Lecture 13: Parkinson's Disease

What is Parkinson’s Disease?

a progressive movement disorder marked by classical symptoms (tremors, muscle stiffness, slow movement, and balance problems) and other heterogenous symptoms

True or False: PD is the second most common neurodegenerative disease and affects 1% of people over age 60.

true

True or False: Most cases of PD are late-onset and sporadic.

true

Which regions are affected during the early/mid stages of PD? What are the functions of these regions?

nigrostriatal: voluntary movement production

Which regions are affected during the late stage of PD? What are the functions of these regions?

• mesocortical: cognition, memory and learning, motivation

• mesolimbic: emotion, perception, reward

• tuberoinfundibular: sensory processing, hormonal regulation, and maternal nurturing

What are the common movement symptoms of PD?

• slowed movements

• muscle stiffness

• tremor

• unstable posture

What are the common non-movement symptoms of PD?

• constipation or urinary urgency

• mood changes

• sleep problems

• loss of sense of smell

What are the main causes of PD?

• progressive loss of dopaminergic neurons in the substantia nigra—leading to dopamine depletion in the striatum

• the presence of intracellular inclusions known as Lewy bodies and Lewy neurites

What are the risk factors of PD?

• aging for a common late-onset form of PD (> 60 yr)

• male > female

• genetic predisposition (PD can run in families)

• environmental exposures (pesticides, manganese) or factors (oxidative stress)

• genetic mutations (familial PD)

What are the genes mutated in familial PD?

• SNCA

• Parkin

• PINK1

• LRRK2

• DJ-1 (PARK7)

• SYNJ1

• DNAJC6

True or False: Emerging evidence shows that the PD-associated genes are involved in synaptic physiology, and mutations in SCNA (alpha-synuclein), LRRK2, and parkin impair synaptic transmission process including synaptic vesicle trafficking.

true

In general, what is the treatment for PD?

a mix of medications, therapy, and supportive care to manage symptoms for daily life

What are the medications for PD? What do these medications do?

• carbidopa-levodopa turns into dopamine in the brain

• dopamine agonists mimic dopamine in your brain

• MAO-B inhibitors slow the breakdown of dopamine in your brain

• anticholinergic medications to reduce tremors

What are the therapies for PD?

• physical therapy

• speech therapy

• infusion therapy (continuous delivery of medication through skin via a pump)

What is the surgery for PD?

deep brain stimulation

Is there a cure for PD?

unfortunately not

What are the cellular processes involved in PD?

• protein aggregation

• protein and membrane trafficking

• neurite structure

• prion-like transmission

• synaptic function and dopamine neurotransmission

• lysosome-autophagy pathway

• mitochondrial function and mitophagy

• ubiquitin-proteasome system

True or False: Abnormal SV trafficking may potentially be a common pathologic mechanism of PD.

true

Where is DJ-1 expressed?

ubiquitously expressed in various tissues, including the brain

What is DJ-1 involved in?

• cell cycle regulation

• oncogenesis

• gene transcription

What does DJ-1 act as?

acts as an antioxidant or sensor of oxidative stress in neurodegeneration

What is the subcellular location of DJ-1?

• mainly the cytosol

• axons

• dendrites

• presynaptic terminals

What does DJ-1 associate with?

the synaptic membrane

What causes the rare, early-onset familial PD?

autosomal recessive mutations I DJ-1 (M26I, E64D, and L166P)

What is unknown in the paper?

the mechanism of DJ-1 and how it causes PD

True or False: DJ-1 is found at presynaptic terminals and associates with synaptic membranes.

true

What is the central hypothesis of the paper?

DJ-1 regulates synaptic vesicle recycling whereas familial PD-associated mutations disrupt this function

What were the methods to test the hypothesis?

• primary cortical neuronal culture, immunostaining, and immunoEM: to test if DJ-1 localize to presynaptic terminal and affect its structures, authors performed immunostaining and immunoEM in primary cortical neurons from DJ-1 KO vs. WT mice; rescue by re-expressing WT DJ-1 in KO neurons

• vGlut1-pHluorin: to examine synaptic vesicle exocytosis and endocytosis, authors performed live imaging of vGlut1-pHluorin which is in synaptic vesicle

• FM1-43 dye: to measure total recycling pool and synaptic vesicle re-availability, authors performed live imaging of neurons loaded with FM1-43 dye

• pharmacology: bafilomycin (blocks re-acidification and thus endocytosis); MCBD and soluble cholesterol to manipulate membrane cholesterol

What are the findings that answer the following question: does DJ-1 localize to presynaptic axon terminals?

