CTR056041 Supplier Grant Application Mayo_Adler.pdf

TABLE OF CONTENTS

Page Number General Information

Review Purposes

Project Abstract

Detailed Project Description

Biographical Sketch(es)

Budget Information

Certifications and Assurances

Attachment C - Grant Offer and Acceptance form shall be completed and signed by authorized representative

Attachment D - Notices, Correspondence, and Payments to the Grantee shall be completed in its entirety

Attachment E - Designation of Confidential Trade Secret & Proprietary Information shall be completed and signed by authorized representative

Attachment F - Participation of Boycott of Israel form shall be completed and signed by authorized representative

Other Attachments – W-9

APPLICATION PACKET

ATTACHMENT B

RFGA NO. BPM000334

General Information

Information about the Applicant organization or individual and the person to contact for any questions during the review process should be clearly listed.

1. Provide the following contact information for the person who is responsible for answering questions related to this Application:

Name Laurie Winslow

Title Sponsored Projects Specialist

Mailing Address 13400 East Shea Boulevard, Scottsdale, AZ 85259

Phone Number 480-301-6734

Email Address Winslow.laurie@mayo.edu

2. Legal name and address of Applicant:

Name Mayo Clinic Arizona

Mailing Address 13400 East Shea Boulevard, Scottsdale, AZ 85259

W-9 Attached See attached

3. Name of Principal Investigator:

Name Charles Adler

Email Address cadler@mayo.edu

Date of terminal degree or completion of medical residency

4. Title of Project

Title Submandibular gland needle core biopsy as a tissue biomarker the diagnosis of Parkinson’s disease and the monitoring of disease progression

5. Total Fund Request for Project Budget Period (identify dollar amount per year):

Year 1 249,048

Year 2 193,665

Year 3 249,608

Total Requested $692,321

Charles Adler, MD, PhD, General Information, RFGA Number BPM000334 1

RFGA NO. BPM000334

REVIEW PURPOSES

1. What field would you like your grant reviewed under? Neuroscience, Neurological, Mental, and Behavioral Diseases and Disorders

1.1. If “Other,” please specify:

Click here to enter text.

2. What is the specialized area of proposed research? Click here to enter text.

We will be studying Parkinson’s disease and REM sleep behavior disorder.

3. Does your proposed research/project include human subjects? Yes

3.1. Is your use of human subjects research exempt? No

3.1.1. If yes, please describe:

4. Is your research an NIH defined clinical trial? No

4.1. If yes, please describe:

5. Does the research involve Vertebrate Animals? No

5.1. If yes, please attach IACUC Approval Animal Welfare Assurance Number.

Charles Adler, MD, PhD, Review Purposes, RFGA Number BPM000334 2

RFGA NO. BPM000334

PROJECT ABSTRACT

All Applicants are required to submit an abstract of the proposed activity suitable for publication. The abstract should be informative to other persons working in the same or related fields and, insofar as possible, understandable to a scientifically or technically literate lay reader. The abstract shall not include any proprietary or sensitive business information as it may be made available to the public.

The abstract shall be within 1,400 characters in length.

Background: We will build on our translational research studying peripheral alpha-synuclein (a-syn) in the submandibular gland (SMG) of patients with Parkinson’s disease (PD) and extend the work to REM sleep behavior disorder (RBD), a prodromal stage of PD. SMG and brain a-syn density correlate but it is unknown if density increases over time.

Research Problem: Determine 1) the magnitude of SMG a-syn density change over time, 2) the improvement in adequate tissue acquisition doing bilateral biopsies, and 3) any laterality differences between glands.

Methods: Perform bilateral SMG needle core biopsies on 30 clinically diagnosed PD and 10 clinically probable RBD. All will have clinical examinations and bilateral SMG biopsies performed at Mayo Clinic Arizona with tissue sent to Banner Sun Health Research Institute for histological staining and evaluation. All subjects will have a second assessment and biopsy 2 years later.

Projected Outcomes: We expect to find an increase in a-syn density over time, improved adequate tissue acquisition, and similar left-right a-syn density.

Project Impact: If SMG a-syn density increases over time this could serve as a tissue biomarker for clinical studies that target a-syn aggregation. We would have powerful preliminary data for planning trials of PD disease-modifying agents and will seek funding and industry partners for these trials in Arizona.

Charles Adler, MD, PhD, Project Abstract, RFGA Number BPM000334 3

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 4

Goals and Objectives Specific Aim 1. Determine, with bilateral submandibular gland (SMG) biopsies in 30 clinically diagnosed subjects with Parkinson’s disease (PD) and 20 non-PD parkinsonian subjects, whether bilateral biopsies improve the rate of acquisition of adequate glandular tissue, whether alpha-synuclein (a-syn) SMG pathology correlates with clinical severity and/or disease duration, whether there is a left-right difference in the density of a-syn pathology, and whether a-syn SMG pathology is specific for PD. This aim will address four important issues. First, a limiting factor with SMG biopsies from multiple centers has been obtaining adequate glandular tissue from unilateral biopsies (~80% at our center).

Bilateral biopsies would theoretically improve this given two chances to obtain adequate tissue. Unpublished data from 7 bilaterally-biopsied PD subjects showed all 7 had adequate glandular tissue from at least one of the two biopsied glands. Additionally, since a-syn pathology may be patchy within the SMG and other peripheral tissue, obtaining bilateral biopsies will improve the sample size and hence reduce variability in density scores. Second, as some subjects will only have adequate tissue from one side, and only one side might have a-syn pathology, establishing whether the density of a-syn pathology is equivalent and/or present bilaterally is important for longitudinal density studies. Third, it is important to determine whether disease duration and/or the clinical severity correlate with a-syn pathology in the SMG. Fourth, it is important to be sure that a-syn pathology is specific for PD.

