Research reportNeurofilament L gene is not a genetic factor of sporadic and familial Parkinson’s disease
Introduction
Recent progress in Parkinson’s disease (PD) research indicates that PD might be caused by protein aggregation due to aberrant protein folding or disturbed protein degradation [reviewed in 18]. This hypothesis has gained major support by the identification of mutations in the α-synuclein and parkin genes, respectively, in some rare forms of inherited PD. Importantly, the gene products of these genes have been identified as (i) components of the characteristic Lewy bodies (LB) in the form of ubiquitinated protein aggregates [α-synuclein (SNCA) and parkin] or as (ii) components of the ubiquitin proteasome protein degradation pathway (parkin). Therefore, we are currently exploring the role of further proteins which aggregate in LB.
Detailed immunohistochemical studies indicate that neurofilaments are major components of LB [16]. Neurofilaments are composed of three neuron-specific proteins with molecular weights of 110 kDa (NF-H), 90 kDa (NF-M) and 61 kDa (NF-L), respectively. Neurofilaments are heteropolymers requiring NF-L together with NF-M or NF-H for polymer formation [10]. All three subunits have been identified in LB [5]. Neurofilaments are the most abundant neuron-specific intermediate filaments [7] and represent—beside microtubules and microfilaments—a major element of the neuronal cytoskeleton. Dopaminergic neurons which are primarily affected in Parkinson’s disease are richly endowed with neurofilaments. Perturbations in phosphorylation or in metabolism of neurofilaments are frequently observed in neurodegenerative disorders [14] and might affect the function of other cytoskeletal proteins such as microtubules and actin filaments. Interestingly, several neuronal kinases including cyclin-dependent kinase 5 (Cdk5) and Ca2+-calmodulin-dependent kinase II [8], [15] have also been identified as components of LB suggesting that impaired protein phoshorylation/dephosphorylation may be a key event in the formation of LB. Recently deregulation of Cdk5 has been shown to cause irreversible damage to the cytoskeleton and neurodegeneration via hyperphosphorylation of neurofilament and tau in vivo [20]. Pollanen et al. suggested a model for LB formation based on three mechanisms: (1) self-assembly and aggregation, (2) post-translational modification (phosphorylation) and (3) proteolysis [16]. Mutations in the genes encoding NF might affect all three steps. Furthermore, decline of the NF-L and NF-H mRNA has been observed in PD [6] potentially altering neurofilament or even neuronal cytoskeleton composition. On the other hand, selective upregulation of NF-L in transgenic mice leads to NF-rich inclusions and neuronal cell death [23]. Despite the mounting evidence for a role of NF in the pathogenesis of PD genetic studies of NF genes have not been published yet. As a first step we therefore investigated the NF-L gene in PD patients by mutation and association analyses.
Section snippets
PD patients
A total of 328 German PD patients (mean age 66.3 years (S.D.±10.8 years); mean age at disease onset 55.9 years (S.D.±11.45 years); males 57.8%, females 42.2%) were evaluated by experienced neurologists and were diagnosed as idiopathic PD based on the UK PD brain bank criteria. These criteria are highly specific to define PD. Based on pedigree analysis 56 patients had a family history of PD, the other patients are thought to be sporadic. None of the patients carried a mutation in the
Mutation analysis
We undertook a detailed search for DNA changes in all four coding exons of the NF-L gene in 328 PD patients using two different SSCP conditions with subsequent sequencing of conspicuous band shifts. Exon 1 was analysed in overlapping PCR reactions and by restriction digestion of the PCR product reducing the PCR fragment length to sizes smaller than 250 bp to achieve highest sensitivity of the SSCP screening. In total three DNA changes (G163A, C224T, C487T) in exons 1 and 3 were identified in
Discussion
Neurofilament proteins have been implicated in the pathogenesis of several neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer disease and Parkinson disease. The common feature in all these disorders is the slow accumulation of neurofilament aggregates in neuronal cells accompanied by cell death. Of the three subunits, NF-L seems to play the most important role in neurofilament assembly, since it is the only neurofilament protein capable of organising filaments by
Acknowledgments
We thank numerous clinicians for providing blood samples of PD patients. The published results are part of the M.D. thesis of NR. This study has been supported by the DFG (Scho754/2-1 to LS and RK) and the BMBF MedNet program (to OR). Data assessment in the MEMO-study was done within the framework of the Cooperative Health Research in the Augsburg Region (KORA). The MEMO-study was supported by the DFG (BE 1996/1-1 to KB).
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