diagnostic guideline criteria creating challenges to clinical care.MethodsThis was a retrospective chart review of patients seen in an outpatient private practice. The objective of this study was to determine the impact of skin biopsy detection of phosphorylated alpha-synuclein (P-SYN) in the clinical care of patients with movement or cognitive symptoms in whom a synucleinopathy was part of the differential diagnosis. The clinical diagnosis and treatment plan before, and after, skin biopsy detection for P-SYN was determined through detailed chart extraction, with ICD-10 codes used to define clinical diagnosis.ResultsOne hundred patients with suspected neurodegenerative disease were included in the chart review, 47 presenting with tremor, 18 with gait dysfunction, 11 with rigidity or bradykinesia and 24 with cognitive dysfunction. After skin biopsy testing for P-SYN, 60% of patients had a change in their ICD-10 documented diagnosis and 91% had a change to their clinical treatment plan.ConclusionIn patients with suspected neurodegenerative disease, skin biopsy detection of P-SYN supported changes in the diagnostic impression or management in the majority of patients in this study. These findings support the growing role of diagnostic biomarkers in the diagnosis and management of patients with suspected neurodegenerative synucleinopathies.

Abstract Introduction Isolated REM sleep behavior disorder (iRBD) is a prodromal neurodegenerative disease characterized by dream reenactment and REM sleep atonia. Pathologically, there is deposition of phosphorylated alpha-synuclein (P-SYN) within the central and peripheral nervous system of patients with iRBD. There is a high risk of phenoconversion from iRBD to a clinically apparent synucleinopathy (Parkinson’s disease (PD), multiple system atrophy (MSA), or dementia with Lewy bodies (DLB)), with >73% of patients phenoconverting over 12 years. We sought: 1) To determine rates of P-SYN deposition in patients with iRBD, 2) To quantify changes in P-SYN deposition over time, and 3) To determine if patterns of P-SYN deposition predict phenoconversion to a specific synucleinopathy subtype. Methods Patients with iRBD (confirmed by polysomnography or validated questionnaire and history) were included in this prospective, blinded multicenter study. Detailed examinations, orthostatic vital signs, and questionnaires were completed. Medical history, ancillary testing, and polysomnograms (PSG) were reviewed, if applicable. Skin biopsies at the distal leg, distal thigh, and posterior cervical sites were collected. Dual immunohistochemical immunostaining for nerve fibers (protein gene product 9.5) and P-SYN were completed with blinded pathological assessment. Results 80 subjects (31% female, age 67.8±8.7 years) were enrolled. P-SYN was detected in 75% (60/80) of subjects at baseline. In a preliminary analysis of the first 49 patients to complete 12-month follow-up, the average P-SYN composite score at baseline was 4.06±4.51, and at the 12-month follow-up was 5.53±5.38 (p>0.05). Four patients that were P-SYN negative at baseline became positive at 12 months. All individuals initially positive for P-SYN remained positive at 12 months. No complications were noted in the biopsy procedure. Conclusion Skin biopsy is a safe and highly sensitive method for detection of P-SYN in prodromal disease. Our quantitative analysis of P-SYN revealed a 36% increase over 12 months. Ongoing longitudinal follow up will determine if P-SYN deposition patterns can predict both time to phenoconversion and synucleinopathy subtype. Support (if any) Funded through an NIH SBIR Grant: R44NS127696

