PD III Flashcards
Why do we care about Lewy Bodies
They are a hallmark of PD
What are lewy bodies
Lewy bodies are cytoplasmic inclusions of misfolded proteins
They have a dense core surrounded by filamentous material
Lewy Body Makeup
Dense core made of : lipids, organelles, membranes intermingled with alpha-synuclein
The core is surrounded by filamentous material
Lewy bodies were described first in ____ as a major ________ of PD brains.
1902; pathological characteristic
Role of α-synuclein
- Major constituent of Lewy bodies
- First protein linked to familial PD (in 1997).
- It is also directly implicated in susceptibility to idiopathic PD (i.e. also has a role in sporadic PD)
Lewy bodies spread from ____ but their role in ____ is still controversial
Lewy bodies spread from the brainstem to striatum and cortex (or even from the gut and nose to the brain!), but their role in disease pathogenesis is still controversial
How Lewy bodies spread
From gut via vagus nerve
and nose via glossopharyngeal nerve
T/F: Disease severity and duration correlate with the number of Lewy bodies
FALSE
Disease severity and duration DO NOT correlate with the number of Lewy bodies, but with the overall α-syn burden.
T/F Lewy bodies are seen in all PD
FALSE
Some familiar forms o PD (parkin- or LRRK2–parkinsonism) have no Lewy bodies.
Lewy bodies originate in ____ explaining these symptoms ______ of prodromal PD
Originate in nose and gut; explaining anosmia, constipation, sleep disorders seen in Stage I/prodromal PD
T/F: α-synuclein burden is directly linked with PD severity
TRUE
α-Synuclein is a ___ kDa _____ that can ______ and has ___ main domains
a-synuclein is a 15 kDa cytosolic protein that can associate to membranes and has three main domains:
3 domains of α-Synuclein
1) N-terminal Amphipathic region (membrane-binding domain)
2) centre hydrophobic region (NAC domain)
3) C-terminal acidic region (calcium-binding domain)
Membrane binding domain of α-Synuclein
2 alpha-helical structure seperated by a break KTEGV motif
On N-terminal end; amphipathic
Allows membrane binding
NAC domain of α-Synuclein
Center of protein
contains Serine 87
Promotes aggregation
Calcium-binding domain of α-Synuclein
C-terminal end; acidic
contains serine 129
C-terminal is acidic and unstructured –> has high (-) charge and may old to hydrophobic end in normal brains to prevent aggregation BUT in acidic conditions (i.e. in lysosomes) the negative charge may promote aggregation
C-terminus of α-Synuclein in normal vs. PD brains
C-terminal is acidic and unstructured –> has high (-) charge and may old to hydrophobic end in normal brains to prevent aggregation BUT in acidic conditions (i.e. in lysosomes) the negative charge may promote aggregation
Missense mutation in ____ domain is associated with ____ PD
membrane-binding domain (N-terminus); familial PD
α-Synuclein NORMAL function
highly enriched in presynaptic terminals, where it associates, in part, to synaptic vesicles, has roles in:
- Regulation of formation of synaptic vesicles from endosomes,
- exocytosis
- regulation of neurotransmitter release
- maturation of presynaptic vesicles
- synaptic vesicle recycling.
In short, normally α-Synuclein has an important role in the _____
SYNAPSE (associates to synaptic vesicles)
Mutations of α-Synuclein linked to familial PD
• missense point mutations (autosomal dominant PD)
• gene duplication and triplication
• polymorphisms in the gene promoter that increase
transcriptional activity
Changes to α-Synuclein in idiopathic PD
In idiopathic PD, a-synuclein undergoes conformational changes, oligomerizes and/or forms protein aggregates (fibrils, Lewy bodies).
