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Flashcards in Neurodegenerative disease Deck (55):
1

What is the epidemiology of Parkinson's disease?

Second most common neurodegenerative disease
1/500 have it
Diagnosis normally in 60s, early onset is 21-40
Duration of illness is 5-15 years
High economic burden

2

What are the symptoms of Parkinsonism?

Motor: Slowness of movement, increased tone and stiffness, resting tremor and loss of postural reflexes
Non-motor: Loss of smell, sleep disorder, constipation (early signs?), dementia, hallucinations, depression, psychosis, anxiety, pain and sensory complaints, fatigue, hyperhidrosis (sweating), urinary urgency (sign of spread of pathology beyond basal ganglia)

3

What are the causes of Parkinsonism?

Parkinson's disease is most common (progressive, spontaneously arising)
Toxins - chemicals interfere with complex I of electron transport chain in mitochondria
Infections - prions or viruses (chronic inflammation causing Parkinsonism)
Treatment with certain medications (non-progressive)
Repeated head trauma
Metabolic disorders

4

What is the pathology of Parkinsonism?

Disease emerges when 60% loss of dopamine producing cells in substantia nigra (SN), 80% of stratal dopamine is depleted. Much of the pathological damage has already occurred.

5

Why does loss function of in the substantial nigra lead to Parkinsons?

Dopamine dampens down movement by slowing down and smoothing out muscle activity triggered by the high speed neurotransmitter acetylcholine. Dopaminergic axons from the substantia nigra innovate the striatum.

6

What is the function of dopamine?

Involved in pleasurable emotions e.g. reward, pleasure, euphoria, compulsion. Also involved in fine tuning motor function and perseveration (learning)

7

What are Lewy bodies?

Eosinophilic bodies. Alpha-synuclein is a major component along with ubiquitin and parkin and others

8

How are Lewy bodies thought to spread?

Braak staging hypothesis: spread from enteric (gut) and peripheral autonomic nervous system up lower brainstem, to midbrain and basal forebrain, finally to neocortex. Thought to follow disease pathology (constipation, sleep disorder, movement)

9

What is DLB?

Dementia with Lewy body. Has Lewy bodies distributed throughout the cerebral cortex (in PD are localised to the sustantia nigra). Resembles the dementia associated with PD

10

What is Multiple System Atrophy?

Another alpha-synucleinopathy disease. Rapid progressive disorder involving failure of the autonomic nervous system. Associated with alpha-synuclein inclusions in glia (not neurones)

11

Is Parkinson's inherited?

Mostly sporadic (idiopathic), but 5% is monogenic forms of disease
Cause is multifactorial - genetics, modifying effects of susceptibility alleles, environment

12

What genes are associated with Parkinson's inheritance?

Only 5% cases are monogenic and inherited
Alpha-synuclein - point mutation or amplification of gene is autosomal dominant
Parkin, DJ-1, ATP13A2, PINK1 - mutations are autosomal recessive

13

What is the structure and associated mutations in alpha-synuclein that lead to PD?

Unstructured zone followed by a hydrophobic alpha helix and an amphipathic helix. Missense mutations that cluster around the N terminus (A53T, A30P etc) former beta sheets. All are dominantly inherited and are linked to a propensity to aggregate. Have increased number of Lewy bodies. Duplication and triplications of SCNA are also not good - not clear which form is toxic vs protective

14

What are the functions of alpha-synuclein?

Located at pre-synapse near nerve terminals
Prefers high curvature membranes (and lipid rafts) and can make membranes tubular (also promotes mitochondrial fission - side effect of remodelling?)
Implicated in membrane trafficking e.g. exocytosis
Suggested to have a role in neurotransmitter release

15

How are levels of alpha-synuclein regulated?

Is responsive to toxic insult and growth factors
Mono-ubiquitination by SIAH-2 (de-ubiquitinated by USP9X) promotes degradation in the proteasome
Degraded by lysosome in monomer and oligomer forms - if defects in lysosomal degradation get bigger risk of PD; potential mechanism

16

What is LRRK2 and how is it involved in PD?

A large protein that is a kinase and GTPase. Mutations found in inherited and sporadic Parkinsons. Mutations lead to decreased GTPase activity and increased kinase activity though the substrates are unknown

17

How does LRRK2 regulate the cytoskeleton?

Regulates cytoskeletal function by regulating actin and tubulin dynamics. Binds microtubules or microtubule proteins (is close). Binds and phosphorylates beta-tubulin and tau. Is a stabiliser for microtubule assembly. Affects neurite outgrowth and axonal transport. Is a link between autophagy and microtubule trafficking due to deficiency in traffic and clearance of autophagic vesicles

18

How is LRRK2 associated with mitochondria?

Associated with outer membrane, protects from toxins. Mutations in LRRK2 show impaired function. Phosphorylates Drp1 which promotes Drp1 translocation to mitochondria to induce fission.