BACKGROUND:Idiopathic rapid eye movement sleep behavior disorder (iRBD) is a prodromal neurodegenerative disease of misfolded alpha-synuclein (P-SYN) with a high risk of phenoconversion to a clinically apparent synucleinopathy (including Parkinson's disease, multiple system atrophy, or dementia with Lewy bodies) over 15 years. OBJECTIVES:To determine rates of cutaneous P-SYN deposition in iRBD, to quantify changes in PSYN deposition over time, and to determine if P-SYN deposition patterns and amounts predict phenoconversion to a specific type of synucleinopathy. CLINICAL TRIAL PROTOCOL:In a prospective, blinded study we will recruit 80 individuals with polysomnography confirmed iRBD or probable RBD using standard diagnostic criteria. Skin biopsies with dual immunohistochemical immunostaining for nerve fibers (protein gene product 9.5) and P-SYN will be completed at 3 sites using standard methodology. Quantitative measures of P-SYN and nerve fiber density will be measured blinded to any clinical data and will be followed longitudinally to determine the final clinical diagnosis. DISCUSSION:Patients with iRBD are an important population to study due to the high rates of phenoconversion to clinically apparent synucleinopathy. Defining the frequency of P-SYN deposition and the risk of phenoconversion will aid in the development of future clinical trials that seek to alter the natural history of synucleinopathies.

OBJECTIVE:To determine the test performance of cutaneous phosphorylated alpha-synuclein (P-SYN) in dementia with Lewy bodies (DLB), individuals with reduced Montreal Cognitive Assessment (MoCA) and healthy controls. METHODS:This is the first subgroup analysis of the Synuclein-One study, a prospective, blinded study evaluating P-SYN detection from skin biopsies in 218 subjects with a referral diagnosis of control (N = 151) and DLB (N = 67). All subjects completed detailed examinations, questionnaires, and had skin biopsies for detection of P-SYN. DLB patients were included if meeting the 4th DLB consensus probable criteria. Control subjects, aged 40-99, had no history, examination findings, or symptoms suggestive of a synucleinopathy or neurodegenerative disease. An expert review panel, blinded to pathological data, determined the final diagnosis. Controls with reduced MoCA (MoCA < 26, N = 26) at screening were analyzed separately. RESULTS:After expert panel review, only 50/67 patients met consensus criteria for DLB, 26/151 controls had a reduced MoCA, and 120/151 controls had a normal MoCA. The proportions of subjects with cutaneous P-SYN detected by skin biopsy were 96.0% (48 of 50) of the DLB group, 31% (8 of 26) of the controls with reduced MoCA, and 3.3% (4 of 120) of the controls with normal MoCA. INTERPRETATION:In this prospective, blinded, cross-sectional study, a high proportion of subjects meeting clinical consensus criteria for DLB had P-SYN detected in skin biopsies. Almost 1/3 of subjects with reduced MoCA testing also had P-SYN detected. These results support a role for skin biopsy detection of P-SYN in patients with DLB. TRIAL REGISTRATION:NCT04700722.

To develop a novel, continuous, quantitative method for analysis of cutaneous phosphorylated alpha-synuclein (P-SYN).

BACKGROUND:Skin biopsies have >95% sensitivity and specificity to detect the presence of phosphorylated alpha-synuclein (P-SYN). OBJECTIVE:To determine the frequency of cutaneous P-SYN in patients with mild cognitive impairment (MCI) due to suspected Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). DESIGN, SETTING AND PARTICIPANTS:The Syn-D study is a multicenter prospective clinical trial from ~10 centers across the United States that includes patients with MCI-AD or MCI-DLB. METHODS:Patients will undergo skin biopsies for detection of P-SYN and plasma biomarkers for ptau-217 to determine biomarker positivity rates at the MCI stage and will be followed longitudinally to determine final clinical diagnosis as defined by an expert panel of clinicians blinded to biomarker results. MAIN OUTCOMES AND MEASURES:The co-primary outcomes include: 1) The sensitivity of skin biopsy detection of P-SYN in patients with clinical diagnoses of MCI due to DLB or AD at baseline and at 12-month follow-up 2) To report the frequency with which cutaneous deposition of P-SYN and blood-based AD biomarkers co-exist in a population of patients with MCI. DISCUSSION:This will be the first in-vivo study to determine the co-existence of biomarkers for both diseases as a surrogate for co-pathology. TRIAL REGISTRATION:NCT05479552.