Some of these forms are toxic to neurons.
a-synuclein and disease spreading
Misfolded a-synuclein can be secreted by neurons and be transferred from neuron to neuron, thus spreading the disease
Loss of function effects of a-synuclein
- impairs microtubule formation and axonal transport
- causes presynaptic dysfunction and abnormal NT release
Gain of function effects of a-synuclein
- can form pores and leak NT from vesicles
- causes mitchondiral dysfunction (increase OXI stress)
- overwhelms Ca-buffering ability
- ER stress
- disrupts ER and golgi trafficking
- impairs proteostasis including protein degradation by ubiquitin-proteasome and autophagy-lysosomal systems
- promotes neuroinflammation (activates microglia)
Targeting α-Synuclein in PD DRUGS
- Clenbuterol –act on transcription
- siRNA–degrade mRNA to prevent translation
- HSPs and Anle138b – prevent misfolding/block aggregation
- Immunotherapy: AFFITOPE PD03A (active) or PRX002 (passive) –degrade extraceullar α-Syn
- Ambroxol hydrochloride, autophagy activators –> dipsoe of α-Syn inside the cell
Targeting α-Syn (where can drugs act)
- production–lower α-Syn levels
- aggregation–prevent misfolding or aggregation (prevent lewy body formation)
- intracellular degradation
- extracellular degradation
- Uptake–reduce α-Syn uptake by neighbouring cells (prevent spread)
Ambroxol hydrochloride
Facilitate α-Syn degradation in lysosome (intracellular degradation)
Autophagy activators
Intracellular degradation of α-Syn
Clenbuterol
prevent α-Syn transcription (DNA –> mRNA)
block α-Syn production
siRNA
degrade mRNA to prevent translation of α-Syn
block α-Syn production
HSP, ANle138b
prevent misfolding/block aggregation and formation of Lewy bodies
prevent aggregation
AFFITOPE PD03A (active) or PRX002 (passive)
Dispose of α-Syn outside of cell (extracellular degradation)
IMMUNIZATION AGAINST α-Syn
RNA interference (RNAi)
Strategy to reduce α-Synuclein
concern: decrease of α-syn could be deleterious
Why could a decrease of α-syn be deleterious
In rat and monkey models where α-syn was decreased by 90%, degeneration of the nigrostriatal pathway was observed
α-syn is one of the most abundant proteins in the brain and is needed for normal functioning
β-2-adrenoreceptors and alpha-syn
Activation of β-2-adrenoreceptors (clembuterol, salbutamol) to reduce α-Synuclein
β-2-adrenoreceptors
- G-protein-coupled receptor activated by epinephrine
- Agonists used for the treatment of asthma and cardiac failure
- Antagonists used for the treatment of hypertension and arhitmia
- receptors found in liver, cardiac myocytes, SM of blood vessels, pancreas–affect depends on tissue
Testing of FDA library compounds against PD
4 passed screening and 3 were beta-agonist and 2 can pass the BBB
Metaproterenol (can’t cross BBB), clenbuterol (BBB permeable), salbuterol (BBB permeable)
Clenbuterol vs saline in PD
Clenbuterol decreases neurodegeneration in a mouse model of PD
Less DA with MPTP admin, but better preserved with clenbuterol admin
MPTP
induces a PD-like phenotype in animals
Norwegian study (beta blockers vs beta agonists)
- Risk of PD is lower in a Norwegian population taking high doses of Salbutamol (Beta-agonist) as treatment for asthma compared to the general population
- Conversely, the risk of PD is higher in a population taking propanolol (beta-blocker)
Drugs that reduce α-Synuclein in PD
- RNA interference (RNAi)
- Activation of β-2-adrenoreceptors (clembuterol, salbutamol)
Drugs that target α-Synuclein aggregation
- Drugs that increase production of Heat Shock Proteins
- Anle128b
Anle128b
- small molecule “modulator” of α-syn aggregation.
- Protective in animal models of PD and tauopathies.
- Still in pre-clinical development.
Anle128b—why use it
- bioavailable, crosses the BBB
- low toxicity
- ameliorates motor deficits in mice
Anle128b FUNCTION
- functions by preventing the formation of more toxic oligomers
- shifts toward less toxic, disordered oligomers (vs, more toxic extended and partially disordered aggregate conformations)
Anle128b discovery as PD drug
found through high-throughput screening of thousands of compounds
Drugs that promote the degradation of α-Synuclein aggregates
- Drugs that increase autophagic-lysosomal degradation of α-syn
- Ambroxol
Ambroxol: what is it
- FDA-approved mucolytic (mucous clearant)
- It promotes folding and stabilization of of the lysosomal enzyme glucocerebrosidase (GBA), as well as its
enrichment in the lysosomes, and promotes α-syn degradation. - basically unorives degration in lysosome
Ambroxol: stage of drug development
Two Phase II clinical trials with ambroxol are testing safety, tolerability and efficacy of the drug.