Hypothesis 1: Performing bilateral SMG biopsies will improve the rate of adequate SMG tissue for individual subjects providing sufficient tissue from more than 90% of subjects.

Hypothesis 2: The density of a-syn pathology will be similar between the left and right submandibular glands.

Hypothesis 3: A-syn density will correlate with PD disease duration and disease severity.

Hypothesis 4: A-syn will not be present is subjects with non-PD parkinsonian disorders.

Specific Aim 2. Determine, with a second SMG biopsy in previously biopsied PD subjects, whether a-syn pathology density increases, decreases, or remains the same after a two year time interval and whether there is a correlation between a-syn SMG pathology and clinical and/or disease progression.

The natural history of peripheral a-syn pathology is unknown. Pilot data from the 7 having had bilateral biopsies noted above, suggests that a-syn density increases a mean of 4 yrs after initial biopsy. But, the increase was only found in 5 subjects, 1 had no change and 1 had a decrease in density. It has been postulated, based on an autopsied cardiac tissue study, that a-syn density might decrease over time due to nerve fiber degeneration. SMG a-syn pathology would be useful as a molecular clinical trial measure if it increased over time, and less specific it it remained static over time, although even then a-syn pathology could be a useful marker of target engagement. All subjects that received bilateral biopsies in Specific Aim 1 will have repeat bilateral biopsies after a 2 year time interval. This will determine any a-syn density change over time and provide useful data for calculating effect size and required subject numbers for clinical trials of a disease-modifying agent. Additionally, changes in a-syn density will be correlated to changes in clinical measures of disease severity and duration, indicating whether progression of SMG a-syn pathology would be a potential biomarker of disease progression.

Hypothesis 5: A-syn pathology will increase by at least 50% over a two year time period.

Hypothesis 6: Change in SMG a-syn density will correlate with clinical change over time.

Specific Aim 3. Determine in subjects with idiopathic REM sleep behavior disorder (RBD), whether there are changes in SMG a-syn density over time and whether there is symmetry in a-syn between right-left SMGs. Only one study of SMG biopsies in RBD has been published, but it has not been replicated, and there is no longitudinal study of SMG a-syn in RBD. As RBD is considered to be prodromal PD with up to 65% of subjects converting to PD within a few year, we will perform bilateral biopsies on 10 clinically probable RBD cases in year 1 and then repeat the biopsies in year 3.

Hypothesis 7: The density of a-syn pathology will be similar between the left and right SMGs.

Hypothesis 8: A-syn density will correlate with RBD disease duration.

Hypothesis 9: There will be an increase in SMG a-syn density over a 2 year time period.

Overall Goals and Objectives. If performing bilateral SMG biopsies increases adequate tissue acquisition, and a-syn pathology increases or remains static over time then SMG biopsies would potentially be a transformative event in the search for a tissue biomarker of disease progression and/or target engagement, and could be used as an outcome marker for anti-synuclein therapeutic trials in PD and RBD. Additionally, a tissue diagnostic test for a-syn pathology in living PD and RBD patients could 1) serve to enrich clinical trials by entering confirmed subjects with a synucleinopathy, 2) become a surrogate for autopsy as a gold-

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 5 standard diagnostic validation of other biomarkers, and 3) could confirm diagnosis in patients being considered for invasive treatments such as deep brain stimulation, focused ultrasound, neural cell transplant, stem cell transplant, or gene therapy. As disease-modifying treatments for PD likely need to be started well before motor parkinsonism begins, better methods for identifying at-risk individuals, such as RBD, is needed.

Background Information and Literature Review

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that impacts patient quality of life, causes disability, and is a huge financial burden to our health care system. PD is a disease of aging.

Approximately 2% of people >65 yrs old have PD and current estimates are there are ~25,000 PD patients in AZ now (~18,000 >65) and by 2050 there will be ~80,000 (~50,000 >65). The US economic burden of PD is currently estimated at ~$25 billion/yr with medication costs alone ~$2,500/year/patient. Research to find a cure is hampered by 1) the ability to adequately identify individuals at-risk for developing PD, so called prodromal PD, 2) the lack of a diagnostic test for PD, and 3) the lack of a tissue biomarker of disease progression. Our group has been leading the research on finding a tissue biomarker.1-4

Clinical trials for disease-modifying agents for PD have been critically hampered by the lack of an in vivo method to confirm the presence of alpha-synuclein (a-syn) pathology, monitor its progression, and measure its response to therapeutic agents. Making an accurate diagnosis of PD is critical for patient care as well as research related to epidemiology, genetics, imaging, biomarker discovery, and both symptomatic and disease-modifying treatments. Methods for diagnosing PD are limited by the lack of a tissue diagnostic test or other definitive biomarker test. Current PD diagnostic criteria use the non-specific clinical findings of rest tremor, rigidity, and bradykinesia, but the gold standard remains neuropathological confirmation. A major goal to improve diagnostic accuracy in living patients has been to find a blood, cerebrospinal fluid (CSF), or tissue biomarker for PD. One inherent complicating factor for finding a biomarker in living PD patients is the potential inaccuracy of a clinical rather than autopsy-confirmed diagnosis.

