Parkinson's Lecture 13 PDF

a-synuclein / Lewy Body Pathology: Braak stageing 2003 Braak & colleagues proposed a Braak stageing (6 stages) for a-synuclein/LB progression in PD that relates to PD symptoms From examining neuropathology in Parkinson’s They proposed but did not prove that: 1. a-syn pathology originates in enteri...

a-synuclein / Lewy Body Pathology: Braak stageing 2003 Braak & colleagues proposed a Braak stageing (6 stages) for a-synuclein/LB progression in PD that relates to PD symptoms From examining neuropathology in Parkinson’s They proposed but did not prove that: 1. a-syn pathology originates in enteric neurons (gut) & olfactory neurons (nose) & spreads to brain neurons via vagus nerve & olfactory bulb neurons respectively 2. a-syn is detected in the normal ENS (enteric nervous system) & olfactory neurons and LB have been detected in these neurons in PD – Braak hypothesised that a-syn pathology instigated at these gut/nasal neurons due to environmental pathogens/infectious agents a-synuclein / Lewy Body Pathology: Braak stageing Stage 1 & 2 Stage 3 & 4 Stage 5 & 6 a-syn/LB pathology in lower Brainstem a-syn/LB pathology enters a-syn/LB pathology enters including DMV- Dorsal motor nucleus of SN(Pc) & midbrain The cerebral cortex vagus (nerve)& olfactory bulb Clinical stage Later Clincal stage Preclinical stage = Premotor phase PD motor symptoms Including cognitive symptoms Affecting gut e.g. constipation & olfaction emerge - loss of smell Note: only 50% of patients have pattern of a-syn/LB pathology consistent with Braak staging a-syn/ LB pathology does not always relate to clinical stages of PD a-syn/LB pathology: a-syn “spread” via connected neuronal circuits Kordower JH et al, Nature Med. 14, 504-6 2008 & Li JY et al, Nature Med. 14, 501-3 2008: Fetal neurons transplanted into Parkinson’s disease brain developed a-syn/LB pathology Images of Lewy bodies in grafted /transplanted dopamine neurons Supports that a-synuclein moves spread stained with antibodies against α- between person’s neurons into transplanted synuclein 16 (left) and 11 (right) years neurons over time after transplantation Adapted from Brundin et al. (2008)  a-synuclein /LB pathology: a-syn “spreads” from gut ENS to brain in models in vivo 2019 Fig from Kim et al., Neuron 103 (2019) Transneuronal propagation of pathological synuclein from gut to brain in models of PD Panel 1 Gut injection of pathologic α-synuclein PFFs propagated upward throughout brain in wild type mice caused PD-like brain pathophysiology including of a-syn, loss of DA neurons & motor defects Panel 2 Propagation occurs via vagus nerve as vagotomy protects from PD brain pathophysiology Panel 3 PD-like pathology and symptoms require endogenous α-synuclein as if SNCA is deleted no PD-like pathophysiology NOTE: Others, including the group of Prof. Cora O’Neill, show only a transient spread of a-syn PFFs to the brain when injected into the gut ENS - which disappears after one month  Possible mechanisms for a-synuclein spread/propagation between neurons / cells Very similar to mechanisms proposed for tau spread Taken from Gallegos et al. 2015 NOTE: a-syn shown recently to move/spread between microglia Takeda - Front. Neurosci., 2019 Scheiblick H et al. Cell 2021 Sept 30 – see later lecture on immune system PD 1. Exosomes, tiny membrane vesicles from endocytic pathway, presence of a-synuclein inside exosomes transmission to recipient cells reported. 2. Exocytosis from one cell and endocytosis including receptor mediated endocytosis by other cell shown 3. a-syn associates with membranes via N-terminal, penetrates membrane and gains access to cytosol 4. Tunneling nanotubes (TNTs) = directly membrane bridges of actin between cells, a-syn spread shown to occur via TNTs in vitro 5. Dying neurons reservoirs of pathological α-synuclein when they expel their contents after degeneration  a-synuclein & mitochondrial function Mitochondrial function can regulate a-synuclein function, a-syn levels/fibril formation MPP+ can increase a-syn levels/aggregation but no LB Conversely a-syn species -monomers oligomers & fibrils regulates mitochondrial