PD Flashcards
briefly outline the physiological role of Pink1 and Parkin and explain how their mutations contribute to the pathology of Parkinsons disease?
Pink1 and Parkin are part of the primary mitophagy pathway.
In this mechanism Pink1 acts as an ignition key and damge detector, under basal conidtion it is taken up by the TOM complex and degraded through a sequence of clevages. However, following mitochondrial damage the mitochondria becomes depolarised and thus cannot take up PINK1 and it begins to accumulate on the OMM due to anchoring by TOM complex subunit Tomm7.
PINK1 fascilitates the colocalisation of Parkin (as shown studies showing ectopic expression of PINK1 on the OMM triggered Parkin recruitment and KO studies showing the loss of Parkin recruitment.) also ectopic expression of parkin alone cannot cause mitophagy.
PINK1 then phophorylates two targets at their serine 65 residues. Ubiquitin and Parkin. This fasciliates the intercation of the 2 proteins. (Parkin is phosphorylated at ubiquitin like site to stabilise its confirmation following ubiquitin binding)
the binding of now pUB to Parkin drives a conformatonal change in which the UBL is released from the parkin core and thus removing the autoinhibition of parkin.
ubiquitinated and activated parkin will now go on to ubiquitinate several down stream targets to aid the seperation of damaged mitochondria from the mitochondria network.. It will also go on to ubiquitinate OMM proteins to build and extend denovo and exisiting phospho-ubiquitin chains. These are recognised by autophagic adaptor proteins to drive the recruitment of the autophagesome machinery.
The autophagesome will then engulf the damaged mitochondria and transport it to lysosomes for degredation.
Hence, mutations in Parkin and PINK1 found in monogenic forms of PD found in a japanese (matsumine) and itallian (valente) respectively cause disrupted mitophagy. This leaves neurons without the appropriate machinery to regulate the quality of their mitochondria resulting in the accumualtion of damage and dysfunction evventually resulting in neuronal death.
Describe the main evidence for and against alpha-synucelins toxicity (differentiate between protofibrills in aggregtaes vs in soluble oligomers
alpha synuclein forms oligomers called photofibrils through fibrillation. The can go on to form large insolu ble aggregate termed lewy bodies.
+A53T and A30P aggregate promoting and can cause monogenic PD.
+ Triplication or duplication of the SNCA gene has been reported in family lines causes dose dependant increase in disease progression. suggesting Key role.
+Dopamine points in another card linked to CMA inhibtion.
+ overexpressing wiltype alpha syn in SH-SY57 cells resulted in increased ROS release shown by oxidant sensitive-dye. This was worsened by overexpressing mutan forms like A53T. (however also can form soluble oligomers like in mazzuli et al 2011)
+ dopamin stabilised mutant forms and increased aggragtions and degeneration in cell lines.
+ve lewy bodies are a primary clinical charcteristic of PD and their spread reliably tracks disease progression in Braak staging, suggestion they cause neuronal death. there, is very little better explantion of spread of disease.
+ve LUK ET AL 2012 direct Unilateral injections of protofibrils into striatum showed progressive formation of cytoplasmic inclusions that devloped into perinuclear inclusions reminiscent in appearance and location to lewy bodies, as well as containing many of the subunits like ubiuitin and Heat shoch proteins that are attriubuted to HUman LBs.
- Inclusions correlated with a porgressive decrease i TH immunorecativity suggesting a Degeneration of dopamienrgic neurons.
- This correlated with a porgressive reduction in motor ability tested on rotarod tests.
- These fetaures where not seen in the other hemisphere which resembled that of monomeric Alpha syn injected controls.
- This sugggests that photofibrils are sufficient to replicate the phenotypes of PD alone.
- ve However, Often neurons with lewy body inclusions survive and studies have shown co-expressing with synphillin which prevents their breakdwon increased survival.- shown by a study using synphillin to prevent the targetting of alpha syn for breakdwon by Blocking STAHI. This increased aggeragtes and increased survival.
- ve expression of mutants in transgenic mice has not been able to induce dopaminergic neuronal death alone despite increasing their suceptibility to oxidative stress.
there is large debate over why wee see this discrepancy many now argue to insoluble aggergates, lewy bodies, are not the primary toxic species. in fact it is the soluble portion that are exttrenmely toxic, hence why we struggle to directly relate alpha synulcein to toxicity. studies expressing E35K and E57K mutations that promote soluble oligomers (Winner et al 2013) found they were more toxic.