Numerous studies report that neuropathological confirmation of a clinical diagnosis of PD may range from 53%-93% depending on the criteria used and the stage of disease. We have recently summarized the work on low diagnostic accuracy5 and a meta-analysis6 has also been published. It is important to note that we are not describing the diagnostic accuracy for patients followed for PD for many yrs, but rather the poor diagnostic accuracy for PD at the first visit.7 This is critical for early intervention studies, especially those needed to assess disease-modifying treatments. To briefly summarize published work on autopsy-confirmed accuracy of a clinical diagnosis of early PD, Rajput et al. followed 43 subjects initially diagnosed with PD to autopsy and only 28 (65%) had path-confirmed PD.8 Multiple studies utilizing the UK Brain Bank have found path-confirmed clinical diagnostic accuracy to be 76-93% accurate depending on the clinical criteria used and the duration of disease.9-11 In the DATATOP study of 13 drug-naïve patients eventually followed to autopsy, only 4 (31%) had PD.12 We have reported that for a clinical diagnosis of PD at the first visit 80/97 (82%) had path-confirmed PD but when divided by disease duration at the time of first visit 72/82 (88%) of those with PD >5 yrs were path-confirmed but only 8/15 (53%) of those with clinically probable PD of <5 yrs duration were path-confirmed.7 While the clinical diagnosis may have changed during the years of clinical follow-up prior to autopsy, the data presented was based on the diagnosis at the first visit. In a meta-analysis of 11 path-confirmed studies with over 2,000 cases from 1988-2014 the pooled diagnostic accuracy was 80.6%: 79.6% at initial visit (but including subjects with longer disease durations) and 83.9% after clinical follow-up.6 Jellinger then reported a 50 year retrospective of his career showing that of 604 autopsied cases of clinical PD only 80.3% had path-confirmed PD.13 In most cases other neurodegenerative disorders, such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA), and Alzheimer’s disease (AD), were found. This low clinical diagnostic accuracy, even with response to treatment, suggests that without tissue biomarker confirmation many subjects enrolled in early treatment trials may not actually have PD. It is not that the subjects did not have a disease, it is just they did not have PD. Dopamine functional imaging improves accuracy but as most misdiagnoses are of another dopamine-depleted condition such as PSP and MSA, this is still inadequate as an inclusion/exclusion criterion.

Just as important has been the search for biomarkers that predate motor parkinsonism. The goal of identifying individuals with prodromal PD is to potentially find disease-modifying treatments that will slow or even prevent the development of PD. Multiple biomarkers have been discussed with the International Parkinson and Movement Disorder Society creating Research Criteria for Prodromal PD.14 The most robust clinical marker, short of finding a genetic predisposition to PD, which is rare, has been the clinical finding of idiopathic REM sleep behavior disorder (RBD)15, but this affects only a minority of PD subjects.

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 6

Our current proposal will build on our translational research investigating peripheral deposition of a-syn in the SMG of PD patients as a tissue biomarker for PD. More importantly the proposed study would not be for diagnosis alone but rather would serve to determine whether biopsy can be used as a possible target engagement biomarker for a tissue-level deposit of a-syn, something that the field critically lacks. We will also validate the only published study of a-syn in the SMG of subjects with RBD,16 and extend their work to determine if a-syn pathology increases over time. In autopsy studies we have previously shown that peripheral a-syn pathology is specific for PD versus other parkinsonian disorders and this study will be the first to assess specificity of biopsy in clinically-diagnosed non-PD parkinsonian subjects.

Using our Arizona Study of Aging and Neurodegenerative Disorders (AZSAND)/Brain and Body Donation Program (BBDP) dataset17 we were among the first to report the widespread presence of a-syn pathology in the peripheral nervous system (PNS), and to investigate biopsy of the PNS as a readily-accessible means to identify a-syn pathology. We initially published an extensive whole-body autopsy survey that identified the SMG as the most promising peripheral biopsy site.18 We further utilized our AZSAND/BBDP autopsy material to study SMG sections from 146 cases with CNS a-syn pathology, composed of 46 PD, 28 dementia with Lewy bodies (DLB), 14 incidental Lewy body disease (ILBD), 33 AD with Lewy bodies (ADLB) and 2 with PSP and Lewy bodies (PSPLB).2 ILBD are subjects that had no evidence of parkinsonism or dementia during life but had CNS a-syn pathology on autopsy; these cases are thought to represent prodromal PD or DLB neuropathologically. Control subjects included 79 normal elderly, 15 AD, 12 PSP, 2 CBD and 2 MSA. SMG a-syn pathology was found in 42/47 (89%) of the PD subjects, 20/28 (71%) DLB, 4/33 (12%) ADLB and 1/9 (11%) ILBD subjects but none of the 110 control subjects. We have established, in our most recent analysis of our autopsy data, that the density of SMG a-syn pathology significantly correlates with UPDRS motor score and disease duration (see data below), indicating potential suitability as a surrogate for clinical disease. Another critical study with autopsy cases was a study of simulated needle biopsy of autopsied submandibular glands from patients with PD; it found that 17/19 needle cores were positive for a-syn staining and thus doing biopsies in living patients should be feasible.2

We then performed two pilot studies of SMG needle biopsy in living PD patients. The first enrolled PD patients with longer disease duration, >5 yrs, to ensure that the clinical diagnosis of PD was as accurate as possible.3 15 PD subjects with mean disease duration of 11.8 yrs and mean age 68.7 had transcutaneous needle core biopsies performed unilaterally. On microscopic examination, three cases did not have any tissue elements consistent with SMG, resulting in a total of 12/15 (80%) cases having identifiable glandular tissue. Adequate tissue was defined, prior to unblinding, as having enough glandular elements for the neuropathologist to have confidence that a-syn staining could be assessed. 9/12 (75%) had positive biopsies.3 All patients tolerated the procedure well with the main side effects being bruising and swelling.

We then investigated unilateral SMG biopsies in 25 early PD patients, < 5 yrs disease duration, and 10 controls.4 Mean age was 69.8 for PD and 64.7 for controls. Mean PD disease duration was 2.6 yrs. Adequate tissue was obtained in 19 of the PD patients and 9 of the controls, 80% in total. Positive a-syn staining was found in 14/19 (74%) PD patients and 2/9 (22%) controls. We have followed one of our positive controls, the other was lost to follow-up, and after three yrs that individual has not developed signs of parkinsonism or dementia. The biopsies were well tolerated with bruising and swelling being the main side effects in 77% of the cases. In unpublished but presented results of the Michael J Fox Foundation (MJFF) Systemic Synuclein Sampling Study (S4) 1/20 controls had positive a-syn staining in the SMG biopsy.

