lecture 23

Question 1

What are clinical symptoms of Parkinson’s disease?

Answer 1

Motor

• slowness
• stiffness
• tremor
• postural instability
• stooped, shuffling gait
• decreased arm swing when walking
• swallowing difficult
• immobile facial expressions

Cognitive

• mood changes
• depression
• anxiety
• pain
• tiredness
• confusion

Sensory

• numbness
• aching
• restlessness
• pain
• anosmia (lack of functioning olfaction, inability to perceive smells)

Autonomic

• hot/cold sensations
• constipation

in patient you’ll see forward tilt of trunk, reduced arm swinging, shuffling gait with short steps, rigidity and trembling of head, rigidity and trembling of extremities

Question 2

What is Parkinson’s disease?

Answer 2

• characterised by severe loss of substantia nigra dopaminergic neurons in the midbrain
• reduced pigmentation within the substantia nigra
• approximately 60 - 70% of substantia nigra dopaminergic cells are lost by the time a patient presents with clinical symptoms

Question 3

What is the function of the substantia nigra?

Answer 3

• controls voluntary movement
• produces the neurotransmitter dopamine
• dopamine regulates mood

Question 4

Where are the substantia nigra located?

Answer 4

• in the midbrain region

- forms part of the basal ganglia

Question 5

What are the basal ganglia?

Answer 5

• collection of nuclei (cluster of neurons) located in white matter of cerebral cortex
• striatum (putamen and caudate nucleus), globus pallidus, substantia nigra and subthalamic nuclei

Question 6

What are the parts of the substantia nigra?

Answer 6

• pars compacta (large pigmented neurons with neuromelanin) and pars reticulata (unpigmented neurons)

Question 7

Where does the pars compacta project?

Answer 7

• mainly to the striatum

Question 8

What is the striatum?

Answer 8

• substantia nigra innervates (connects), via its dopaminergic neurons, into the striatum
• the striatum has a major role in the planning and modulation of movement pathways

Question 9

What is the neuronal loss in PD?

Answer 9

• neuronal loss mainly affects ventrolateral area of SN - region that projects to striatum
• approximately 60-70% of the SN dopamine cells are lost by the time a patient presents with clinical symptoms

Question 10

What causes Parkinson’s disease?

Answer 10

Sporadic/idiopathic PD
- majority of PD is idiopathic i.e. occurs sporadically
- suggested mediators of PD include:
> toxins such as pesticides
> metals
> drugs MPTP (by-product of synthetic opiate 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP))

Familial PD

• inherited as either autosomal dominant or recessive
• a number of disease causing genes identified:
• • α-synuclein (SNCA)
• • parkin (PRKN)
• • Leucine-rich repeat kinase (LRRK2)
• • DJ-I
• • PINK I

Question 11

What is the pathology of PD?

Answer 11

• Lewy bodies and Lewy Neurites - Lewy pathology can occur in 5-20% of non-symptomatic individuals > 60 years
• lewy pathology can occur in the absence of neuronal loss and gliosis - these cases may be pre-clinical cases… unclear

Question 12

What is the main component of Lewy bodies?

Answer 12

• α-synuclein
• natively unfolded
• linked to learning, development and synaptic plasticity associated with vesicles
• • possible regulator of vesicular transport and dopamine release
• 140 amino acids
• 7 repeats across the protein
• hydrophobic region in the centre
• • non A-beta component (can also occur within amyloid beta plaques of alzheimer’s patients)
• acidic domain that is mainly made up of negatively charged amino acids
• also has a region rich in basic amino acids that has a high tendency for α-helical formation
• α-synuclein is an aggregating protein
• using electron microscopy you can see the development of fibrils
• thioflavin t can be used to measure formation of amyloid by measuring its fluorescence
• lag period and then growth phase

Question 13

What is Braak staging?

Answer 13

Braak et al (2003) classified the evolution of Lewy Body (LB) pathology in the brains of PD patients and those with incidental PDs

proposed that LBs begin to accumulate well before diagnosis, and well outside of the substantia nigr, and that other neurotransmitter systems, other than the dopaminergic systems are affected

Pathological process proceeds in six stages:

1: dorsal motor nucleus of the vagal nerve; anterior olfactory structures
2: lower raphe nuclei; locus coeruleus (located in brain stem control responses to stress and panic)
3: substantia nigra; amygdala; nucleus basilis of Meynert (part of the basal ganglia - clinical diagnosis)
4: temporal mesocortex (memory and emotions)
5: temporal neocortex (memory and language, sensory association and premotor areas)
6: neocortex; primary sensory and motor areas

diagnosis is typically delayed until stage 3, further implying a long preclinical period of neuronal decline

Question 14

Which is the toxic species?

Answer 14

• α-syn > partially folded intermediate > oligomer > larger oligomers > aggregate (amyloidogenic structure - beta sheet rich)
• somewhere along this pathway it is believed there is a toxic species that contributes to parkinson’s disease

Question 15

What factors modulate the aggregation of α-synuclein?

