Two novel point mutations of mitochondrial tRNA genes in histologically confirmed Parkinson disease.

Mutations in mitochondrially encoded tRNA genes have been described in a variety of neurological disorders. One such mutation, the A to G transition at nucleotide position 4336 of the mitochondrial tRNA(Gln) gene, has been associated with both Alzheimer and Parkinson disease. We have now performed a complete sequence analysis of all 22 mitochondrially encoded tRNA genes in 20 cases of histologically proven idiopathic Parkinson disease. Genomic DNA extracted from the substantia nigra of frozen or formalin-fixed and paraffin-embedded brains was used for amplification by polymerase chain reaction followed by automated sequencing. Two new homoplasmic point mutations were detected in the genes for tRNA(Thr) (15950 G/A) and tRNA(Pro) (15965 T/C) in 1 patient each. Restriction enzyme digestion revealed absence of the 15950 G/A mutation in 96 controls and in 40 cases of neuropathologically confirmed Alzheimer disease. The 15965 T/C mutation was shown to be absent from 100 control subjects and 47 Alzheimer cases. In addition to the two novel mutations, six known sequence variants were detected in a total of 6 different patients in the genes for tRNA(Asp) (G7521A, 1), tRNA(Arg) (T10463C, 1), tRNA(LeuCUN) (A12308G, 2), and tRNA(Thr) (A15924G, 1; G15928A, 2), including 1 patient carrying the tRNA(Gln) (A4336G) mutation. The G15950A transition affects position 70 of the aminoacyl acceptor stem of tRNA(Thr), which has been implicated as a recognition element for threonyl-tRNA synthetase and, at least in some tRNAs, in the processing of primary mitochondrial transcripts. The T15965C point mutation in the mitochondrial tRNA(Pro) gene alters position 64 of the TpsiC stem. The corresponding nucleotide in bacterial aminoacyl-tRNAs is involved in the interaction with elongation factor Tu. Thus, the two novel mutations are likely to be of functional relevance and could contribute to dopaminergic nerve cell death in affected individuals.

Microglial activation in Alzheimer disease: Association with APOE genotype.

Microglial cells are considered to play an important role in the pathogenesis of Alzheimer disease. Apart from producing the Alzheimer amyloid precursor (APP) as an acute phase protein, microglial cells seem to be involved in the deposition of its amyloidogenic cleavage product, the amyloid-beta peptide (Abeta). Abeta is bound by apolipoprotein E (APOE) in an isoform-specific manner, and it has been demonstrated that inheritance of the AD susceptibility allele, APOE epsilon4, is associated with increased deposition of Abeta in the cerebral cortex. However, the relationship between APOE epsilon4 gene dose and microglial activation is unknown. Using microglial expression of major histocompatibility complex class II molecules as a marker, we have performed a quantitative genotype-phenotype analysis on microglial activation in frontal and temporal cortices of 20 APOE genotyped AD brains. The number of activated microglia and the tissue area occupied by these cells increased significantly with APOE epsilon4 gene dose. When a model of multiple linear regression was used to compare the relative influence of APOE genotype, sex, disease duration, age at death, diffuse and neuritic plaques as well as neurofibrillary tangles on microglial activation, only APOE genotype was found to have a significant effect. Thus, the APOE gene product represents an important determinant of microglial activity in AD. Since microglial activation by APP has been shown to be modulated by apoE in vitro, a direct role of microglia in AD pathogenesis is conceivable.

Novel mutations of mitochondrial complex I in pathologically proven Parkinson disease.

Complete sequence analysis of all mitochondrial complex I genes was performed in 22 cases of neuropathologically confirmed idiopathic Parkinson disease (PD). DNA from the substantia nigra was used as a template for polymerase chain reaction-based genomic sequencing. Seven novel mutations causing the exchange of amino acids were detected in subunit genes ND1 (3992 C/ T, 4024 A/G), ND4 (11253 T/C, 12084 C/T), ND5 (13711 G/A, 13768 T/C), and ND6 (14582 T/C). In addition, five known missense mutations affecting the ND1 (3335 T/C, 3338 T/C), ND2 (5460 G/A), ND3 (10398 A/G), and ND5 (13966 A/G) genes as well as three secondary LHON mutations (4216 T/C, 4917 A/ G, 13708 G/A) were found in the PD group. Among the novel mutations, the 11253 T/C transition which changes a conserved isoleucine residue into threonine is most likely to be of functional relevance. Furthermore, 43 synonymous polymorphisms were detected in PD brains, including 20 novel sequence variants. Haplogroup analysis revealed that most unique missense mutations were found in PD cases belonging to the D(c) haplogroup. Our data are in line with the view that PD is not a single disease entity but comprises a genetically heterogeneous group of disorders. The results of our study further suggest that 90% or more of all idiopathic PD cases are not due to sequence variation of mitochondrial complex I, but that mitochondrial mutations may play a pathogenic role in a subset of PD patients.

On the question of apoptosis in the parkinsonian substantia nigra.

Apoptosis has been postulated as a mechanism of nerve cell death in Parkinson's disease. In the present study, the substantia nigra of 22 neuropathologically confirmed Parkinson cases and 8 control brains was studied using the in situ end-labeling (TUNEL) method. About 50% of parkinsonian brains showed a small number of TUNEL-positive glial cells in the substantia nigra, whereas no neurons showed convincing TUNEL positivity or any morphological signs of apoptosis. No correlation was observed between the number of TUNEL-positive glial cells and microglial activation. Our results fail to demonstrate apoptosis as a mechanism of cell death in Parkinson's disease.

Detoxification of cyclophosphamide by human aldehyde dehydrogenase isozymes.

In in vitro studies, no turnover of aldophosphamide and mafosfamide was observed with the tumor-specific aldehyde dehydrogenase 3 isozyme (ALDH3) isolated from human stomach mucosa as well as from lung (A549) and pharynx (UMSCC2) carcinoma cell lines. Only the human liver cytosolic ALDH preparation (ALDH1) showed any significant oxidation of aldophosphamide and mafosfamide.