The use of SMG biopsies for PD was independently confirmed in a study of unilateral biopsies of 24 PD patients, 21 patients with REM sleep behavior disorder (RBD), and 18 controls.16 Positive a-syn staining was found in 8/12 (67%) PD cases, 8/9 (89%) RBD patients, and 0/18 controls. The main side effects were localized pain and subcutaneous hematoma. This has been the only study of patients with RBD and suggests SMG biopsy might be valuable for identifying subjects at-risk for developing PD. A discouraging result was that despite using ultrasound guidance when doing the biopsies, not done at Mayo Clinic which used palpation alone, adequate tissue was obtained in only 43% of the RBD patients and 50% of the PD patients as well as all the controls. Given all the control data cited above we have removed the proposed biopsies of controls in our LOI but kept the comparison with non-PD parkinsonian subjects as this has never been done with clinically-diagnosed subjects.

To date there are no published studies of laterality of a-syn density in the two SMG glands or most importantly the possible changes in a-syn density over time. These are critical issues for future studies of disease progression and response to anti-alpha synuclein therapies. We now have preliminary, unpublished data, comparing a-syn density in both SMGs of 30 autopsy confirmed PD cases. One SMG was fixed at the time of autopsy and the other was frozen and then fixed later. The cases had mean age of 80.9 and mean disease duration of 12.5 yrs. In this study, 25/30 (83%) of the cases had at least one positive SMG and 22/30

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 7 had both glands positive for a-syn staining bilaterally. The mean a-syn density score was exactly the same bilaterally, with good correlation within individuals between the two glands (r= 0.63, p<.001). For the originally fixed SMG there was good correlation with both total brain a-syn density score and disease duration (r= 0.49, p= 0.006). Of note three subjects included in this autopsy study had previously undergone unilateral SMG biopsies. Using H&E staining on serial sections through the entire gland there was no evidence of any damage to the previously biopsied SMG.

We also have a second unpublished pilot study which investigated performing a second SMG biopsy in previously unilaterally biopsied patients and doing so bilaterally to improve tissue acquisition. Seven PD patients were included, mean age at time of second biopsy was 76 yrs and mean time between biopsies was

4.1 yrs. All subjects had sufficient SMG tissue on at least one side with 12/14 (86%) individual glands having sufficient tissue for assessment. All 12 glands were positive for a-syn. There was a 4x increase in a-syn density on average in the repeat biopsy, with 6 having an increase and one a decrease in density when comparing ipsilateral SMGs. Side effects were similar to previously reported: bruising, swelling, slight bleeding. Although a small pilot, it’s the first to show 1) bilateral biopsies are safe and improve adequate tissue acquisition, 2) have similar mean a-syn density bilaterally, and 3) increased a-syn density over time.

We have focused our work on the SMG given the extensive autopsy and living clinical data available.

Other potential biopsy sites have been suggested, notably colon and skin. It is not possible to completely review this literature in the present proposal, however, we showed, in our original, extensive, whole-body survey, that other tissue sites had markedly lower prevalence and density of a-syn pathology.18 We were leaders of two multicenter blinded-panel studies sponsored by the Michael J Fox Foundation (MJFF) that have shown that colonic biopsies have low sensitivity in PD. Although some single-center studies have reported high sensitivity and specificity for skin biopsies in PD subjects, these results did not have enforced blinding and clinical trial-like management, as has been done for the colon studies and for the MJFF Systemic Synuclein Sampling Study (S4). In the latter study, preliminary data presented at two international conferences (but not yet published in full) has shown that the sensitivity of a-syn in skin is less than half of that for SMG, with nearly identical very high specificity. Importantly, these MJFF-sponsored studies were also preceded by comprehensive blinded assessment of multiple different histological methods.19

Preliminary Work (see summary above and reference list below- our work is highlighted)

Experimental Methodology

Specific Aim 1: Thirty clinically diagnosed PD patients will be recruited from Mayo Clinic Arizona, Banner Sun Health Research Institute (BSHRI), and Barrow Neurological Institute (BNI). All will sign written informed consent approved by the Mayo Clinic Institutional Review Board. The recruiting physicians (Drs. Adler, Mehta, Caviness, Driver-Dunckley, Shill, Walsh, and Shprecher) are close collaborators in the AZSAND. The institutions have pre-established reciprocal data sharing and intellectual property agreements. All subjects will undergo clinical examination to include the MDS-UPDRS, Montreal Cognitive Assessment (MoCA), Mayo Sleep Questionnaire, and a University of Pennsylvania Smell Identification Test (UPSIT).

In the first year of funding all 30 PD subjects will have bilateral SMG biopsies. The transcutaneous needle core biopsies will be performed by board-certified ENT surgeons, Dr. Michael Hinni, Dr. David Lott, or their colleagues during a single outpatient office visit at Mayo Clinic Arizona. This is the same team that has performed over 60 unilateral and 7 bilateral biopsies in our previously cited studies.3, 4 Tissue from left and right SMG biopsies will be immediately placed in separate standard plastic cassette between two sponges, immersed in 70 ml of 10% NBF (Fisher Scientific, Kalamazoo, MI) and transported the same day from Mayo Clinic Arizona to Dr. Thomas Beach at BSHRI (30 minute drive). All patients will receive a phone call from the study coordinator the day after the biopsy to assess any side effects. If side effects occur then follow-up phone calls by the study coordinator will be made until the side effect(s) have resolved, and both the type of side effect, the severity (mild, moderate, severe), and the duration will be recorded.