Glucocerebrosidase (aka: GBA, GCase)
- a lysosomal enzyme
- GB1 gene encodes for GBA
GB1
- encodes for GBA
- Mutations result in Gaucher disease, a lysosomal storage disease (in homozygous patients)
- Heterozygous carriers of GBA1 mutations have a 20-30 fold increased risk to develop PD.
- 7-10% of PD patients carry mutations in this gene.
Relationship between GBA and α-syn
- Increased GBA activity = decreased levels of α-syn and aggregates (and therefore decreased toxicity)
- And decreased GBA = increased α-syn
decreased GBA (aka GCase)
increased alpha-synuclein –> α-syn overexpression cells, PD triplication cels, PD brian–> cycles to decrease GBA –> worsens PD (by increasing α-syn)
How α-syn affects GBA (cyclic)
α-syn impairs normal GBA trafficking from ER (where it is translated) –> prevent protein maturation + localization into lysosomes –> decreased GBA in lysosomes –> glucoceramide (Glc Cer) accumulates –> stabilizes α-syn oligomers and promotes α-syn fibrilation –> some of these oligomers escape the lysosome –> cycles
AMBROXOL can prevent this
Immunotherapy against α-syn
- Passive immunotherapy: PRX002
- Active immunotherapy: AFFITOPE PD03A
HOW: Immunotherapy against α-syn
- Passive and active immunotherapies have been experimented in animal PD models with success (decreased central levels of α-syn, reduction of
symptoms and lack of toxicity).
Potential concerns related to immunotherapy
- limited penetration of antibodies into the CNS
- potential off-target responses and inflammatory
reactions
Passive immunotherapy: PRX002
- Humanized antibodies against αsyn. It decreases plasma levels of α-syn by >90%.
- CNS penetration demonstrated in Phase I trial (but may not pass through in large amounts)
- Currently in Phase II trial
Active immunotherapy: AFFITOPE PD03A
- Vaccine containing α-syn synthetic peptide.
- In Phase I clinical trial in a small number of PD patients
- create anti-α-syn antibodies
PRX002 CNS penetration
CNS penetration demonstrated in Phase I trial
BUT may not pass through in large amounts –> not a super-strong decrease in α-syn –> good because α-syn is important and don’t want to completely deprive it
Glial-derived neurotrophic factor (GDNF)
- promote the survival of DA neurons
- In PD animal models, GDNF was shown to be neuroprotective
Glial-derived neurotrophic factor (GDNF) HOW
- Dimeric GDNF interacts with two molecules of GDNF family receptor α (GFRα1) co-receptor
- The complex recruits the Ret receptor tyrosine kinase, promotes its dimerization and downstream signalling
- GDNF-mediated signalling regulates various cellular functions, including survival and adhesion, it decreases DAT activity and promotes dopamine release
- Severe neurodegeneration in the substancia nigra occurs when GDNF is reduced by 60% in animal models
GDNF has a role in promoting ___ release by ____
- DA release
- decrease DAT activity (increase striatal DA)
- increased phosphorylation of TH (first enzyme in DA synth) –> activate TH and therefore activates DA synth
Failure of GDNF in clinical trials–background
Open-label trials that administered GDNF into the putamen reported improvement of symptoms BUT
A larger double-blind study showed no improvement
Failure of GDNF in clinical trials WHY
Potential reasons for trial failure
- development of anti-GDNF antibodies
- accumulation of α-synuclein in PD neurons disrupts GDNF signaling (Decressac et al, 2012)
- Decreased levels of gangliosides in PD neurons disrupts GDNF signaling (Hadaczek et al., 2015)