function Including: Complex 1 activity ; ATP-synthase function Induction of mitophagy/ fragmentation and energy depletion a-syn can bind mitochondrial membranes via N-Terminus One theory Monomeric α-synuclein interacts with ATP synthase and improves the efficiency of ATP synthesis. Oligomeric α- synuclein also interacts with ATP synthase but conversely, impairs respiration, and depolarises mitochondria -> activating cell death pathways Ludtmann 2018 Nat Commun  PD mechanism: Mitochondrial defects History of focus on mitochondria in PD 1983 – Langston et al., reported young patients who developed acute levodopa responsive parkinsonism very rapid but indistinguishable from PD Caused S.Nigra degeneration, no LB formation Owing to self -administration of toxic heroin analogue MPTP – 1 methyl-4 phenyl-pyridinium MPP+ = toxic species destroys dopaminergic neurons  PD mechanism: MPTP toxin highlights how mitochondrial defects can lead to PD MPTP à crosses BBB à enters brain à enters glial cells à converted to MPP+ using enzyme MAOB (monoamine oxidase B) in glial cells MPP+à exits glial cells à taken up selectively into DA neurons by DA transporter (DAT) as MPP structure similar to DA MPP+ à mitochondria in DA neurons à inhibits mitochondrial Complex I activity and impairs mitochondrial function / bioenergetics ATP generation/electron leakage/oxidative stress/inflammation Supporting this 30-40% decrease Complex I activity detected SNpc neurons of PD patients Nature Reviews Neuroscience 4; 365-375 (2003); doi:10.1038/nrn1100 Targeting programmed cell death in ND MPTP/MPP+ does not result in LB in people or animal models but can increase a-synuclein levels and fibril formation SNCA (a-syn) gene deletion can protect v/s MPTP in some models  PD mechanism: mitochondrial defects & defective Complex I since MPTP discovery – extensive search for other environmental toxin working like -but slower than- MPTP to block Complex I /or overall mitochondrial function that could cause PD – not generally successful- BUT Rotenone Insecticide/pesticide derived from plants Well known inhibitor of Complex I Lipophilic can cross membranes & BBB Greenamyre group showed chronic infusion of very high doses of rotenone in rats – that would never happen in reality- could reproduce PD pathophysiology S. nigra degeneration, a-syn pathology motor defects, Gut / peripheral defects rotenone can also induce PD-like pathophysiology if administered to gut  PINK1: PD causing mutations ~>40 mutations identified point, deletions, whole gene heterozygous deletions to cause Parkinsonism (2004) all predicted to cause loss of function Muquit et al., Protein Phosphorylation Unit, Dundee University e.g. G309D and L347P missense in kinase domain inhibit the function of PINK1 by blocking its kinase activity W437OPA- nonsense truncates last 145 amino acids of kinase domain C-terminus – inactive PINK1 produced and is unstable therefore, loss of PINK1 function causes Parkinsonism  PINK1 function and mitochondrial health PINK1 REGULATES MAJOR MITOCHONDRIAL FUNCTIONS mitophagy, mitochondrial fission/fusion, clear role for PINK1 in: MDVs control of mitochondrial function/health Presentation of Mitochondrial cell mitochondrial death /survival protecting neurons from antigens pathways mitochondrial & oxidative /immune damage & promotion of cell survival in response to stress loss of these functions damaging to neurons especially DA neurons à PD Mitochondrial Bioenergetics transport in Complex I axons/neurons regulation PINK1 function: deletion of PINK1 Mice null models/ knock out - no major PD or other phenotype Matheoud 2019 Nature: Intestinal infection triggered PD-like symptoms in Pink1−/− mice Model proposed: Intestine exposed to gram negative bacterium à dendritic immune cells without PINK1 present a mitochondrial antigen à recognized by cytotoxic T cells which kill DA neurons by “autoimmune attack” Primate PINK1 -/- via CRISPR/Cas9 show neurodegeneration. Li et al., 2019 Cell Res  PINK1 and Parking interaction: mitophagy Geisler et al, Nat Cell Biol. 2010 Narendra et al., PLoS Biology 2010 More recent papers See Youle as author healthy