Exploring Parkinson’s Disease New Areas of Research for Treatment and Prevention Mary Kate Miller Academic Excellence Showcase May 26, 2016

Overview Introduction to the Disease Possible Causes Current & Common Treatment New Research for Treatment & Prevention Looking Forward 1) Intro to disease including symptoms and hallmarks

I. What is Parkinson’s Disease? First account by James Parkinson (1817) Progressive neurodegenerative disease Movement (motor) disorder Many common patterns of progression; also varies greatly Prevalence thought to increase due to industrialization 2nd most common neurodegenerative disease to Alzheimer's Although symptoms can vary greatly from person to person, there are stages that are seen in most cases and aid in diagnosis -Premotor phase, first subtle motor signs, typical motor signs, advanced stages Image from: https://upload.wikimedia.org/wikipedia/commons/c/c3/Parkinson,_An_Essay_on_the_Shaking_Palsy_%28title_page%29.png

I. Motor Symptoms Primary Secondary Resting tremor Bradykinesia Rigidity Postural Instability Secondary Freezing Micrographia Mask expression Unwanted acceleration Motor symptoms are essentially symptoms that involve movement or motor control, the primary motor symptoms are the most common and most obvious, the secondary motor symptoms are less common and less obvious. Non-motor symptoms are those not associated with movement

I. Non-Motor Symptoms Cognitive changes Focusing, planning, attention, thought, language and memory Sleep disorders Loss of sense of smell Soft voice Speech/swallowing problems Orthostatic hypotension Many More There are many non-motor symptoms that vary greatly with each case -cognitive changes could be due to the damage in the brain -sleep disorders are often seen in premotor phase -sense of smell from Braak’s hypothesis which says the earliest signs of PD are seen in the enteric nervous system, the medulla and specifically the olfactory bulb, which controls your sense of smell -soft voice could also be cognitive -orthostatic hypotension, balance issues, dizzy when standing up -there are even more symptoms experienced, most without a clear cause

I. Dopamine Neurotransmitter Pathways Excess Dopamine Reduced Dopamine Cognition Motor control Reward/Pleasure Excess Dopamine Addiction, Psychosis Reduced Dopamine Depression, Parkinson’s Disease Image from: https://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/Dopamine2.svg/2000px-Dopamine2.svg.png and https://assets-production-webvanta-com.s3-us-west-2.amazonaws.com/000000/47/62/original/images/img_80_antiparkinson/dopamine1.png

I. Hallmarks of Parkinson’s Degeneration of dopaminergic neurons Reduced dopamine signaling & release -The primary hallmark is the issues associated w/ dopamine -many neurons in the substantia nigra project toward the striatum, pathway associated w/ dopamine and motor control (DA levels of 80% in striatum show clinical symptoms) -substantia nigra is in midbrain, which is in part responsible for motor control/sleep-wake -aggregates seen in substantia nigra -not understood if Lewy bodies play role in cell death of if they are the body’s response to the disease - Image from: https://upload.wikimedia.org/wikipedia/commons/thumb/d/de/Dopamine_pathways.svg/1280px-Dopamine_pathways.svg.png

I. Hallmarks of Parkinson’s Degeneration of dopaminergic neurons Reduced dopamine signaling & release Aggregates of α-synuclein called Lewy bodies Basal Ganglia Striatum Substantia Nigra -The primary hallmark is the issues associated w/ dopamine -many neurons in the substantia nigra project toward the striatum, pathway associated w/ dopamine and motor control (DA levels of 80% in striatum show clinical symptoms) -substantia nigra is in midbrain, which is in part responsible for motor control/sleep-wake -aggregates seen in substantia nigra -not understood if Lewy bodies play role in cell death of if they are the body’s response to the disease - Image from: https://upload.wikimedia.org/wikipedia/commons/thumb/d/de/Dopamine_pathways.svg/1280px-Dopamine_pathways.svg.png