Describe the primary findings and aplications from Mazzuli et al 2011
Mazzuli et al 2011-
This stduy investigated Gauchers disease a lysosomal disroder in which 20% of pateints can display parkinsonism. This is in cases type 2 and 3 where there in nerualdegeration. they wanted to investigate whether neuronal death could be atribbuted to alpha synuclein.
Firstly they use short hairpin RNA to knock down glucocerebrocidase (Gcase) the protein that is KO in GD. they found this caused a increased neurotoxicity and concomitant increase in alpha synuclein expression in cell cultures.
secondly they investigated how it increased alpha syn toxicity. They carried out Gcase KD in cells expressing the A53T mutant (increased fibrilliation) or artificially fibrilation inept delta (triangle) 71-82 alpha syn. They found that Gcase KD only increased toxicity with A53T showing that fibrillation was neccessary.
Western blot- Further investigation showed Gcase KD lead to a particular increase in insoluble fibrils and soluble oligomers. In particular their was an increase in S soluble high weight molecular form. To show this was the result of LOF of Gcase and no just lysosomal dysfunction they using leupeptidin to cause lysosomal dysfuntion and reported neither increased toxicity of a HMW alpha syn.
Looking at elution profile on SDS page- following from this they tried to explain it from the ffects of LOF of Gcase this being the accumualtion of its substrate Glucosylceramide (CLcer). They found Glcer accumulation stablised the soluble oligomeric forms of alpha syn prolonging conversion into fibrils. showing that purified solutions with higher proportions of Glcer would increase alpha syn soluble accumulation compared to controls.
SUGGESTS increased toxicity is primrily soluble forms.
Looking in human samples with GBA1 mutant linked Pakinsonism they used Mab like syn211 or pathogenic oligomer slective SYN303 to report increased abunance of toxic oligomer alpha syn not seen in healthy controls.
To assess this invivo they investigated the effects in GD mouse models with Gcase defficiency. They reported accumualtion of alpha syn species and neurodgeen suggested from eosinopillic cells found in areas like the SNpc.
To investigate the impact of alpha syn accumualtion oN Gcase function they again expressed A53T or defficient 71-82 alpha syn in inducible H4 cells. they then assessed for the abundance of pre and post-ER Gcase (immature and mature) (assessed using endo-H analysis). They found a inhbition of Gcase maturation which was fibrillation dependant.
Thus they believe to have idnetified a toxic pathogenic loop in which Gcase defficieincy or the accumualtion o toxic soluble alpha synuclein oligomers can trigger the other contributing to neurodegen and pottentially explain the comorbidity of enruooxic forms of GD and PD.
Describe and assess at least 4 pieces of evidence for and against the role of disrupted mitophagy in PD pathology.
Monogenic PD- a mutation in japanese, matsumine et al, and a itallian fammily, valente et al. were discovered causing monogenic EOPD. this truned out to be muations in Parkin, an E3-ubiquitin ligase. and PTEN induced putative kinase 1 (PINK1). these are now recognised as core parts of the prinmary mitophagy pathway.
Vincow et al 2013- they investigated the impacts of Parkin KO and general autphagy KO through ATG7 in the runover of mitchondrial proteins. expectedly ATG7 having both general and effects downstream of Parkin had a larger overall effect. However, in 53% of all MRC proteins inlcuding all 5 complexes the trunover was more dependant on Parkin (KO led to longer turnover). simmilar but less significant findings were found for Pink1. this isndicates the the parkin-pink1 mitophagy pathway has a speccific role in regulating MRC function. Given the link of mitochondrial dysfinction to PD, particualrlly complex 1 efficieny indicated by scahpira et al this again gives an intircate link between mitophagy and dysfunction seen in PD.
PINK1 and complex 1 efficacy. KO of PINK1 in drosophilla has been shown to decrease the functionality of complex 1 and decrease the repiratory capacity of the the MRC. Villain et al 2012 also used the expression of yeast complex 1 to reverse the effects o PINK1 KO again making this link.
Pickrell 2015- invented the most conivincing invivo recapitualtion of PD. he crossed a Parkin-ko AND the mutator-mouse (polg mutation) to produce te Parkin-KO mutator. Both phenotypes were shown to be essential to the phenotype. Motor defficiency shown on pole tests, the first model to show dopaminergic neurodegen shown by TH staining and nissle stain counts, slectiv degen as shown by normal number in Neun marking in other cortical areas. Meets PD criteria as after 4 weeks of L-dopa reatments the motordeficits were much improved. Hence, this showed bpoth mitochondrial damage and deffiecient mitophagy were needed for neurodegen. As the myuatotor pheotype confers an accumualton of mtDNA mutations and thus mitpohcnodrial dysfunction something that been shown to occur over normal life with againg this can be seen as acclerating te nrmla animals aging process and thus suggesting the loss of mtiophjagy is the key factorin inducing neuronal detah throigh the risk of age in PD.