mitochondria- PINK1 maintained at low levels mainly in cytosolic part of cell /neurons & mitochondrial PINK1 cleaved for degradation malfunctioning mitochondria with associated depolarisation of the mitochondrial membrane potential à PINK1 is recruited to mitochondria and stabilized PINK1 recruits Parkin to the mitochondrial surface à phosphorylates both Parkin and ubiquitin end-result à disposal of the damaged MT by ubiquitinylation & mitophagy à through Parkin activity - both UPS & EAL pathways implicated Note: PINK1 can also function in mitophagy without Parkin via other ubiquitin ligases e.g. ARIHI promotes survival in cancer cells (Cell 2017 Sept) Parkinson’s mechanisms Protein clearance: the endosomal autophagic lysosomal system  Parkinson’s: molecular mechanism: impaired clearance of damage proteins Progressive synaptic transmission failure Diminished trophic / metabolic support, Mitochondrial In PD, a-synuclein accumulates in soluble defective calcium & defects & oxidative stress oligomers, fibrils and aggregates survival signalling Implicating impaired function of protein clearance pathways & Inflammation & proteostasis in PD neurons defective immune Defective: protein response clearance, vesicular trafficking, autophagy Protein clearance pathway failure can also cause a-synuclein “spread”  Parkinson’s: molecular mechanism: impaired clearance of damage proteins Major routes for protein clearance in cells B. Lysosomal degradation system more broadly A. The Ubiquitin Proteasomal System termed EAL Endosomal Autophagic Lysosomal UPS system or simply AUTOPHAGY EAL EAL defects more strongly implicated in PD than UPS defects Parkinson’s: molecular mechanism: impaired clearance of damaged proteins focus on EAL: autophagy 3 4 1 2 5 Model for macroautophagy = major form of autophagy. 1 and 2 A portion of cytoplasm enclosed by autophagic isolation membrane, membrane elongates & encapsulates cell components for degradation 3. eventually results in formation of a double membrane structure = autophagosome. 4. Outer membrane of autophagosome fuses with the lysosome to form autolysosome 5. the cytoplasm-derived materials are degraded by lysosomal enzymes Note: Many key proteins and also membrane lipids are essential for this process What is autophagy? Autophagy is essential for a cell to survive and function. Recycles damaged cell parts into fully functioning cell parts. Gets rid of nonfunctional cell parts that take up space and slow performance. Destroys pathogens in a cell that can damage it, like viruses and bacteria. Regulation of autophagy Starts with phagophore formation, elongation, and maturation of autophagosome, until the autophagosome-lysosome fusion. Building an autophagososme The phagophore is the first autophagy-related organelle to be formed, prior to autophagosome, in response to a variety of stresses, including nutrient deprivation. The phagophore most probably grows via lipid acquisition Despite the identification of most of the regulatory proteins involved in phagophore formation, closure, and maturation, the origin(s) of the membrane(s) that directly participate in its biogenesis is still unclear. A consensus suggests that the omegasome, an ER membrane transient subdomain, serves as an assembly platform to promote phagophore biogenesis. In addition to the omegasome, many endomembranes have been linked directly or indirectly to the phagophore biogenesis: Golgi vesicles, endosomes and endosomal associated vesicles and tubules, mitochondria, lipid droplets, and plasma membrane. While a lot of proteins have been identified, it is still unclear how an autophagosome forms. Nobel prize for discovery of mechanisms for autophagy 2016  2016 October Nobel Lecture Autophagy in Health and Disease Cells use autophagy to eliminate damaged proteins, lipids, organelles, essential to counteract aging especially in postmitotic neurons 3 4 1 2 eliminate invading intracellular bacteria / viruses 5 sculpt systems during embryonic development Respond to stress/starvation provides fuel & building blocks § Mutations in autophagy genes / disturbance in system linked to many diseases of