IntroductionThe diagnosis of diseases known as synucleinopathies, Parkinson’s disease (PD), multiple system atrophy (MSA) and Lewy body dementia (DLB), is predominantly based on clinical criteria. However, diagnostic uncertainty may persist until late in the disease process leading to delays in diagnosis and medical mismanagement. Skin biopsy detection of phosphorylated alpha-synuclein (P-SYN) is a sensitive and specific technique that increases diagnostic sensitivity of synucleinopathies, although the clinical utility of this test has not been fully explored.MethodsTo determine the role of skin biopsy in the diagnosis of synucleinopathies we performed a retrospective chart review of patients who underwent skin biopsy for detection of P-SYN in the evaluation of neurodegenerative disease at a tertiary care academic institution to investigate the change in diagnosis and medical management based on the results of skin biopsy detection of P-SYN.ResultsWe included 97 patients suspected to have a synucleinopathy: 54 with PD, 19 with DLB and 24 with MSA. After skin biopsy testing for P-SYN, 78% of patients had a change in their clinical care with 66% having a change in their diagnosis and 55% having a change in their treatment. Changes in diagnosis were most common in patients with parkinsonism with prominent action tremor (93%), lower-extremity predominant parkinsonism (postural instability and gait dysfunction) (90%), and parkinsonism with predominant cognitive dysfunction (76%).DiscussionIn patients with suspected synucleinopathies, skin biopsy detection of P-SYN had a high level of clinical utility leading to changes in clinical diagnosis and treatment.

To describe results of skin biopsy for phosphorylated alpha-synuclein (P-SYN) in patients with essential tremor (ET) with parkinsonism.

Importance:Finding a reliable diagnostic biomarker for the disorders collectively known as synucleinopathies (Parkinson disease [PD], dementia with Lewy bodies [DLB], multiple system atrophy [MSA], and pure autonomic failure [PAF]) is an urgent unmet need. Immunohistochemical detection of cutaneous phosphorylated α-synuclein may be a sensitive and specific clinical test for the diagnosis of synucleinopathies.Objective:To evaluate the positivity rate of cutaneous α-synuclein deposition in patients with PD, DLB, MSA, and PAF.Design, Setting, and Participants:This blinded, 30-site, cross-sectional study of academic and community-based neurology practices conducted from February 2021 through March 2023 included patients aged 40 to 99 years with a clinical diagnosis of PD, DLB, MSA, or PAF based on clinical consensus criteria and confirmed by an expert review panel and control participants aged 40 to 99 years with no history of examination findings or symptoms suggestive of a synucleinopathy or neurodegenerative disease. All participants completed detailed neurologic examinations and disease-specific questionnaires and underwent skin biopsy for detection of phosphorylated α-synuclein. An expert review panel blinded to pathologic data determined the final participant diagnosis.Exposure:Skin biopsy for detection of phosphorylated α-synuclein.Main Outcomes:Rates of detection of cutaneous α-synuclein in patients with PD, MSA, DLB, and PAF and controls without synucleinopathy.Results:Of 428 enrolled participants, 343 were included in the primary analysis (mean [SD] age, 69.5 [9.1] years; 175 [51.0%] male); 223 met the consensus criteria for a synucleinopathy and 120 met criteria as controls after expert panel review. The proportions of individuals with cutaneous phosphorylated α-synuclein detected by skin biopsy were 92.7% (89 of 96) with PD, 98.2% (54 of 55) with MSA, 96.0% (48 of 50) with DLB, and 100% (22 of 22) with PAF; 3.3% (4 of 120) of controls had cutaneous phosphorylated α-synuclein detected.Conclusions and Relevance:In this cross-sectional study, a high proportion of individuals meeting clinical consensus criteria for PD, DLB, MSA, and PAF had phosphorylated α-synuclein detected by skin biopsy. Further research is needed in unselected clinical populations to externally validate the findings and fully characterize the potential role of skin biopsy detection of phosphorylated α-synuclein in clinical care.