19

What is Parkin?

A RING E3 ubiquitin ligase. Causes autosomal recessive juvenile parkinsonism. Mutations can be found across the gene, knock out gene function e.g. indel

20

What is PINK1?

PTEN-induced kinase 1. Found in autosomal recessive parkinsonism. Mutations can be found across the gene, knock out gene function e.g. indel

21

How does PINK1 activate Parkin?

PINK1 is stabilised on damaged mitochondria by binding Tom7 (is normally translocated into matrix and degraded). PINK1 phosphorylates Parkin at the N terminal to relieve auto inhibition, then it phosphorylates ubiquitin to further activate Parkin - feed forward.

22

What does Parkin do?

Once activated on damaged mitochondria by PINK1 it catalyses addition of ubiquitin chains to many cytosolic and outer mitochondrial membrane proteins. The OMM proteins are ERAD-extracted and degraded, promoting mitophagy. Has de-ubiquitinases to antagonise - inhibiting these promotes mitophagy (and could be a drug target for defective Parkin)

23

What is Fbxo7?

A protein that interacts with Parkin and PINK1 and mutations have been associated with PD. Fbxo7 targets substrate to a ubiquitin ligase (SCF, Cul). It helps recruit Parkin to mitochondria under stress, and seems to be involved with ubiquitination of Parkin (and another undefined E3 ub. ligase) substrates. Works downstream of PINK1, but is not phosphorylated by PINK1 itself - may need substrate phosphorylation for function.

24

What has the study of PARK genes taught us about causes of PD?

Cellular systems that are implicated are interconnected:
Mitochondrial function, ubiquitin proteasome system, inflammation, trafficking

25

Why might poor mitophagy lead to PD?

Retention of depolarised mitochondria could lead to increased ROS, defective energy production and cell death

26

How can PD be treated?

L-dopa: the precursor to dopamine
Dopamine agonists
MAO-B inhibitors
Deep brain stimulation
Cell transplants (don't think we are quite there yet)

27

How is L-dopa used as a drug in PD?

Is the precursor to dopamine. Is given with Carbidopa which prevents decarboxylation of Levodopa to dopamine whilst in circulation.

28

How are MAO-B inhibitors used to treat PD?

Monoamine oxidase type-B metabolises dopamine. Inhibitor can be given on its own or with L-dopa.

29

How can deep brain stimulation be used to treat PD?

Stimulation of the subthalamic nucleus with high frequency through implanting lead wires can reduce tremors and restore motor function - potentially knocks out over excitation signals

30

Describe the autophagy-lysosome pathway

Isolation membrane wraps around an area of cytoplasm (random in most cells, polarised in neurones). Trafficked on microtubules to the MTOC where it fuses with a lysosome and breaks down contents.

31

What is the role of AMPK in triggering autophogy?

Responds to stress and starvation (e.g. low ATP:AMP ratio) to inhibit mTORC1 (an inhibitor of ULK by phosphorylation) and activate ULK by phosphorylation (and therefore inhibiting mTORC1 inhibitory phosphorylation). This activates the ULK-ATG1 complex which leads to autophagy

32

What is the role of mTORC1 in triggering autophagy?

Inhibits activation of the ATG1-ULK1, thus inhibiting autophagy. mTORC1 is inhibited by AMPK and other stress conditions

33

What is the role of the ATG1-ULK1 complex in triggering autophagy?

Positively regulates activity of the VPS34 complex via phosphorylation. Provides PIP3 to the phagophore which aids LC3 lipidation by recruiting the ATG12-ATG5-ATG16L1 complex to the membrane.

34

What is the role of VPS34 in triggering autophagy?

Phosphorylates phosphatidylinositol to phosphatidylinositol 3 phosphate (PI3P) which aids LC3 lipidation by recruiting the ATG12-ATG5-ATG16L1 complex to the membrane.

35

What does LC3-II do in triggering autophagy?

Is conjugated to PE (a phospholipid) and inserted into the phagophore membrane. Cargo-specific proteins bind LC3-II through LIR and UBA domains.

36

Describe the formation of an autophagosome

Activated by AMPK in low energy conditions. Leads to nucleation of an isolation membrane and formation of a phagophore (crescent shaped double membrane vesicle) which then fuses to form the double membraned autophagosome

37

Where does the membrane for an autophagosome come from?

Endoplasmic reticulum is site of formation of omegasomes which are essential for phagophore formation and elongation. Mitochondria grow upon starvation and supply lipid vesicles to the phagophore. Golgi is essential for trafficking Atg9-containing vesicles to the phagophore. The plasma membrane also contributes under basal and starvation conditions

38

How can proteins be identified as an autophagy substrate?

Inhibit or enhance autophagy and observe effect on protein levels.

39

How does autophagy triggered by glucose starvation work?

