NDD - PD Flashcards
Polymeropoulos et al 1997
- they identified the first genetic aberrantion linked to PD in a large Italian kindred and in some Greek familial cases. The mutation corresponded to a C209A substitution in the SNCA gene encoding for a-syn, resulting in a A53T amino acid change (this is important as used in the animal models that will further be discussed). The pattern of inheritance was AD, and the disease was early-onset, usually in the 40s
Singleton et al 2003
Linked SNCA to PD through a different mechanism - identified triplications of the SNCA locus in separate families with an AD inheritance pattern
Maraganore et al 2006
This was a large metanalysis. Focussed on the Rep1 polymorphic region located approx 10 kB upstream of the SCNA transcriptional start site. Looked at 2700 cases and 2700 controls - They found SCNA REP1 alleles differed in frequency for cases and controls (P
Nalls et al 2019
This was a meta-analysis of GWASs - largest so far. Looked at 37000 patients, 17000 so-called proxy cases (individuals with a parent with PD) and 1.4 million controls. Identified 90-independent risk hits. 38 of these were newly identified. Therefore, this study nearly doubled the number of known PD risk variants. Big step forward, Among the identified loci were previously identified rare variants - these included SNCA, LRRK2, CBA The heritable component of PD due to common genetic variability was estimated to be around 22%, and the GWAS loci identified to date explain just a fraction of this. Thus, many more risk variants are yet to be discovered. Further calculations estimate that to make major progress in resolving the basis of this heritable component, we need to almost triple the number of cases used in GWASs. Efforts to extended sample collection genotyping have been successful for other diseases (such as CVD) and have yeilded novel loci - is PD next?
Braak et al 2003
They used a-syn immunohistochemistry in a large number of autopsy cases. Based on a-syn aggregation, they suggested a six-stage scheme/patholgy timelime. Proposed to begin in the lower brainstem and olfactory bulb. Then spreads. Proposed to enter sub nig at stage 3 and by stage 6 the diseases has fully invaded the neocortex, spreading into sensory and motor areas of the brain
Matheoud et al 2019 Nature - interesting
“Showed that intestinal infections in mice engineered to lack the gene Pink1 led to the development of motor symptoms similar to those of pts with PD as well as loss of dopaminergic axonal varicosities in the mouse striaum. Previously, was known that PINK1 had a role in adaptive immunity, repressing presentation of mitochondrial antigens Showed that the infection triggered an immune response in PINK1 KO mice - the gram neg bacterium engages mitochondrial antigen presentation that elicited the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and brain - used ELISPOT analyses - showed anti-mitochondrial specific CD8+ T cells were observed sig upregulated only in brains of the PINK1 KO mice. They then observed the capacity for these specific CD8+ T cells to kill dopaminergic neurons in vitro. They showed that when PINK1 KO neurons and astrocytes were incubated with mitochondrial-antigen-specific CD8+ T cells, the dopamine neurons, but not the non-dopamine neurons (TH+/-) were depleted. This potentially implicates the mitochondrial antigen presentation in the development of PD. “ Limitations - Did not demonstrate that cytotoxic T cells killed the DA neurons in vivo Future directions - Investigate whether in vivo cytotoxic T cells specific for mitochondrial antigens infiltrate the brain and target DA neurons in the sub nig. Should investigate whether Pink1-KO mice show upregulated MHC class I molecules in DA neurons - Matheroud only shows this in culture.
Nemani et al 2010
- cotransfected WT alpha-synuclein with VGLUT1(vesicular protein)-pHlourin into postnatal cultures from ventral rat midbrain.
- pHlourin’s flouresence is quenched by the acidic vesicular environment, fluorescence increases upon exocytosis due to an increased pH and then decreases again during endocytosis and reacidification.
- Therefore, it was used to monitor vesicle exocytosis/endocytosis. Can see it using optical imaging.
- Cotransfected VGLUT-pHlourin with either WT human asyn or empty vector into the postnatal cultures from the rat ventral midbrain.
- The peak fluorescence was significantly lower in neurons overexpressing alpha-synuclein, which indicates a defect in exocytosis.