Sun et al 2015- used the mt-keima mouse model to track basal levels of mitophagy/.
showed that it was heterogeneous acorss the brain with ares liek the DG having high levels and areas like the the SNpc having intermediate levels. Interestingly KO of autophagy via ATG5/7 KO led to almost complete KO in the intermediate areas but some remained in others. hence, this implies a varienace in redundnacy which may explain slective degen in diurupted mitopahgy.
showed that mitophagy declined with life between a 3month and 21 month old neuron. again supporting this idea that a loss of mtiophagy function was key to the risk of age in PD. this is furter supported by the expression of the mutator phenotype increasing mitophagy rate showing that our cells usually deal with increased stress and thus the loss of mtiophagy is more significant in terms of toxicity
is there any indication of an overlap between alpha-synuclein pathology and disrupted mitophagy in PD,
it is possible that alpha syn mediates its toxicity through disrupting mitophagy.
LI etal 2019 recently showed that the toxicity of defficent Gcase function i partly mediated through a loss of mitophagy function shown using mt-keima trangenic mice expressing a common Gcase mutation (L444P) homozygously. they were able to attribute this to reduced expression of proteins involved in mitochondrial priming, showed by wetstern blot, and deffiecieny in the induction of autophagy, shown using rapamycin to induce autophagy through te inhibition of MTOR and showing a reduced increase in the formation of autophagic vacuoles in GD modles than controls. (60 vs 25%)
Given the existence of a Gcase alpah syn pathogenic loop, identified by mazzuli et al 2011 this could present a pathogenic mechanism for this.
Disrupted mitophagy may fascilitate alpha syn acucmualtion.
Chung et al 2016 showed that in IPSCS PINK1 over expression led to a reduction in alpha syn accumualtion.
Schlossmacher et al reported that Parkin colocalised with Alpha syn in lewy bodies.
FARRER ET AL Hence it is possible mitophagy mechanisms play a defensive role against the toxic species of alpha syn, this would explain why Farrer et al reportred in some Parkin lof exmaples no lewy bodies are seen. this being because the parkin sequestering role is lossed.
oultine the primary pathology and symptoms of parkinsons disease.
Parkinsons stems There is progressive degeneration of dopaminergic neurons (DAs) in the Substantia nigra pars compacta (SNpc) (Gröger et al,.2014), this results in the loss of dopaminergic activity in the Nigrostriatal pathway projecting to the striatum ; Existing as both the posterior head of the caudate and the putamen, the striatum is the hub that drives wanted movement and prevents those unwanted. Nigrostriatal activity at D2 dopamine receptors usually excites the direct pathway to disinhibit the motor relay in the thalamus. Activity at D1 receptors, inhibits the obstruction of motor drive through the indirect pathway. Therefore, motor drive is obstructed in PD (Hisahara and Shimohama, 2011).
This results in a deffiency in voluntary movements. Patients often show slow distorted movement bradykinesia, cog like rigid joints, classic resting tremor starting unilateal and progressive to a bilateral experience.
LEWY BODY DEPOSITIONS.
there are also pre and post-motor symtpoms
pre- prior to motor symptoms 90% will experince olfactory deficits and 50% can be depressed.
Post- after onset of motor sympotms patients can develop dementia further down the line and many develop a bent over posture.
what form of neurodegenrative disease does PD fall under?
PD is a sycleineophathy
what ar lewy bodies? state the two disease they are best associted with.
Lewy bodies are alrge insoluble aggregates of alpha synuclein primarily and many othe proteins such as Parkin and ubiquitin. they are primarily asociated with the sequestering of toxic alpha syn species.
They are associated with PD and dementia with lewy bodies.
outline the 3 pathways modulating motor activity through the striatum?
Direct- This promotes movement. receieving excitatory input from the cortex the GABAergic neurons in the striatum prpject to and inhibit the internal segemtnof the globus plaidus. this usually prjects to an inhibits the thalamus and thus thispathwa dishinibits the thalamus fascilitatijng the relay of motor comands and drive to the motor cortex. this is modulated by the SNpc through the nigrostriatla pathway. dopamiergic neurons acts of D1 receptors to excite the drive of movement.