All tissue staining procedures will be performed similarly as for our autopsy studies and our three previous needle biopsy clinical studies. After 24 hours fixation at 4oC, the tissue cores will be dehydrated in alcohols, infiltrated with paraffin and serial sections cut with a rotary microtome. Every section will be mounted on a separate slide, beginning with the first appearance of tissue fragments and ending when tissue fragments are no longer visible. The primary antibody will be a polyclonal antibody raised against a-syn phosphorylated at serine 129 that has been extensively characterized in our previously cited studies. The method is highly specific for pathological a-syn deposits as, in our autopsy studies, we have never found immunohistochemically-positive peripheral nervous system elements in subjects that lack CNS a-syn

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 8 pathology. As in our three clinical studies of SMG needle biopsy, every other section (every other slide) will be stained with an anticipated average of 60 stained slides per subject. Staining every other section will reduce the number of sections to be screened and will protect against technical failure during staining, by reserving half of the sections. Each subject (in all aims) enrolled will be given a subject ID by the Mayo Clinic statistician such that Drs. Adler and Beach will remain blinded through the period of data analysis.

In year 2 of the study 20 subjects with non-PD parkinsonian disorders will have bilateral SMG biopsies.

All procedures will be as detailed above. These subjects will not have a second biopsy. Subjects will be recruited from the centers listed above.

Specific Aim 2: All 30 PD subjects will undergo a second bilateral SMG biopsy ~2 yrs later. All 30 subjects will have had their second biopsy by the end of the 3 year study. Each biopsy visit will be preceded by a clinical examination to include the MDS-UPDRS, Mayo Sleep Questionnaire, and MoCA. We will not perform an UPSIT a second time as studies have shown no significant worsening in PD patients in this time frame.

All biopsy procedures and tissue preparation and assessment procedures will be as described above.

Specific Aim 3: Ten clinically probable idiopathic RBD subjects (RBD without any other neurodegenerative disease diagnosis) will be recruited from the centers discussed in Aim 1. All RBD subjects will undergo an initial bilateral SMG biopsy in year 1 and then a repeat biopsy in year 3. All will have the same clinical assessments as the PD subjects discussed in Specific Aims 1 and 2. We will perform a second UPSIT with the RBD subjects as rate of change of the olfactory test may be a biomarker for eventual conversion to PD.

All biopsy procedures and tissue preparation and assessment procedures will be as described above.

Data Analysis

Statistical analyses will be performed using SAS by a professional statistician employed by the Mayo Clinic.

Proportional differences and correlations will be tested using chi-square, Fisher’s Exact test, Pearson or Spearman methods as appropriate. Although sample sizes are relatively small, our prior published and unpublished biopsy studies indicate sufficient power for the cross-sectional studies. These studies are only exploratory in that they are intended to serve as feasibility tests and as preliminary data with which to calculate needed subject numbers for clinical trials of anti-synuclein agents.

Specific Aim 1: The analyses will determine 1) if there is a significant difference in a-syn pathology density in left versus right SMG, 2) what is the adequacy of tissue acquisition with bilateral biopsies, 3) are there correlations between a-syn density and clinical measures (motor, sleep, olfaction, cognitive) as well as disease duration, 4) is a-syn pathology specific for PD or also found in non-PD parkinsonian disorders.

Specific Aim 2: The analyses will determine 1) whether a-syn pathology density increases, decreases, or remains static after a two year time interval, 2) what is the adequacy of tissue acquisition with bilateral biopsies, 3) is there a significant difference in a-syn pathology density in the left versus right SMG, and 4) are there correlations between changes in a-syn density and changes in clinical measures (motor, sleep, olfaction, cognitive) as well as disease duration.

Specific Aim 3: The analyses will determine 1) if there is a significant difference in a-syn pathology density in left versus right SMG in RBD, 2) what is the adequacy of tissue acquisition with bilateral biopsies, 3) are there baseline and/or followup correlations between cross-sectional or longitudinal measures of a-syn density and clinical measures (motor, sleep, olfaction, cognitive) as well as disease duration, and 4) does a-syn pathology density increase, decrease, or remain static after a two year time interval.

Limitations: While the methods and procedures proposed have been utilized by the investigators the key limitation has been subject numbers. While the number is small, given the preliminary data we have cited regarding bilateral biopsies and second biopsies in the 7 PD cases, we believe adequate information regarding tissue acquisition, laterality, and progression of a-syn pathology will be obtained, enabling useful subject number estimation for clinical trials of anti-synuclein agents. While only 10 RBD cases are proposed, this is intended only as an initial exploratory study and would double the current world literature on SMG biopsies in RBD and expand this to second biopsies which has never been reported.

Timeline Year 1: A RedCap database will be created to record all demographic, clinical and pathology data. All 30 PD and 10 RBD subjects will have their clinical examinations and bilateral SMG biopsies performed at Mayo

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 9

Clinic Arizona. Tissue will be processing will begin at Banner Sun Health Research Institute (BSHRI).

Year 2: All 20 non-PD parkinsonian subjects will have their clinical examinations and bilateral SMG biopsies performed at Mayo Clinic Arizona. Data analysis will be completed for the initial biopsy data.

Year 3: All 30 PD and 10 RBD subjects will have their second clinical examinations and bilateral SMG biopsies performed at Mayo Clinic Arizona. Tissue will be processing will occur at Banner Sun Health Research Institute (BSHRI) followed by data analysis.

Evaluation Dr. Adler will be responsible for oversight of the entire research project. Dr. Adler will have a Mayo Clinic Arizona team, including a research coordinator, to ensure all subjects have their clinical examinations, biopsies, and follow-up for any side effects. The AZSAND clinical group, led by Dr. Adler, meets on a monthly basis and will discuss study progress and subject recruitment as specified in Specific Aims 1 & 3 as well as for the follow-up in Specific Aims 2 & 3. Dr. Adler will be in communication, by email/phone/in person meetings, with Dr. Beach at BSHRI regarding progress on the pathology components of the study. Dr. Adler and Dr. Beach meet on a regular basis as they are co-PIs of the AZSAND program. Dr. Adler will also be responsible for working with the Mayo Clinic statisticians to insure data entry and analysis is completed.