- To determine whether changes in fusion kinetics or a reduction in the pool of vesicles was responsible, neurons were loaded with the styryl dye FM 4-64 (basically just stains vesicles and amount of dye released by stimulation provides and measure of the vesicle poool available for release).
- Uptake was sig reduced (50% less dye uptake); however, no difference in dye efflux between transfected and control neurons.
- Therefore, alpha-synuclein overexpression seems to reduce the available pool of vesicles Cav - Rat ventral midbrain is only 90% DA neurons
Abeliovich et al 2000
Generated single alpha-synuclein KO mice. Striatal brain slices from WT and KO mice, containing DA neuron terminals from the nigrostriatal pathway were electrically stimulated using paired pulses. Peak extracellular dopamine levels were measured using cyclic voltammetry. WT mice, peak DA levels following the second stimulus were lower than those following the first stimulus for intervals less than 60s. The authors designated this paired pulse inhibition. However, in slices from KO mice, paired pulse inhibition is reduced and recovery of DA release is much faster, which indicates enhanced recycling of vesicles and greater vesicle availability
Lee et al 2001
They demonstrated coimmunoprecipitation of aSYN and DAT (dopamine transporter removing it from cleft) in rat striatal tissue, indicating interactions between these two proteins - Moreover, expression of aSYN, DAT or both proteins in Ltk cells revealed that DAT is localised diffusely when expressed alone, with a significant proportion present intracellularly - However, when coexpressed with aSYN, this intracellular pool was significantly diminished, indicating that aSYN enhances translocation of the transporter to the cell membrane - Furthermore, 3[H] dopamine uptake was significantly greater in coexpressing cells; radioactivity was measured using a scintillation counter - Significantly greater apoptosis was also detected in cells coexpressing aSYN and DAT than cells only expressing DAT upon administration of DA, and this was abolished by uncoupling the a-SYN-DAT interaction
Fountaine and Wade-Martins 2001
RNAi-mediated knockdown of alpha-syn reduced DAT activity in SH-SY5Y neuroblastoma cells. 3[H]-dopamine uptake velocity was significantly reduced in cells with alpha-syn knockdown (up to 50% decrease in DA uptake velocity via DAT) , as measured using scintillation counting. After exposure to the toxin MPP+ which needs DAT to enter cells, cell survival was significantly greater in cells with alpha-syn knockdown (measured with MTT assays and counting live cells) - therefore aSYN knockdown protects from MPP+ toxicity. Further links aSYN to DA transport Cav - RNAi only achieved an 80% protein knockdown of aSYN
Wersinger et al 2003
Demonstrated reduced DAT activity by coexpression w aSYN. - Used very similar experimental set-ups including Ltk- cells, similar transfection protocols and 3[H]-dopamine to measure DA uptake. - The reason for conflicting results with Lee et al is unclear
Fountaine et al 2008
Knockdown of aSYN in SH-SY5Y cells significantly increased VMAT2 number (calculated by ImmunoGold staining). Therefore, aSYn seems to have a negative regulatory effect on VMAT2, reduced DA sequestration. (They also found knockdown using RNAi reduced availabily of DAT on neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of VMAT2 molecules by 2.8 fold)
Xilouri et al 2009
Showed that neuronally-differentiated SH-SY5Y cell lines overexpressing A53T aSYN had impaired chaperone-mediated autophagy (measured using radioactive pulse chase experiments), which is a lysosome-dependent process
- This was associated with toxicity and death, measured using ethidium homodimer labelling
- They found survival of these neurons was increased with pharmacological or molecular inhibitors of macroautophagy (used 3-MA or ATG5-siRNA) - lead to the conclusion that ASYN induces autophagy death in human differentiated SH-SY5Y cells, a death that depends on the activation of macroautophagy
- Authors concluded that expression of mutant A53T leads to CMA dysfunction that leads to a compensatory induction of macroautophagy.