II. Possible Causes Early Onset: genetic mutation Late Onset: no single cause, increased risk with age Oxidative stress Environmental toxins Industrialization PD can be seen as both early and late onset, early typically means before 50 and late after, early onset has been asspciated w/ certain genetic mutations that could be familial, late onset seems to have a more complicated explanation that is still very unknown but age does play a role in the disease Many parts of proposed mechanisms of PD lead to oxidative stress has been shown to related to onset of PD, inability of body to detoxify ROS which can be produced from dopamine metabolism, dysfunction in the mito of increased Ca influx Research shows risk from certain environmental factors (industrialization), could be interesting to see prevalence in our generation as we age -MPTP is a prodrug, oxidized by MAO to the neurotoxin MPP+ after crossing BBB, high affinity for DA receptors -In 80’s heroin addicts used illicitly developed synthetic substitute that caused PD type symptoms, widely used in animals for PD -Speculation that environmental equivalent could lead to PD With so many possible but mainly unofficially described causes, treatment/diagnosis is difficult but leaves the door open for many treatment research methods MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) Image from: https://upload.wikimedia.org/wikipedia/commons/thumb/a/a6/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine_200.svg/184px-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine_200.svg.png

III. Current Treatment Goal: Improve dopamine levels Treatments: Levadopa MAO-B Inhibitors COMT Inhibitors Problems: Only reduce symptoms temporarily Long-term side effects (dyskinesia) Tyrosine Tyrosine hydroxylase -Improving DA levels improves release/signaling -Levadopa is a precursor for DA that can cross the BBB, DA cannot -MAO-B is an enzyme that metabolizes DA, you want as much to stick around as possible MAO-B activity increases w/ age -COMT is an enzyme that breaks down L-dopa and dopamine, again want DA to stick around -Long term use has shown dyskinesia, hallucinations and on-off effects, could be due to excess stimulation of receptors, as disease progresses it is more difficult to treat so higher doses needed and side effects increase -New research in non-dopaminergic treatment to follow Levadopa Dopa Decarboxylase Dopamine Images from :https://upload.wikimedia.org/wikipedia/commons/thumb/d/d0/Tyrosin_-_Tyrosine.svg/290px-Tyrosin_-_Tyrosine.svg.png, https://upload.wikimedia.org/wikipedia/commons/thumb/c/cf/3,4-Dihydroxy-L-phenylalanin_%28Levodopa%29.svg/2000px-3,4-Dihydroxy-L-phenylalanin_%28Levodopa%29.svg.png,https://upload.wikimedia.org/wikipedia/commons/thumb/e/e7/Dopamine_degradation.svg/2000px-Dopamine_degradation.svg.png

IV. Adenosine Receptor Adenosine receptor 2a (A2A) Adenosine = neuromodulator High density in striatum and basal ganglia Coupled to stimulatory G-protein Form heteromer with dopamine D2 receptor, opposing affects -Adenosine is a neuromodulator that coordinates response to dopamine and other NTs in areas of brain responsible for motor function, mood and learning/memory -A2a and D2 receptors have opposing effects, so inhibiting A2a receptors enhances D2 signaling D2 A2a Adenylyl Cyclase ATP cAMP G-Protein Image from: https://www.google.com/search?q=adenosine&biw=1280&bih=672&source=lnms&tbm=isch&sa=X&ved=0ahUKEwj259PalLMAhUS42MKHeB8B1sQ_AUIBigB#q=adenosine&tbs=sur:fc&tbm=isch&imgrc=WR5Mml6iUrIYlM%3A

IV. Glutamate Receptor Metabotropic glutamate receptor 5 (mGluR5) Basal ganglia and glial cells Coupled to G-protein Over expressed in Parkinson’s May form heteromeric complexes with A2a and D2 receptors Found in basal ganglia where they’re thought to play a role in motor loops and also on glial cells where they could play a role in neuroinflammation associated w/ PD -Over expression could play a role in motor symptoms, induced dyskinesia and excitotoxic mechanisms that sustain neurodegeneration mGlu Phospholipase C Adenylate Cyclase G-Protein PIP2 IP3 ER Ca2+

IV. Dual Target Strategy Targeting mGluR5 and A2a Receptors Evaluate neuroprotective and behavioral effects on Parkinson’s Rodent model using Oxidopamine Uses a rodent model of PD to evaluate neuroprotective and behavior effects of targeting mGlu and A2a receptors -Looked at combination of antagonizing both receptor targets along with use of L-Dopa and also looked at the antagonists alone