• endogenous DJ-1 partially co-localizes with the presynaptic marker endogenous synaptophysin

• transfected GFP-tagged DJ01 co-localizes with the co-transfected presynaptic marker VAMP2 which is tagged with mCherry

• immunogold EM shows DJ-1 gold particle labeling at the axonal nerve terminal in WT mice but not KO mice

What are the findings that answer the following question: does loss of DJ-1 change the structure of the axon terminal?

WT and KO neurons show similar synapse morphology, synaptic vesicle (SV) number and size, or active zone length

What are the findings that answer the following question: does loss of DJ-1 alter the total recycling vesicle pool?

• FM1-43 loading and unloading to measure the total population of function SVs released during neuronal activities

• neurons maximally loaded with FM1-43 via prolonged stimulation (900 AP at 10 Hz to label total recycling at SVs) were subjected to sequential stimulation (1,200 AP and 600 AP at 10 Hz) for unloading of FM1-43 via recycling of synaptic vesicles

What are the findings that answer the following question: does loss of DJ-1 alter the synaptic vesicle (SV) endocytosis?

• cultured WT and KO neurons transfected with vG-pH were stimulated at 100 AP

• the pHluorin fluorescence decay in DJ-1 KO neurons is 2-fold longer than that of WT neurons

• reintroducing (transfecting) wild-type DJ-1 completely rescued this KO-induced defect

• cultured WT and KO neurons transfected with vG-pH were stimulated at 300 AP with or without Bafilomycin (BAF) which blocks re-acidification and endocytosis

• BAF treatment increases vGlut1-pHlourin fluorescence in WT but not DJ-1 neurons upon neuronal stimulation, suggesting that DJ-1 KO neurons have reduced SV endocytosis

What is vGlut1-pHluorin?

a pH-sensitive GFP reporter on SV exo- and endocytosis

True or False: GFP fluorescence rises by exocytosis and decays as endocytosis re-acidifies vesicles.

true

What are the findings that answer the following question: does endocytic defects in DJ-1 KO neurons affect other synaptic physiology?

• measure vG-pH fluorescence during repeated stimulation with 100 AP and 300 AP to examine sustained synaptic vesicle release

• DJ-1 KO neurons showed progressively reduced vG-pH fluorescence during repeated stimulation than WT and Rescue (transfection of DJ-1 in KO neurons)

• fewer synaptic vesicles returned to the releasable pool—indicating impaired vesicle re-availability

What are the findings that answer the following question: does endocytic defects in DJ-1 KO neurons affect SV re-availability?

• DJ-1 KO neurons showed a significant decrease in the release of FM1-43 compared to WT neurons

• impaired synaptic vesicle re-availability

What are the findings that answer the following question: do familial PD mutations disrupt the function of DJ-1 in regulating synaptic vesicle recycling?

• express WT DJ-1 or mutant DJ-1 containing a familial mutation in DJ-1 KO neurons

• examine SV exo- and endocytosis using vGlut1-pHluorin imaging

• the pHluorin fluorescence decay in KO neurons expressing PD mutant DJ-1 is 2-fold longer than that of KO neurons expressing WT DJ-1

• mutant DJ-1 proteins are expressed at presynaptic terminals

What are the findings that answer the following question: is impairment in SV endocytosis in DJ-1 KO neurons caused by the changes in membrane cholesterol levels?

• treat neurons with MBCD to deplete membrane cholesterol and examine SV endocytosis using vGlut-pHluorin

• depleting cholesterol (MBCD) increases the time constant in WT but not DJ-1 KO neurons

• KO is already “cholesterol-low”

• supplement soluble cholesterol (SC) to neurons and examine SV endocytosis using vGlut1-pHluorin

• adding SC decreases the time constant in KO neurons to the level of WT neurons, suggesting that SC rescues SV endocytosis in KO neurons

What are the conclusions and models from the paper?

• DJ-1 localizes to nerve terminals but is not needed for synapse structure or exocytosis

• loss of DJ-1 selectively slows synaptic vesicle endocytosis

• loss of DJ-1 reduces the retrieval and re-availability of synaptic vesicles, especially during repeated activity

• PD-linked familial mutants disrupts SV endocytosis

• defects in SV endocytosis in DJ-1 KO neurons could be due to low levels of membrane cholesterol

Did the authors prove their hypothesis?

yes, the authors prove the central hypothesis that DJ-1 regulates synaptic vesicle recycling whereas familial PD-associated mutations disrupt this function

What are the caveats of this paper?

• PD is characterized by the progressive loss of dopaminergic neurons in the SN. However, the authors used cortical glutamatergic neurons to study DJ-1 function in SV endocytosis and the effects of familial PD mutations of DJ-1

• in dopaminergic neurons, vesicular monoamine transporter 2 (VMAT2) is responsible to package dopamine into the SVs, not vGlut. The authors did not directly measure dopamine release. It is unclear how reduced cholesterol can alter SV endocytosis.