References

*1. Beach TG, Adler CH, Dugger BN, et al. Submandibular gland biopsy for the diagnosis of Parkinson's disease. J Neuropath Exp Neurol 2013;72:130-136.

*2. Beach TG, Adler CH, Serrano G, et al. Prevalence of Submandibular Gland Synucleinopathy in Parkinson's Disease, Dementia with Lewy Bodies and other Lewy Body Disorders. J Park Dis 2016;6:153.

*3. Adler CH, Dugger BN, Hinni ML, et al. Submandibular gland needle biopsy for the diagnosis of Parkinson’s disease. Neurol 2014;82:858-864.

*4. Adler CH, Dugger BN, Hentz JG, et al. Peripheral Synucleinopathy in Early Parkinson's Disease:

Submandibular Gland Needle Biopsy Findings. Mov Disord 2016;31:250-256.

*5. Beach TG, Adler CH. Importance of low diagnostic accuracy for early Parkinson's disease. Mov Disord 2018;33:1551-1554.

6. Rizzo G, Copetti M, Arcuti S, Martino D, Fontana A, Logroscino G. Accuracy of clinical diagnosis of Parkinson disease: A systematic review and meta-analysis. Neurol 2016;86:566-576.

*7. Adler CH, Beach TG, Hentz JG, et al. Low clinical diagnostic accuracy of early vs advanced Parkinson disease: clinicopathologic study. Neurol 2014;83:406-412.

8. Rajput AH, Rozdilsky B, Rajput A. Accuracy of clinical diagnosis in parkinsonism--a prospective study.

Can J Neurol Sci 1991;18:275-278.

9. Hughes AJ, Ben Shlomo Y, Daniel SE, Lees AJ. What features improve the accuracy of clinical diagnosis in Parkinson's disease: A clinicopathologic study. Neurol 1992;42:1142-1146.

10. Hughes AJ, Daniel SE, Kilford L, Lees AJ. The accuracy of the clinical diagnosis of Parkinson's disease:

a clinicopathological study of 100 cases. J Neurol Neurosurg Psych 1992;55:181-184.

11. Hughes AJ, Daniel SE, Lees AJ. Improved accuracy of clinical diagnosis of Lewy body Parkinson's disease. Neurology 2001;57:1497-1499.

12. Jankovic J, Rajput AH, McDermott MP, Perl DP. The evolution of diagnosis in early Parkinson disease.

Parkinson Study Group. Arch Neurol 2000;57:369-372.

13. Jellinger KA, Logroscino G, Rizzo G, et al. Accuracy of clinical diagnosis of Parkinson disease: A systematic review and meta-analysis. Neurol 2016;87:237-238.

14. Berg D, Postuma RB, Adler CH, et al. MDS research criteria for prodromal Parkinson's disease. Mov Disord 2015;30:1600-1611.

15. Postuma RB, Gagnon JF, Bertrand JA, Genier Marchand D, Montplaisir JY. Parkinson risk in idiopathic REM sleep behavior disorder: preparing for neuroprotective trials. Neurol 2015;84:1104-1113.

16. Vilas D, Iranzo A, Tolosa E, et al. Assessment of a-synuclein in submandibular glands of patients with idiopathic rapid-eye-movement sleep behaviour disorder: a case-control study. Lancet Neurol 2016;15:708.

*17. Beach TG, Adler CH, Sue LI, et al. Arizona Study of Aging and Neurodegenerative Disorders and Brain and Body Donation Program. Neuropathol 2015;35:354-389.

*18. Beach TG, Adler CH, Sue LI, et al. Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders. Acta Neuropathol 2010;119:689-702.

*19. Beach TG, Serrano GE, Kremer T, et al. Immunohistochemical Method and Histopathology Judging for the Systemic Synuclein Sampling Study (S4). J Neuropathol Exp Neurol 2018;77:793-802.

Project Resources

1. Laboratory

The Civin Laboratory of Neuropathology at Banner Sun Health Research Institute (BSHRI) is the site of the Brain and Body Donation Program. It is run by Thomas Beach, MD, PhD and his staff. All supplies and methods are already in place for performing submandibular gland tissue processing, staining, and analysis for alpha-synuclein pathology. Biopsy samples sent from Mayo Clinic Arizona have been processed in 3 previous studies. The tissue storage areas are 1600 sq. ft. and the histology lab is 1890 sq. ft.

2. Clinical

Charles H. Adler, MD, PhD will oversee the entire clinical program proposed. He is one of 4 movement disorders specialists (also Drs. Shyamal Mehta, Erika Driver-Dunckley, and John Caviness) at Mayo Clinic Arizona that will recruit subjects. All subjects will have their clinical examinations (by one of these doctors) and biopsies at Mayo Clinic Arizona. Movement disorders specialists at Barrow Neurological Institute (Drs.

Holly Shill and Ryan Walsh) and at Banner Sun Health Research Institute (Dr. David Shprecher) will recruit subjects and refer them to Mayo Clinic Arizona for clinical examination and biopsies. All biopsies will be performed at Mayo Clinic Arizona by Dr. David Lott, Dr. Michael Hinni, or one of their colleagues. This is an outpatient procedure. All 3 clinical sites are within 25 miles of one another. The drive between Mayo Clinic Arizona, Phoenix campus where biopsies will be performed, and BSHRI, is only 30 minutes and samples will be sent immediately following biopsy.