ageing: neurodegeneration including PD/AD; cancer, diabetes § Autophagy being targeted in several disease including PD & AD PD mechanisms Focus on protein clearance defects EAL and UPS systems  Parkinson’s: molecular mechanisms focus on endosomal-autophagic lysosoma (EAL) autophagy § a-synuclein impairs lysosomal function & impaired lysosomal function increases a-synuclein build up, aggregation & spread § Activation of TFEB (Transcription factor EB): increases lysosomal biogenesis & lysosomal activity § clears a-synuclein pathology & improves PD symptoms in animal models § EAL being targeted therapeutically in attempts to normalise EAL in PD § Neurons & synaptic vesicle release requires very effective EAL systems associated with mitochondrial efficiency & protein health. § The interplay between these systems may “miswire” à a-synuclein accumulation a-syn fibril formation/ spread which in turn can impair these systems à progressive degeneration of DA neurons/ circuits in PD Parkinson’s: molecular mechanisms focus on EAL: autophagy § Number of PD risk genes (familial & idiopathic) encode proteins linked to lysosomal function e.g. LRRK2, lysosomal enzyme b-glucocerebrosidase (GBA), PINK1, PARKIN, SNCA , VPS35 - SEE LATER LECTURES ON RISK GENES Beilina and Cookson J Neurochem. 2016 review links between genes for Parkinson's (red) and the autophagy– lysosomal system. They propose many of PD genes can be assigned to pathways that affect (I) turnover of mitochondria via mitophagy (II) turnover of several vesicular structures via macroautophagy or chaperone‐mediated autophagy or (III) general lysosome function ESCRT mediated cargo sorting Ubiquitylated (Ub) cargoes are recognized by the ESCRT proteins. ESCRT 0,1 and 2 recruit ubiquitylated proteins into a subdomain. Finally, ESCRT 3 spiral filament surrounds the cargo, and its assembly brings about dissociation of the early ESCRT complexes. Growing ESCRT-III filament buckles from a flat structure into a three-dimensional spring, thereby deforming the membrane in to the budding ILV. Parkinson’s: molecular mechanisms focus on UPS: Ubiquitin Proteasomal System UPS § major non-lysosomal route for degrading misfolded proteins In the UPS pathway energy from ATP is used to tag an unwanted protein with a chain of ubiquitin proteins (PINK) = ubiquitination this marks it protein for destruction –IMAGE AND TEXT Michal Sharon’s lab, Weizmann Inst. SEE VIDEO U TUBE: SCOTISH ENTERPRISE UBIQUITIN PROTEASOME SYSTEM  Parkinson’s: molecular mechanisms focus on UPS: Ubiquitin Proteasomal System UPS system § highly ordered process § Lys on target protein (green) tagged by multiple ubiquitin proteins (pink) § 3 step enzymatic (E) process E1, E2, E3 needed for each Ubiquitin added E1: Ub-activating enzyme E2: Ub-carrier protein Ub-conjugating enzyme E3 : Ub-protein ligase Ubiquitin tagged proteins are tagged for destruction undergo proteolytic degradation by 26S proteasome (red and blue) 2004 Nobel prize Chemistry “discovery to ubiquitin mediated protein degradation” Aaron Ciechanover, Avram Hershko & Irwin Rose  Parkinson’s: molecular mechanisms focus on UPS: Ubiquitin Proteasomal System 1. a-synuclein aggregates are surrounded by ubiquitin in LBs => defects in a-synuclein clearance by ubiquitin 2 in turn a-synuclein fibrils may also impair aspects of UPS 3. mutations in genes encoding proteins that work in UPS cause familial PD e.g Parkin is an E3 ubiquitin ligase -see later lectures 4. Parkin ubiquitin ligase activity is essential for clearing damaged mitochondria = mitophagy linking UPS defects directly to mitochondrial dysfunction in familial PD One possible model for idiopathic PD Loss of UPS function à decreased mitophagy à Defective mItochodrial function à including increase ROS à a-syn fibril formation à may also induce vicious cycle to impair aspects of UPS Parkinson’s: molecular mechanisms Progressive synaptic transmission failure Diminished trophic / metabolic support, Mitochondrial defective calcium & defects & survival signalling oxidative stress Inflammation & defective immune Defective: protein response clearance, vesicular trafficking, autophagy