Antagonists MPEP: mGluR5 negative allosteric modulator MSX-3: A2a antagonist ANR 94: A2a antagonist Both MSX and ANR are different in structure but both have a purine feature that corresponds to being an adenosine antagonist (purine) -MPEP has been shown to improve akinesia associated w/ L-Dopa and reduce toxin induced neurodegenerative processes in rats -A2a antagonists have shown to improve motor symptoms and reduce off time w/o increasing dyskinesia in advanced patients w/ Ldopa treatment --Studied each other alone or in combination with each other and or L-Dopa Images from: https://www.google.com/search?q=2-Methyl-6-%28phenylethyny%29pyridine&biw=1280&bih=672&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjZ_PiHv8TMAhUNwmMKHQVtBUoQ_AUIBigB#imgrc=fMGZbJWvo0DSQM%3A , http://www.sigmaaldrich.com/catalog/product/sigma/m3568?lang=en&region=US, http://www.apexbt.com/anr-94.html

Neuroprotection Combinations Right striatum injected, 28 day period MSX-3, ANR, MPEP, MPEP + MSX-3, MPEP + ANR 94 Right striatum injected, 28 day period Studied a group of 49 rats treated with 6-OHDA injected into right striatum which causes slow/progressive loss of DA neurons, doses ranged from 1-2mg/kg/day for 28 days -Results percentages represent TH positive striatal nerve terminals (converts tyrosine to Ldopa) -Initial treatment to induce PD reduced neurons to 45.9% of original

Neuroprotection Initial 45% reduction of tyrosine hydroxylase (TH) surviving neurons Studied a group of 49 rats treated with 6-OHDA injected into right striatum which causes slow/progressive loss of DA neurons, doses ranged from 1-2mg/kg/day for 28 days -Results percentages represent TH positive striatal nerve terminals (converts tyrosine to Ldopa) -Initial treatment to induce PD reduced neurons to 45.9% of original Conclusion: Stronger neuroprotection when mGluR5 and A2a are targeted together

MPEP + Low ANR 94 + Low Levadopa Behavior Analysis Combinations Each alone, Each + Levadopa, Each combination ± Levadopa Medial forebrain bundle injection, acute treatment This section looked at 80 rats treated w/ 6-OHDA in left medial forebrain bundle which causes rapid neuronal loss, two weeks later primed w/ LDOPA and then 3 days later treated acutely, range of 2-3mg/kg/day with 2 being a “low dose”, rats showed 15% neuronal survival on lesioned side -Turning behavior is induced by acute treatments that improve motor deficits or increase efficacy of L-DOPA -Some induced slight motor stimulation but not significant anti-parkinsonian effect -While giving ANR and MSX individually with L-Dopa showed significant behavior, the combination of MPEP with low ANR and low L-Dopa shows significance in the ability to potentiate the effect of L-Dopa with lower doses Significant Turning Behavior ANR 94 + Levadopa MSX-3 + Levadopa MPEP + MSX-3 MPEP + Low ANR 94 + Low Levadopa Conclusion: Targeting both receptors simultaneously yields higher efficacy of Levadopa

Dual Target Conclusions Targeting both mGlu and A2a receptor shows more significant benefits than targeting individual receptors Low doses which reduce neuronal loss and improve motor symptoms may lower long term side effects More information needed to find optimal doses and window of opportunity This study revealed how targeting both receptor types simultaneously yields better neuroprotection and motor performance than when either are targeted alone -The low doses used in this trial provide hope that long term side effects would be reduced (currently studying in lab) -As the disease progresses the needs of the patient change so it is critical to find treatment options that correspond to the stage of the disease and make them available in the clinical setting -For that reason, this research provides great non-dopaminergic treatment possibilities but finding the right window of opportunity and optimal dose is still under investigation

IV. Targeting α-synuclein Image from Kardani et al, 2015

Targeting α-Synuclein A well-known A2a Antagonist… Images from : https://upload.wikimedia.org/wikipedia/commons/thumb/a/a1/Koffein_-_Caffeine.svg/2000px-Koffein_-_Caffeine.svg.png and https://pixabay.com/static/uploads/photo/2015/02/10/21/28/coffee-631767_960_720.jpg