3. Animal

Not applicable

4. Computer

All Mayo Clinic sites have desktop computers available in every examination room and physician office and the investigators also have Mayo Clinic issued laptop computers. The statistician at Mayo Clinic also has a desktop computer. Mayo Clinic Arizona has an electronic medical record where all clinical data will be entered. A RedCap database will be created at Mayo Clinic Arizona with access granted to Dr. Beach and his group at BSHRI to enter all pathology data. The BSHRI has eleven desktop computers in the pathology section, nine of which are connected to the Banner Health network. Banner Health provides servers and network protection in compliance with HIPAA regulations.

5. Office

Dr. Adler has an office (80 sq ft) and multiple examination rooms available for clinical examinations. There are multiple examination/procedure rooms at Mayo Clinic Arizona available for the otolaryngologists to perform the biopsies. The BSHRI pathology area has a total of 800 sq ft of office space, consisting of 260 sq.

ft. for diagnostic microscopy and five additional offices ranging from 130 to 260 sq. ft. The largest of these is used by the Tissue Bank Manager, and also contains a file room for charts containing medical records, research documentation and BSHRI assessment results for deceased BBDP subjects.

6. Major Equipment

No major equipment is required.

7. Other

Major equipment needed for diagnostic and research histology includes a Nikon Eclipse 30i research and teaching microscope with four teaching heads and fluorescence illumination, an Olympus BH-2 compound light microscope with teaching head, a Nikon Optiphot compound fluorescence microscope, Leica CM3050S automated cryostat, four rotary paraffin microtomes, Sakura Tissue Tek VIP5 tissue processor, paraffin embedding station and dedicated plumbed-in ultrapure water system (reverse osmosis with molecular filter).

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 10

The tissue bank’s frozen tissue is contained within 32 ultralow temperature freezers protected by multiple redundant systems, including two redundant temperature-sensitive alarms that automatically telephone, text and email maintenance and BBDP personnel, online monitoring of current and historical freezer temperatures, CO2 tanks that are automatically triggered for backup cooling, and a backup diesel-generated power supply. Other shared major equipment within BSHRI includes ultracentrifuges, microscopes, blot imaging instruments, ELISA plate scanners and photographic darkrooms.

Charles H. Adler, M.D., Ph.D., Detailed Project Description, RFGA No. BPM000334 11

OMB No. 0925-0001 and 0925-0002 (Rev. 09/17 Approved Through 03/31/2020)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Adler, Charles H.

eRA COMMONS USER NAME (credential, e.g., agency login): CADLERAZ10

POSITION TITLE: Professor of Neurology, Mayo Clinic College of Medicine

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if applicable)

Completion Date

MM/YYYY

FIELD OF STUDY

Temple University, Philadelphia, PA B.A. 5/1980 Biology

NYU School of Medicine, New York, NY M.S. 5/1984 Pharmacology

NYU School of Medicine, New York, NY Ph.D. 5/1986 Pharmacology

NYU School of Medicine, New York, NY M.D. 5/1986

Manhattan VA/NYU Medical Center, New York, NY Internship 6/1987 Hospital of the University of Pennsylvania, Philadelphia, PA Residency 6/1990 Neurology

Graduate Hospital, University of Pennsylvania, Philadelphia, PA Fellowship 8/1991 Movement Disorders

A. Personal Statement I have specialized in the diagnosis and treatment of Parkinson’s disease, and performed multiple research studies of patients with Parkinson’s disease, Alzheimer’s disease, dementia with Lewy bodies, incidental Lewy body disease, REM sleep behavior disorder for the past 30 years. I serve as Co-PI and Clinical Core Director on a multi-institutional study implemented by the Arizona Parkinson’s Disease Consortium (APDC) and the Arizona Study of Aging and Neurodegenerative Disorders program. I am responsible for overseeing clinical assessments and then establishing the final movement disorder and clinicopathologic diagnoses for all subjects that come to autopsy. I have led multiple studies of submandibular gland biopsies in patients with Parkinson’s disease over the past several years.

B. Positions and Honors 1986 National Board of Medical Examiners 1987 – Present Member, American Academy of Neurology; Fellow 2004-Present 1989 – 1990 Instructor in Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 1990 – Present Member, Movement Disorder Society; Chair, Education Committee 2015-2017 1991 American Board of Psychiatry and Neurology 1991- Present Arizona Medical License #20278 1991 – 1994 Senior Associate Consultant, Department of Neurology, Mayo Clinic, Scottsdale, AZ 1993 – 1997 Assistant Professor of Neurology, Mayo Clinic College of Medicine, Scottsdale, AZ 1994 - 2014 Florida Medical License #ME0067071 1994 – Present Consultant, Department of Neurology, Mayo Clinic, Scottsdale, AZ 1994 – Present Co-Director, Parkinson’s Disease and Movement Disorders Center, Mayo Clinic in Arizona 1996 – 2001 Special Project Associate, Sun Health Research Institute, Sun City, AZ 1996 – 2002 Adjunct Appointment to Supervisory Committees, Interdisciplinary Doctoral Program in

Speech and Hearing Science, Arizona State University, Tempe, AZ Charles Adler, MD, PhD, Biographical Sketches, RFGA Number BPM000334 12

1997 – 2001 Associate Professor of Neurology, Mayo Clinic College of Medicine, Scottsdale, AZ 2000 Doctor of Excellence Award, Arizona Dystonia Support Group 2001 – 2008 Chair, Mayo Foundation Division of Movement Disorders, Department of Neurology, Mayo Clinic, Scottsdale, AZ 2001 – Present Professor of Neurology, Mayo Clinic College of Medicine, Scottsdale, AZ 2002, 2007 Outstanding Faculty Member of the Year, Mayo School of Continuous Professional

Development 2005 – Present Co-Principal Investigator, Arizona Parkinson’s Disease Center Consortium, Scottsdale, AZ 2006 Distinguished Mayo Clinic Investigators Award, Mayo Clinic in Arizona 2015 – 2017 Chair, Movement Disorder Society Education Committee 2017 – Present Movement Disorder Society International Executive Committee

