A Study of alpha-Synuclein, Parkin and TDP-43 ; Proteins Implicated in Neurodegenerative Disease.

Foulds, Penelope (2008) A Study of alpha-Synuclein, Parkin and TDP-43 ; Proteins Implicated in Neurodegenerative Disease. PhD thesis, Lancaster University.

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Abstract

alpha-Synuclein (alphaS) is a 14.5 kDa neuronal protein associated with Parkinson's disease (PD) and related disorders. Aggregated protein inclusions called Lewy bodies found in the brain in PD contain alphaS. Rare inherited forms of early-onset PD are caused by autosomal dominant mutations in alphaS or by autosomal recessive mutations in parkin, a 53kDa E3 ubiquitin ligase. alphaS and parkin have been reported to interact functionally, with parkin ubiquitinating a glycosylated, heavier 22-24 KDa form of alphaS (alphaSp22). ELISA assays were developed to investigate if parkin interacts with alpha-synuclein in human blood plasma. Immunoprecipitation followed by immunoblotting techniques were utilised to first capture and then assess the molecular weight of the complex. Ion exchange chromatography and gel-filtration chromatography allowed the complex to be further isolated and characterised. ELISA assays detected the aS/parkin complex in plasma from patients with PD and controls. The alphaS/parkin complex was isolated from plasma by immunoprecipitation, and immunoblotting revealed the alphaS to be the normal 14.5 kDa form. Application of plasma to an ion exchange column allowed isolation of the complex. Autopsy studies have shown that about 55% of patients with frontotemporal lobar degeneration (FTLD) and 25% of patients with Alzheimer's disease (AD) harbour TDP-43 immunoreactive pathological changes in their brains. Again, ELISAs were developed to investigate whether TDP-43 was present, or indeed increased amounts detected, in plasma. Elevated levels of TDP-43 protein were detected in plasma of 46% patients with FTLD with clinical frontotemporal dementia (FTD) and 22% patients with AD, compared to 8% of control subjects. The proportions of patients with FTD and AD showing raised plasma TDP-43 levels correspond closely to those proportions known from autopsy studies to contain TDP-43 pathological changes in their brains. Raised TDP-43 plasma levels may thereby reflect TDP-43 pathology within the brain. This study has confirmed that alphaS and parkin do interact in biological samples. However, the target for parkin does not appear to be the reported 22-24 kDa glycosylated isoform of alphaS, but the unmodified protein. For the first time, the existence of parkin and the complex has been demonstrated in human plasma. Plasma TDP-43 levels may be a biomarker that can provide a laboratory test capable of identifying the presence of TDP-43 brain pathology in neurodegenerative disease during life. These findings may prove to be useful in the diagnosis of neurodegenerative diseases.