Answer 15

Genetics:

• mutations in α-synuclein
• via copy number variation of α-synuclein gene (overexpression of protein) (familial parkinson’s)

Dopamine
- inhibits aggregation

Exposure to these factors can promote aggregation

• iron
• oxidants
• nitration
• exposure to environmental toxins

Question 16

How do metals affect α-syn aggregation?

Answer 16

• if you dilute out the concentration of α-syn you don’t get aggregation –> lag of aggregation in control
• increase concentration = increase aggregation
• adding metals accelerates aggregation of α-synuclein

Question 17

What is the relationship between α-synuclein and dopamine?

Answer 17

• the substantia nigra is significantly affected in PD
• the substantia nigra neurons have a high concentration of dopamine
• is there a physiological/pathological interaction between α-synuclein and dopamine? - yes
• dopamine induced α-synuclein oligomers are not Thioflavin T reactive –> abolished ability to form amyloidogenic forms of α-synuclein

Question 18

What are α-synuclein aggregation pathways?

Answer 18

• α-syn monomer α-syn olgimers insoluble α-fibrils (ThT positive)

or Off pathway

• α-syn + dopamine SDS-resistant soluble α-syn oligomers oxidised methionines (ThT negative) (i.e. stops formation of Beta-sheet structures)
• – dopamine
• ThT positive insoluble α-syn fibrils (takes many weeks)
• adding dopamine to insoluble fibres can cause them to dis-aggregate and form soluble oligomers

Question 19

What are familial PD mutations?

Answer 19

• A30P
• E46K
• A53T
• mutations in α-syn (familial PD) - dominantly inherited
• overexpression of α-syn via copy number variation: triplication and duplication of α-syn gene (Devine et al. 2011)
• point mutations change the aggregation properties of α-syn
• A53T makes it come off lag phase earlier and aggregates to higher level
• A30P also shortens lag period but reaches same amount of aggregation as wild type after 4 days
• an increase in α-syn gene dosage leads to increased levels of α-syn protein
• higher α-syn concentration will promote aggregation
• shortens time to plateau

Question 20

What are transgenic α-syn mouse models (with mutant α-syn)?

Answer 20

Giasson et al. 2002
- neuronal-Synucleinopathy with Severe Movement Disorder in Mice Expressing A53T Human α-Synuclein

• promoter: prion protein (PrP) with A53T
• look for expression in these mice
• seen in cerebellar nuclei, striatum, ventral horn of spinal cord
• transgenic mice overexpressing A53T α-synuclein get a motor deficit
• looked at the percentage of mice that got this motor deficit of hind limbs
• as they start to age you get 100% at 16 months with the mutation acquiring this particular phenotype
• not transgenic animals don’t have this particular motor deficit
• this provides strong evidence of α-syn promoting/causing PD

Question 21

What are transgenic α-synuclein models that overexpress wild type α-synuclein?

Answer 21

Masliah et al. 2000
- Dopaminergic loss and inclusion body formation in α-synuclein mice: implications for neurodegenerative disorders

• promoter for human platelet derived growth factor-beta
• wild-type α-syn gene
• created a number of different lines where each had different numbers/copies of the gene
• line D had high expression
• A, B, and M had intermediate expression
• C had low expression
• non transgenic don’t show any inclusions (obviously)
• transgenic mice expressing wild type α-syn have reduced striatal tyrosine hydroxylase levels
• 50% reduction in line D of tyrosine hydroxylase levels
• this doesn’t marry up with the reduction in TH activity (still lower in Line D but not half)
• transgenic mice expressing wild-type α-syn have a movement disorder
• spend less time on rotorod

Question 22

What is tyrosine hydroxylase?

Answer 22

an enzyme involved in the synthesis of dopamine

- dopamine is a central molecule in PD - it is a loss of dopamine that causes PD

Question 23

How was the propagation of α-synuclein investigated?

Answer 23

• intracerebral inoculation of pathological α-syn initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice
• injected brain homogenate from symptomatic M83 mice into non-symptomatic M83 mice (M83 = transgenic mouse over-expressing α-syn A53T gene)
• injected α-syn spread throughout the brain and caused deposition in different locations in a non-transgenic animal (propagation)
• injected saline solution into another showed no deposition at the same time points
• injected recombinant myc-tagged α-syn preformed fibrils (PFF) into non-symptomatic M83 mice
• injected into cortex and striatum
• Syn^120 = residues 1-120 α-syn
• WT syn = wild type α-syn
• deposition in these brain regions
• spread to other areas where it wasn’t injected
• also done with full length WT syn PFF and similar pathology occurred
• less in non-injected
• also seen when injected into non-transgenic animals

Question 24

What is cellular release of α-synuclein?

Answer 24

• can be secreted from neurons
• • classical secretory vesicle pathway
• • can end up by the golgi into endosomes –> cycling endosomes release to extracellular space
• • or off endosomes can form multi vesicular bodies –> release molecules from cells via exosomes

i.e. uses three different secretory pathways to release α-synuclein from neurons

this extracellular α-syn (either monomeric or aggregated forms) can interact with other cells such as microglia and astrocytes, activating these cells and causing inflammatory processes
(inflammation is one of the pathologies of parkinson’s disease)
- released α-syn can also be taken up by bystanding neurons and then further released to other neurons - propagated and transferred
- once internalised can form lewy bodies

Question 25

Does injected/propagated α-syn affect lifespan?