Kamp et al 2010
Overexpressed alpha-syn in SH-SY5Y cells. Using a PEG fusion assay with mito-GFP and mito-DsRed-labelled mitochondria - overexpression of alpha-syn sig decreased mitochondrial fusion. Also showed, by monitoring changes in mitochondrial morphology via imaging of cells transfected with mito-GFP, that increased mitochondrial fragmentation was observed in SH-SY5Y overexpressing alpha-syn. They further found thay in C.elegans, age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier timepoint upon expression of exogenous alpha-syn. The mitochondrial fragmentation induced by expression of aS is rescued by coexpression of PINK1, parkin or DJ-1 with alpha-syn. The PD-associated mutations of these genes did not do this.
Park et al 2006
They generated drosophila with LOF PINK1 mutations. - Found these flies had locomotor deficits - defects in flight ability and slower climbing speed (rescued by PINK1 expression) - found a significant decrease in the number of dopaminergic neurons in the brain in the mutants (using immunostaining with a TH antibody) - they also examined TH-mitoGFP lines, were mitochondria-targeted GFP was expressed in DA neurons, to examine the mitochondria. Using TEM, they revealed grossly enlarged mitochondria. - They note that the phenotypes of the PINK1 mutants were highly reminiscent of parkin mutants generated by the same (and other) groups - Wanted to test whether there were any possible interactions between the two genes - Expression of parkin ameliorated the phenotype in PINK1 mutants significantly; loss of dopaminergic neurons and mitochondrial enlargement were almost completely rescued - This indicated that parkin and pink1 act along the same pathway involved in mitophagy, with parkin acting downstream of pink1; other experiments have shown that pink1 does not rescue the phenotype of parkin mutants
Wade Martins group (2015)
LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function
- Investigated the role of LRRK2 in primary neurons expressing two different LRRK2 mutations of human WT LRRK2.
- They found increased lysosomal pH, decreased lysosomal protein degradation capacity as well as altered lysosomal calcium dynamics in cortical neurons from BAC transgenic rats expressing R1441C LRRK2.
- Importantly, Western blots showed that a1 subuniit was downregulated in LRRK2-R14441C cultures as opposed to WT.
- They then found using coimmunoprecipitations that hWT-LRRK2 and the other mutation bind the a1 subunit of vATPase, which is abolished by the LRRK2-R1444C mutation.
- This v-ATPase is regulates lysosomal pH levels. And it was this interaction that was proposed to increase lysosomal pH and dysregulate Ca2+ dynamics.
Mittal et al 2017
They found the B2 agonists (such as salbutamol) could reduce a-syn gene expression by modulating transcription through alterations of histone deacetylase activity at the a-syn gene promoter and enhancer regions. First, they did an unbiased drug screen to identify B2 adrenoreceptor as a regulator of SNCA gene. Then confirmed this in vivo. They injected a B2 agonist intraperiotoneally into mice and this resulted in sig reduction in nigral a-syn protien and mRNA levels. They also found that B2AR regulates transcription of SCNA - found reduced acetylation across the promotors following B2AR compared to vehicle. Consistent w this, a Norwegian epidemiological study incl 4 million people reported that treatment with albutamol against asthma corresponded with a reduced lifetime risk of developing PD, whereas B2AR antagonist propanolol increased PD risk Limitations Association does not equal causation. Their screen taregtted neuronal SCNA, however B2AR may have additional benefits on glia/inflammation
PRX001
Results from phase 1a = 96.5% reduction in the con of free serum a-syn
Now phase 2
Ghosh et al 2016
Showed MSDC-0160 protected against MPP+ insult in murine and culture human midbrain dopamine neurons and in an a-syn-based C.elegans model. They found in vovo that in MPTP-treated mice, MSDC improved locomotor behaviour, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels and reduced neuroinflammaiton.
Duan and Mattson 1999
Mice were on DR or AL diets for 3 months and then administered MPTP. 24h post MPTP, there was sig impairment of motor function in both groups of mice (decrease in time peirod they were able to stay on the rotary rod). Whereas the deficit in motor function was maintained through the 7 day time point in DR mice, the motor function recovered to near normal levels by 48h in AL mice. Then mice killed after 7 days and brain sections stained with a TH antibody. In AL mice, MPTP caused a hgiih signifcant loss of RH positive neurons compared to saline administered mice. However, there was a sig preservation of TH positive neurons in SN of MPTP-treated mice maintained on DR.
