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4.11.1 Alpha synuclein, Parkin and UCH-Ll-are they all involved in a common pathway in PD?

Several investigators have speculated on the fact that Parkin, a-synuclein and UCH-Ll may be interacting proteins in a common pathway. Mutations in these genes encode altered proteins which may then give rise to the clinical phenotype of PD. Environmental factors may also play a possible role in gene expression. Studies in normal human brain show that expression of each of these three genes was predominantly neuronal. Alpha-synuclein and Parkin mRNAs were expressed in a restricted number of brain regions, whereas UCH-Ll mRNA was more uniformly expressed throughout the brain. The melanin-containing

dopamine neurons of the substantia nigra had particularly robust expression. The

expression pattern of a-synuclein and Parkin mRNAs were similar, suggesting that the two proteins may be involved in common pathways contributing to the pathophysiology of PD

such as ubiquitination (Solano et al., 2000). UCH-Ll mutations may alter ubiquitin

recycling.

4.11.2 Genes, the ubiquitin-proteosome pathway and nigral degeneration

Ubiquitin, a small protein consisting of 76 amino acids, has been found in all eukaryotic cells studied. It is one of the most conserved proteins known. Ubiquitin is required for ATP-dependent, non-lysosomal intracellular protein degradation, which eliminates most intracellular defective problems as well as normal proteins with a rapid turnover (Baker and Board, 1987). Degradation involves covalent binding of ubiquitin to the protein to be degraded, through isopeptide bonds. The function of ubiquitin is presumed to be labeling of the protein for disposal by intracellular proteases. Ubiquitinated

histones are present primarily in actively transcribed chromosomal regions, so ubiquitin^ may play a role in regulation of gene expression (Baker and Board, 1987).

Animal models of neurodegenerative diseases have also been associated with

progressive accumulation of ubiquitinated protein conjugates (Yamazaki et al., 1988).

findings suggested that altered function of the ubiquitin system may directly cause neurodegeneration (MacDonald, 1999). The role of ubiquitin in a variety of

widely supported (Lowe et al., 1988). Since most misfolded proteins are degraded via the ubiquitin-proteosome pathway, defects in this pathway may have a central role in neuronal degeneration. Specifically, impaired proteosomal degradation of abnormal proteins, such as alpha-synuclein and Parkin, or defects in the pathway by, for instance, mutant UCH-Ll, may underlie the pathogenesis of PD.

Shimura et a l, 2 0 0 1 showed evidence of a functional link between mutant parkin and

a-synuclein in the cell via the process of ubiquitination, an intracellular mechanism for targeting proteins for degeneration. They proposed that an accumulation of non- ubiquitinated a-synuclein aSp22 may lead to cellular toxicity. If nigral neurons are particularly sensitive to these accumulations, the end result could be nigral degeneration and PD. A summary of the main evidence provided is as follows: a) Parkin is an E3

ubiquitin ligase, recruiting ubiquitin conjugating enzymes UbcH7 and UbcH8 as well as

recognising the target for ubiquitination; b) Lewy bodies contain Parkin, ubiquitin and poly ubiquitinated a-synuclein, but LBs are not found in brains of patients with parkin mutations; c) Parkin colocalises with a-synuclein and UbcH7 in purified presynaptic fractions of brainstem; d) Parkin co-immunoprecipitates (co-IPs) UbcH7 and alpha- synuclein, including a novel glycosylated 22kDa isoform of a-synuclein (aSp22) in these fractions, but does not co-IP these substrates in homogenates of ARPD brains. Therefore, the authors proposed that mutant Parkin either: a) fails to recognise and bind aSp22 at parkin’s N-terminal Ubl domain, or b) fails to recruit UbcH7 via parkin’s RING box. The result is an accumulation of nonubiquitinated aSp22 leading to accelerated neural toxicity. In other forms of PD, functional Parkin mediates

ubiquitination of aSp22 and other substrates, but these fail to be appropriately targeted to the proteasome (for other genetic or environmental causes, such as mutant a- synuclein) and accumulate in Lewy bodies. Although there are likely to be additional targets for Parkin in brain, it seems to show remarkable specificity, as beta-synuclein is not targeted for ubiquitination. Other E2 proteins may also be recruited by Parkin, as a Parkin pool is found at postsynaptic terminals which do not contain UbcH7 or a-

synuclein (Shimura et al., 2001). Mutations in the ubiquitin C-terminal hydroxylase,

UCH-Ll may also cause peturbations in this pathway. Figure 4.10 shows the hypothetical pathway for the proposed interaction of Parkin with Ubiquitin. It is

proposed that the C-terminal RING-box domain of the Parkin protein recruits a specific E2 enzyme (UbcH7) and the N-terminal Ubl domain required for recognition of the

target protein, designated ‘X ’, for ubiquitination before proteosomal degradation (see Figure 4.10).

Synuclein pathology appears to be a prominent feature of some neurodegenerative diseases (such as PD, MSA), yet a secondary pathology in others (such as AD). Lewy bodies occur in families with either amyloid precursor protein (AFP) or presenilin

mutations (Lippa et a l, 1998). This surprising observation has been extended to prion

diseases where Lewy bodies have been described in association with tangle/tau

pathology (Piccardo et a l, 1998). Farrer et al., 1999 suggested that, with respect to

AD at least, it seems as if the tangle/tau pathology of the Lewy body/synuclein pathology are alternative responses to the primary lesion, because cases of AD with little tau pathology have much synuclein pathology and vice versa. From the limited components identified to date, it is possible to speculate that these proteins may functionally overlap in a common pathway of cytoskeletal maintenance and

intracellular vesicle transport. The normal function of a-synuclein may depend on an ability to undergo a conformational change in the presence of specific phospholipids

(Perrin et al., 2000). Nitrated a-synuclein is present in the major filamentous building

blocks of Lewy bodies, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of a-synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and

progression of neurodegenerative synucleinopathies (Giasson et al., 1999).

Despite experimental progress, how a-synuclein, Parkin and UCH-Ll interact remains largely speculative at present although the discovery of mutations in all three genes, other PD loci and recent biochemical studies have enhanced molecular exploration of PD and may also lead to a future revolution in our understanding of other

neurodegenerative diseases that are characterized by involvement of abnormal protein handling including MSA, AD, other tau-opathies, GAG repeat disorders and

amyotrophic lateral sclerosis. Novel genes are still to be discovered which will further enhance our study of the aetiology of PD as described in chapter 5.

F ig u re 4 .1 0 M o d el o f the in v o lv e m e n t o f Parkin in the ubiquitin ation p ath w ay. S e v e r a l other proteins are also lik e ly to be in v o lv ed . Key: U b , ubiquitin; E l , u b iq u itin -a ctiv a tin g en zy m e; E 2 , u b iq u itin -con ju gatin g en zy m e; U b l, u b iq u itin -lik e d om ain. “X ” is a

p u tative target protein.

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CHAPTER 5: A CLINICO- GENETIC AND PATHOLOGICAL STUDY OF