We provide an initial description and validation of some public domain patient-reported outcome (PRO) items to assess cancer symptom burden to address immediate barriers to symptom assessment use in clinical practice and facilitate future research. We created the Open Symptom Framework (OSF), a flexible tool for clinical cancer-related symptom assessment. The items comprise six components: recall period, concept, symptom, qualifier(s), a definition, and a 5-point Likert-type response. We recruited patients receiving cancer therapy in the United States and United Kingdom. We assessed external construct validity by comparing OSF scores to the PRO-CTCAE measure and assessed reliability, scalability, dimensionality, and item ordering within a non-parametric item response theory framework. We tested differential item functioning for country, age, gender, and level of education. We developed a framework alongside clinical and psychometric experts and debrieifed with 10 patients. For validation, we recruited 331patients. All items correlated with the PRO-CTCAE equivalents (r = 0.55–0.96, all p < 0.01). Mokken analysis confirmed the scalability and unidimensionality of all symptom scales with multiple items at the scale (Ho = 0.61–0.75) and item level (Hi = 0.60–0.76). Items are interpreted consistently between demographic groups (Crit = 0 for all groups). The public domain OSF has excellent psychometric properties including face, content, and criterion validity and can facilitate the development of flexible, robust measurements to fulfil stakeholder need. The OSF was designed specifically to support clinical assessment but will function well for research. Further work is planned to increase the number of symptoms and number of questions per symptom within the framework.

We aimed to describe the clinical features of patients with pure autonomic failure (PAF) preceding phenoconversion that could be useful as predictive markers for advancing alpha-synuclein-associated neurodegeneration of the brain.Patients diagnosed with PAF were evaluated at eight centres (seven US-based and one European) and enrolled in a longitudinal observational cohort study (NCT01799915). Subjects underwent detailed assessments of motor, sleep, olfactory, cognitive and autonomic function and were followed prospectively to determine whether they developed parkinsonism or dementia for up to 10 years. We identified incident cases of Parkinson's disease (PD), dementia with Lewy bodies (DLB) or multiple system atrophy (MSA) and computed hazard ratios for phenoconversion as functions of clinical features.A total of 209 participants with PAF with a median disease duration of 6 years (IQR: 3-10) were enrolled. Of those, 149 provided follow-up information at an office or telemedicine visit. After a mean follow-up duration of 3 years, 48 (33%) participants phenoconverted (42% to PD, 35% to DLB and 23% to MSA). Faster phenoconversion from study enrolment to any diagnosis was associated with urinary and sexual dysfunction [hazard ratio (HR) 5.9, 95% confidence interval (CI): 1.6-22 and HR: 3.6, 95% CI: 1.1-12] followed by subtle motor signs (HR: 2.7, 95% CI: 1.2-6), trouble swallowing (HR 2.5, 95% CI: 1.4-4.5) and changes in speech (HR:2.4, 95% CI:1.1-4.8) at enrolment. Subjects reporting deterioration of handwriting were more likely to phenoconvert to PD (HR: 2.6, 95% CI: 1.1-5.9) and those reporting difficulty handling utensils were more likely to phenoconvert to DLB (HR: 6.8, 95% CI: 1.2-38). Patients with a younger age of PAF onset (HR: 11, 95% CI: 2.6-46), preserved olfaction (HR: 8.7, 95% CI: 1.7-45), anhidrosis (HR: 1.8, 95% CI: 1-3.1, P = 0.042) and severe urinary problems (HR 1.6, 95% CI: 1-2.5, P = 0.033) were more likely to phenoconvert to MSA. The best autonomic predictor of PD was a blunted heart rate increase during the tilt-table test (HR: 6.1, 95% CI: 1.4-26).Patients with PAF have an estimated 12% (95% CI: 9-15%) per year annual risk following study entry of phenoconverting to a manifest CNS synucleinopathy. Millar Vernetti et al. describe the clinical features of patients with pure autonomic failure that are associated with the highest risk of imminent phenoconversion to Parkinson's disease, multiple system atrophy, and Lewy body dementia. Specific markers of autonomic, olfactory, cognitive, and motor function can help in predicting the final diagnosis.