C. Contributions to Science

1. I have worked to develop a diagnostic tissue biopsy test for Parkinson’s disease (PD) and Dementia with

Lewy bodies (DLB). This work began with determining that PD is a systemic disorder with pathologic alpha-synuclein being found in multiple organs. Our group then determined that all autopsied PD subjects had synuclein staining in the submandibular gland and that simulated needle biopsies of the gland were found to have the alpha-synuclein staining. I then was PI for a study of submandibular gland needle biopsies in living patients with advanced PD where we found 9/12 cases to be positive for alpha-synuclein staining. I was also PI on our study in early PD found that 14/19 PD cases had positive needle biopsies for alpha-synuclein staining as did 2/9 healthy controls. This work has led the field of finding a tissue biomarker for PD. Further studies are being done. Relevant papers include:

a. Beach TG, Adler CH, Sue LI, Vedders L, Lue L, White Iii CL, Akiyama H, Caviness JN, Shill HA, Sabbagh MN, Walker DG, Arizona Parkinson's Disease Consortium. Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders. Acta Neuropathol. 2010 Jun; 119(6):689-702. Epub 2010 Mar 21. PMCID: PMC2866090

b. Adler CH, Dugger BN, Hinni ML, Lott DG, Driver-Dunckley E, Hidalgo J, Henry-Watson J, Serrano G, Sue LI, Nagel T, Duffy A, Shill HA, Akiyama H, Walker DG, Beach TG. Submandibular gland needle biopsy for the diagnosis of Parkinson disease. Neurology. 2014 Mar 11; 82(10):858-64.

Epub 2014 Feb 05. PMCID: PMC3959757

c. Adler CH, Dugger BN, Hentz JG, Hinni ML, Lott DG, Driver-Dunckley E, Mehta S, Serrano G, Sue LI, Duffy A, Intorcia A, Filon J, Pullen J, Walker DG, Beach TG. Peripheral synucleinopathy in early Parkinson’s disease: Submandibular gland needle biopsy findings. Mov Disorders Jan 22. doi:

10.1002/mds.26476, 2016.

d. Beach TG, Adler CH, Serrano G, Sue LI, Walker DG, Dugger BN, Shill HA, Driver-Dunckley E, Caviness JN, Intorcia A, Filon J, Scott S, Garcia A, Hoffman B, Belden CM, Davis KJ, Sabbah MN.

Prevalence of submandibular gland synucleinopathy in Parkinson’s disease, dementia with Lewy bodies and other Lewy body disorders. J Parkinson Dis doi: 10.3233/JPD-150680, 2016

2. My work in the field of essential tremor (ET) is leading the way in showing that this disorder is not linked to Parkinson’s disease. We have shown this in a number of different clinical and pathologic papers. This work has been critical in trying to define what causes ET as well as in establishing the clinical progression of patients with ET. I have served as co-PI of the Arizona Study of Aging and Neurodegenerative Disorders where this work has been performed. Relevant papers include:

a. Shill HA, Adler CH, Sabbagh MN, Connor DJ, Caviness JN, Hentz JG, Beach TG. Pathological findings in prospectively ascertained essential tremor subjects. Neurology. 2008 Apr 15; 70(16 Pt 2):1452-5. Adler CH, Shill HA, Beach TG. Essential tremor and Parkinson's disease: lack of a link.

Mov Disord. 2011 Feb 15; 26(3):372-7. Epub 2011 Jan 31. PMCID: PMC3070808

b. Adler CH, Hentz JG, Shill HA, Sabbagh MN, Driver-Dunckley E, Evidente VG, Jacobson SA, Beach TG, Boeve B, Caviness JN. Probable RBD is increased in Parkinson's disease but not in essential tremor or restless legs syndrome. Parkinsonism Relat Disord. 2011 Jul; 17(6):456-8. Epub 2011 Apr 08. PMCID: PMC3119772

c. Shill HA, Adler CH, Beach TG, Lue LF, Caviness JN, Sabbagh MN, Sue LI, Walker DG. Brain biochemistry in autopsied patients with essential tremor. Mov Disord. 2012 Jan; 27(1):113-7. Epub 2011 Oct 28. PMCID: PMC3261329

3. I have worked on the topic of Prodromal Parkinson’s disease including clinical and pathologic findings in autopsied subjects with incidental Lewy body disease. In order to develop treatments that would potentially

Charles Adler, MD, PhD, Biographical Sketches, RFGA Number BPM000334 13 slow or stop the progression of PD, it is critical that the disease be identified at an earlier stage.

Establishing biomarkers for prodromal PD may do that. Additionally this work has led us to developing a novel staging system for Lewy body disorders. I have served as co-PI of the Arizona Study of Aging and Neurodegenerative Disorders where this work has been performed. Relevant papers include:

a. Beach TG, Adler CH, Sue LI, Peirce JB, Bachalakuri J, Dalsing-Hernandez JE, Lue LF, Caviness JN, Connor DJ, Sabbagh MN, Walker DG. Reduced striatal tyrosine hydroxylase in incidental Lewy body disease. Acta Neuropathol. 2008 Apr; 115(4):445-51. Epub 2007 Nov 06PMCID:PMC2724592

b. Beach TG, Adler CH, Lue L, Sue LI, Bachalakuri J, Henry-Watson J, Sasse J, Boyer S, Shirohi S, Brooks R, Eschbacher J, White CL III, Akiyama H, Caviness J, Shill HA, Connor DJ, Sabbagh MN, Walker DG. Unified staging system for Lewy body disorders: Correlation with nigrostriatal degeneration, cognitive impairment and motor dysfunction. Acta Neuropathol (Berl). 2009 Jun;

117(6):613-34. PMCID: PMC2757320

c. Adler CH, Connor DJ, Hentz JG,…