Answer 25

• yes it does
• M83 mice injected with M83 lysate die at ~280 days
• M83/non-injected live to about 470/80
• if you inject lysate at birth they die about 125 days

Question 26

What are the genetics of PD?

Answer 26

Gene, pathological aggregates, comments:

• α-synuclein, lewy bodies, dominant point mutations, onset 30-60 years, duplication onset 40-50s, triplication onset 30s
• Parkin, substantia nigra degeneration, occasionally lewy bodies, recessive, onset 10-50 years
• PINK-1, Lewy body pathology found, recessive, onset 30-50 years
• DJ-1, No pathology reported, recessive, onset 20-40 years
• ATP12A2, Lewy body pathology found, recessive, young onset 10-22 years
• LRRK2, usually lewy bodies, dominant mutations onset 30-50 years

Question 27

What is Parkin?

Answer 27

• autosomal-recessive juvenile Parkinsonism
• parkin mutations are the second most common genetic cause of L-dopa-responsive parkinsonism
• more than 100 mutations identified
• loss-of-function
• cytosolic protein that may function in the cellular ubiquitination/protein degradation pathway as a ubiquitin ligase
• suggested role in PD is that a loss of parkin function results in non-ubiquitinated substrates accumulating in neurons and this alteration in protein turnover affects neuronal function

Question 28

What is PINK-1?

Answer 28

• autosomal recessive juvenile Parkinsonism
• PINK1 - 581 amino acid ubiquitous protein, with an N-terminal mitochondrial targeting motif (MTS) and a conserved kinase domain
• the majority of the identified mutations are in the kinase domain, suggesting PINK1 enzymatic activity is relevant to PD pathogenesis
• a substrate of PINK1 is the the TNF receptor associated protein 1 (TRAP1)
• TRAP1 is a mitochondrial molecular chaperone and PINK1 phosphorylates TRAP1. phosphorylation is increased in response to oxidative stress
• PD-linked PINK1 mutants abolish kinase activity, but do not affect either PINK1-TRAP1 interaction or their mitochondrial co-localisation

Question 29

What is LRRK2?

Answer 29

• LRRK2 gene most common cause of autosomal-dominantly inherited Parkinson’s disease
• precise function remains unknown
• contains a protein kinase domain of the MAPKKK class, suggesting a role in intracellular signalling pathways
• at least 20 LRRK2 mutations identified
• pathogenic mutations may be associated with increased kinase activity
• LRRK2 promotes mitochondrial fragmentation
• pathogenic mutations promote this activity further

Question 30

What is DJ-1?

Answer 30

• autosomal recessive Parkinsonism
• more than 10 DJ-1 mutations reported
• normal function of DJ-1 and its role in dopamine cell degeneration is unknown
• DJ-1 is involved in modulating the oxidative stress response and mitochondrial function
• during oxidative stress, DJ-1 translocates to the outer mitochondrial membrane

Question 31

What is the spectrum of familial vs sporadic forms of PD?

Answer 31

• known genetic variants in familial
• early age onset for familial
• occurrence of sporadic forms increases with age
• believed that with sporadic form of the disease, environmental and unknown genetic variants play a much more prominent
• some particular polymorphisms don’t cause disease but modulate it

Question 32

What are some of the pathways that can lead to disease or neuronal dysfunction?

Answer 32

• we think that as α-syn aggregates and accumulates in cells that is probably a key component of the pathogenic pathway
• so what ever can regulate α-syn aggregation is probably going to modulate disease
• toxins interact with and affect α-syn (e.g. metals) (dopamine drives in opposite direction)
• glucocerebroside is a neurochemical which can go on to form ceramide
• glucocerebroside can stabilise α-syn oligomers
• mutations in GBA (glucocerebrosidase) seem to be linked to PD (causes an accumulation of glucocerebroside instead of forming ceramide - interacts with α-syn instead
• parkin mutations and the ubiquitination of proteins –> degradation
• loss of parkin activity will affect the degradation of proteins via the proteasome = accumulation of oligomers
• oligomers can interact with golgi and cause it to fragment
• LRRK2 mutations can also interact with golgi and cause golgi fragmentation
• this leads to increase in formation of oligomers

Question 33

What is the relationship between α-syn (and other proteins) and the mitochondria?

Answer 33

• α-syn interacts with and inhibits complex I of electron transport chain
• this results in a decrease of the energy capacity of cells
• α-syn, LRRK2 promote fragmentation of mitochondria
• mitophagy
• PINK1, DJ-1, Parkin all seem to prevent fragmentation of mitochondria and by preventing this the cell retains intact mitochondria and adequate energy levels
• biogenesis and maintenance of mitochondria and mitochondrial activity
• mutations could lead to lack of mitochondria, energy deficit and cells dying