Indirect- This inhibits movement, This time the striatum projects to the external segemnt of the globus pallidus this time disinihibiting the subthalamic nuclei. The subthalmaic neucleis excitatory projects can then excite the internal segemnt of the globuc pallidus to inhibit the thalamus and stop movement. This is also modulated by the SNpc, this time dopaminergic neurons acts on inhIBITORY D2 receptors to turn off this pathway.
Hyper direct- inhibits movement through direct projections form the coretx to the subtlamic nuclei.
Given an example of how the reduced dopamine population of the SNpc is demonstrated through imaging
Brook et al using single positron emission computed tomography (SPECT) to mark for the dopamine uptaker showing a much small level in the SNpc of PD patients. other studies have used a the sma method to demonstarated the progressive decline.
provide support for mitochondrial dyfunction in PD (in particular mitochondrial resppiratory chain (MRC)
Schapira et al reported reduced Complex 1 activity in the PD brain.
MtDNA muations- studies investigating the brain in postmortem samples have reported a life long accumualtion fo mtDNA mutations demonstarted by increased levels of fractionated MtDNA. These muattions were correlated with a defficieny in the function of cyclooxidase (COX) a key MRC mediator.
Bender et al have shown that the SNpc of PD patients has significantly higher mtDNA muations. hence this correlates with disease and MRC dyfunction.
MPTP example- MPTP is a contaminant stemming from the incorrect production of the recreational drug MPPP. this drug when used is taken up and metabolised by mono amine oxidase B forming the toxic protein MPP+. This has an very high affinity for the dopamine uptake transporter and thus selectively accumulates in dpaminergic neurons. it then will translocate to the mitochindira where it will inhibit complex-1 of the MRC halting ATP production and resulting in neuronal dysfunction and death specifically in dopaminergic neurons.
What features of SNpc DA neurons might make them particuarly sucetpible to mitochondrial dysfunction?
Like all neurons that produnction of APS and maintencae of the membrane pottential is an energ demnading process requring ATP. the same can be said for axonal transport.
Dopamine neruons in particular have a autonomous activation know as pace maker activity. this produces slow wave APs uniquely reliant on calcium influc. calcum in toxic if not remove dand thus is through active mechnaims making the nergy demnd even high..
when the MRC produced ATP it also [produced dmaging reactive oxygen species like superoxides which can damge the mitchondira and mtDNA. this increases oxidative stress and reduces the efficacy of the MRC. hence dopaminergic neruons are more succeptibel to this.
outline the role of oxidative stress and ROS release may cause in disease? how can this be dealt with?
ROS release can damge cell proteins, mitochondrial walls and mtDNA. this causes oxidative stress which fascilktates more dmage, more ROS production and more dysfunction. ROS molecules can also act as signalling molecules between mitochnidra inducing a wave of mitochondria release troughout the mitochjndrial network, this is ROS induced ROS rlease.
to stop this and maintain neruonal health dmaged mitochindria must be seperated from the mitochindrial network and destroyed via mitophagy.
ANTIOXIDANTS like superoxide dismutase 1 (SOD1) are vital here. SOD1 converts superoxide to hydrogen peroxide.
Defficiencies of antioxidants SOD1 and Glutathione
increased iron levels, failed iron chelation linked to the conversion of hydrogen peroxide to toxic species.
What is PARK2 and what is PARK6
Park2 is the loci of Parkin and Park6 is the loci of Pink1 (PTEN induced putative kinase)
Outline the primary findings from FIESEL 2015. how does this support disrupte mitophagy in PD?
fiesel used Pub as a marker of the parkin-pink1 pathwy. he used the mitochondrial uncupler CCCP to depolarise mitochnodria and mimic damage .he showed that pink1 was neccesary for accumuatlion of Pub, this makes sense with the pathway as UB is a PINK1 substrate. Parkin was not neccesary but boosted accumualtion which is intouch with parkin parimily being a Pub substrate and the feedforward pottentiation.He also showed the stress induced accumualtion of Pub co localised with stains of Parkin and of TOM complex markers for mitochindria, so the pathway is functional in humans
invetsigating PUB ACCUMUALTION SIN diseaseowed accumualtion in normal PD patients and controls but in PINK1 KO mutants there were no snPC accumualtions. ence this is is evidence that this pathwa is infact deffiencient in at least monogenic PD.