Is VPS34 independent. Could be due to a VPS34 independent source of PI3P, or PI5P (the mirror of PI3P). Has been found that PI5P can bind the same effectors as PI3P and has a similar effect.

40

What are the effects of enhancing autophagy?

Reduces levels of intracytoplasmic aggregate prone proteins and various pathogens
Reduces susceptibility to apoptosis
Protects against huntington's disease models and others

41

What is lysosomal storage disease?

Lysosomal hydrolyse mutation, often X-linked and recessive. Build up along autophagy pathway leads to an early death, but are asymptomatic in the early years of life.

42

What mutations affect autophagy induction?

Decreased edlin 1 levels (in Alzheimers), WIPI4 mutations in beta propeller protein associated neurodegeneration, Laforin and Malin mutations in Lafora disease

43

What mutations affect sequestration of autophagy substrates?

Adaptor protein nutations e.g. p62 and optineurin in ALS

44

What mutations affect mitophagy?

PINK1/Parkin in parkinson's

45

What mutations affect autophagosome trafficking?

Dynactin and profilin mutations in ALS

46

What mutations affect lysosomal function?

PS1 mutations in Alzheimers.

47

How is the plasma membrane involved in phagophore formation?

Morozova 2015: Atg16L positive vesicles bud inwardly and give rise to phagophores through homotypic fusion or heterotypic fusion with Atg9 positive vesicles.
Fusion of homotypic vesicles is mediated through the SNARE VAMP7 and Annexin A2, likely as a bridge with other proteins (2x S100). Fusion of heterotypic vesicles is through SNARE VAMP3

48

What does annexin A2 do?

Morozova 2015: Budding of Atg16 positive vesicles from the PM is aided by annexin A2 which promotes enrichment of phosphotidylinositol and phosphotidylserine micro domains at the PM (calcium dependent) and subsequent budding of Atg16 positive vesicles
Annexin A2 also aids fusion of homotypic Atg16 positive vesicles. Annexin A2 acts as a heterotetramer with 2 annexin A2 and 2 S100 proteins. Annexin A2 binds phosphotidylserine whilst S100 stabilises the annexin dimer. This requires calcium.

49

How does mutations in VPS35 (NOT 34) affect parkinson's disease?

Zavodszky 2014: VSP35 is part of the retromer complex. A rare mutation in VSP35 prevents retromer association with the WASH complex, impairing vesicle traffic from recycling endosomes to the TGN. Atg9 is internalised from the PM by clathrin mediated endocytosis, and with impaired traffic can't do heterotypic fusion with Atg16 vesicles, thus impairing autophagy and leading to accumulation of alpha-synuclein

50

What does CALM do?

Is an Alzheimers risk locus and is associated with autophagy. Regulates endocytosis and therefore Atg5/atg12/atg16L vesicle formation. Also influences maturation and enlargement of phagophore precursors by regulating SNARE (VAMP2 and VAMP3) endocytosis. The SNAREs associate with pre-phagophore vesicles in a CALM dependent manner and are required for optimal fusion of homotypic atg16L (VAMP2) and heterotypic atg16L and atg9 (VAMP3). This causes tau accumulation (a good measure of function in AD), modulating its toxicity

51

How does autophagy impact stem cell differentiation?

Is involved in degradation of the receptor Notch1. It is taken up in Atg16L positive vesicles. Therefore, impairment of autophagy leads to Notch hyper activation and defects in differentiation - cells are held in the early stage.

52

What is ataxin 3?

A deubiquitinating enzyme that has a polyQ tract in which expansions are a cause of spinocerebellar ataxia type 3 (SCA3) neurodegeneration.

53

How does expansion of the polyQ tract in ataxin 3 cause SCA3?

Ashkenazi 2017: In wild type ataxin 3 the polyQ tract interacts with beclin 1 and stabilises it by protecting it from proteasome mediated degradation. Stabilisation of beclin 1 enables autophagy (beclin 1 regulates starvation induced autophagy). Expansion of the polyQ tract decreases its deubiquitinase activity to beclin 1, decreasing starvation induced autophagy

54

How do polyQ diseases influence autophagy?

Ashkenazi 2017: Huntingtin polyQ competes for binding of beclin 1 with ataxin 3. Ataxin 3 binds by a polyQ stretch and stabilises beclin 1 and therefore positively influences autophagy. Competition of polyQ proteins reduces beclin 1 stability, impairing starvation induced autophagy. The longer the polyQ, the worse the impairment
Also interaction with atropin 1/androgen receptor with beclin 1, both of which are mutated in polyQ disease DRPLA and spinal and bulbar muscular atrophy respectively

55

How is autophagy associated with neurodegeneration?

Autophagy up regulation results in removal of toxic aggregate-prone proteins, protects against apoptosis. It protects against disease in models for huntingtons etc. If autophagy is compromised get enhanced aggregate formation and cell stress. Is seen in other neurodiseases