The Synuclein-One study is an NIH-funded 30-site trial that included 33 participants with pure autonomic failure (PAF). We report the detection and quantitation of cutaneous phosphorylated alpha-synuclein (P-SYN) in patients with suspected PAF.

Background: ATTR (ATTRv) amyloidosis neuropathy is characterized by progressive sensorimotor and autonomic nerve degeneration secondary to amyloid deposition caused by a misfolded transthyretin protein (TTR). Small nerve fiber neuropathy is an early clinical manifestation of this disease resulting from the dysfunction of the A delta and C small nerve fibers. Tafamidis, a selective TTR stabilizer, has proven its efficacy in the earlier stages of hATTR. Objectives: To evaluate the clinical course and utility of cutaneous pathological biomarkers in patients with ATTR amyloidosis treated with tafamidis compared to control patients. Methods: Forty patients diagnosed with early stages of ATTRv amyloidosis (polyneuropathy disability [PND] scores 0-II) underwent small and large nerve fiber neurological evaluations, and annual skin biopsies for intraepidermal nerve fiber density (IENFD) and amyloid deposition index (ADI) estimation. Thirty patients were allocated to receive tafamidis, and 10 patients served as controls. Tafamidis pharmacokinetics analysis was performed in patients who received the treatment. Results: At baseline, 12% of patients in stage PND 0 and 28% in PND I displayed small nerve fiber denervation in the distal thigh, whereas 23% and 38%, respectively, in the distal leg. Similarly, 72% and 84% had amyloid deposition in the distal thigh and 56% and 69% in the distal leg. Following 1 year of treatment, the tafamidis group showed significant clinical improvement compared to the control group, revealed by the following mean differences (1) -9.3 versus -4 points (p = <.00) in the patient's neuropathy total symptom score 6 (NTSS-6) questionnaire, (2) -2.5 versus +2.8 points (p = <.00) in the Utah Early Neuropathy Score (UENS), and (3) +1.2 degrees C versus -0.6 (p = .01) in cold detection thresholds. Among the patients who received tafamidis, 65% had stable or increased IENFD in their distal thigh and 27% in the distal leg. In contrast, all patients in the control group underwent denervation. The ADI either decreased or remained constant in 31% of the biopsies in the distal thigh and in 24% of the biopsies in the distal leg of the tafamidis-treated patients, whereas it rose across all the biopsies in the control group. At the 4-year follow-up, the tafamidis group continued to display less denervation in the distal thigh (mean difference [MD] of -3.0 vs. -9.3 fibers/mm) and the distal leg (mean difference [MD] -4.9 vs. -8.6 fibers/mm). ADI in tafamidis-treated patients was also lower in the distal thigh (10 vs. 30 amyloid/mm2) and the distal leg (23 vs. 40 amyloid/mm2) compared to control patients. Plasma tafamidis concentrations were higher in patients with IENFD improvement and in patients with reduced amyloid deposition. Patients without amyloid deposition in the distal leg at baseline displayed delayed disease progression at 4 years. Conclusions: Cutaneous IENFD and amyloid deposition assessments in the skin of the distal thigh and distal leg are valuable biomarkers for early diagnosis of ATTR amyloidosis and for measuring the progression of small nerve fiber neuropathy. Early treatment with tafamidis slows the clinical progression of the disease, skin denervation, and amyloid deposition in the skin. Higher plasma concentrations of tafamidis are associated with better disease outcomes, suggesting that increasing the drug dose could achieve better plasma concentrations and response rates. This study describes the longest small nerve fiber neuropathy therapeutic trial with tafamidis and is the first to report small fiber symptoms, function, and structural assessments as outcomes.