α-Synuclein Aggregation In vitro incubation of purifiedα-synuclein to study aggregation kinetics Time Aggregation State Saturation Phase Growth Phase Lag Phase Purified α-synuclein in vitro to study kinetics of aggreagate formation -Initial phase of incubation (lag), natively disordered α-synuclein and no aggregation -Sudden increase fibrillar aggregates in log phase -Reached plateau (saturation) indicating fibrillation reached stationary phase -Aggregation follows nucleation dependent process, sigmoidal curve with distinct nucleation, growth (fibrillation) and equilibrium (saturation) Lag = Natively disordered Growth = Oligomers/Protofibrils Saturation = Fibrils

Caffeine & α-Synuclein Aggregation Effects of Caffeine on α-synuclein Increase extent of aggregation Decreased lag time Rapid formation of oligomers and conversion to fibrils Increase in extent of aggregation in concentration dependent manner Conclusion: Increasing conversion of oligomers to fibrils reduces toxicity Image from Kardani et al, 2015

Caffeine & α-Synuclein Binding Properties α-synuclein acquires alpha helical formation upon binding of phospholipids Changes to binding properties could change function Particular affinity for phospholipid membranes --Analyzed lipid interacting property of aggregates by incubating w/ nitrocellulose membrane pre-spotted with certain phospholipids, protein-lipid complexes were identified by immunoblotting w/ a α-synuclein specific antibody Image from: https://www.google.com/search?q=alpha+synuclein&biw=1400&bih=956&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjq6ez_jM7MAhUExWMKHaxsB-YQ_AUIBigB#q=alpha+synuclein&tbm=isch&tbas=0&imgrc=LHCvTDXLIj6VyM%3A

Caffeine & α-Synuclein Native α-synuclein binds acidic phospholipids like PS and PtdIns Phosphatidylserine (PS) Interestingly, as seen in the glutamate receptor pathway… IP3 = inositol 1,4,5-triphosphate PIP2 = phosphatidylinositol 4,5-bisphosphate Phosphatidyl Inositol (PtdIns) Both important in cell signaling, membrane trafficking and apoptosis https://www.google.com/search?q=lag+log+and+saturation+phase&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjQnpX9usnMAhVE-2MKHYNKChAQ_AUIBygB&biw=1280&bih=672#q=phosphatidylserine&tbm=isch&tbs=sur:fc&imgrc=R-UJ7b0ipKJWiM%3A and http://www.cyberlipid.org/images/pict87.jpg

Caffeine & α-Synuclein Native α-synuclein binds acidic phospholipids like PS and PtdIns mGlu Phospholipase C G-Protein Interestingly, as seen in the glutamate receptor pathway… IP3 = inositol 1,4,5-triphosphate PIP2 = phosphatidylinositol 4,5-bisphosphate PIP2 IP3 ER Ca2+ 25 Both important in cell signaling, membrane trafficking and apoptosis https://www.google.com/search?q=lag+log+and+saturation+phase&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjQnpX9usnMAhVE-2MKHYNKChAQ_AUIBygB&biw=1280&bih=672#q=phosphatidylserine&tbm=isch&tbs=sur:fc&imgrc=R-UJ7b0ipKJWiM%3A and http://www.cyberlipid.org/images/pict87.jpg

Caffeine & α-Synuclein Incubated with nitrocellulose membrane, analyzed with immunoblotting Aggregates w/o Caffeine No interaction with PtdIns and negligible binding to derivatives Aggregates w/ Caffeine Retain higher lipid binding ability to PtdIns and derivatives Conclusion: Caffeine helps maintain lipid binding ability of α-synuclein aggregates

Research Conclusions Targeting mGlu and A2a receptors in combination yields stronger neuroprotection and improved motor deficits Lower doses can be used when targeting mGlu and A2a possibly leading to fewer long-term side effects Caffeine may reduce toxic Lewy Bodies and maintain normal functions of α-synuclein

V. Looking Forward Non-dopaminergic treatments show great promise Istradefylline (A2a antagonist), Japan 2013 Important to focus on multiple mechanisms of a disease Caffeine may help in prevention Image from: https://upload.wikimedia.org/wikipedia/commons/thumb/f/fa/Istradefylline.svg/507px-Istradefylline.svg.png

Disclaimer… Images from:https://news.starbucks.com/uploads/images/Beverages/Caramel_Cocoa_Cluster_Low_Res.JPG, https://pixabay.com/static/uploads/photo/2015/02/10/21/28/coffee-631767_960_720.jpg

Remember… “Always be kind, for everyone you meet is fighting a battle you know nothing about.”