Mitochondrial dysfunction is linked with the etiopathogenesis of Alzheimer disease and Parkinson disease. Mitochondria are intracellular organelles essential for cell viability and are characterized by the presence of the mitochondrial (mt)DNA. DNA methylation is a well-known epigenetic mechanism that regulates nuclear gene transcription. However, mtDNA methylation is not the subject of the same research attention. The present study shows the presence of mitochondrial 5-methylcytosine in CpG and non-CpG sites in the entorhinal cortex and substantia nigra of control human postmortem brains, using the 454 GS FLX Titanium pyrosequencer. Moreover, increased mitochondrial 5-methylcytosine levels are found in the D-loop region of mtDNA in the entorhinal cortex in brain samples with Alzheimer disease-related pathology (stages I to II and stages III to IV of Braak and Braak; n = 8) with respect to control cases. Interestingly, this region shows a dynamic pattern in the content of mitochondrial 5-methylcytosine in amyloid precursor protein/presenilin 1 mice along with Alzheimer disease pathology progression (3, 6, and 12 months of age). Finally, a loss of mitochondrial 5-methylcytosine levels in the D-loop region is found in the substantia nigra in Parkinson disease (n = 10) with respect to control cases. In summary, the present findings suggest mtDNA epigenetic modulation in human brain is vulnerable to neurodegenerative disease states.

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DNA methylation occurs predominantly at cytosines that precede guanines in dinucleotide CpG sites; it is one of the most important mechanisms for epigenetic DNA regulation during normal development and for aberrant DNA in cancer. To determine the feasibility of DNA methylation studies in the postmortem human brain, we evaluated brain samples with variable postmortem artificially increased delays up to 48 hours. DNA methylation was analyzed in selected regions of MAPT, APP, and PSEN1 in the frontal cortex and hippocampus of controls (n=26) and those with Alzheimer disease at Stages I to II (n=17); Alzheimer disease at Stages III to IV (n=15); Alzheimer disease at Stages V to VI (n=12); argyrophilic grain disease (n=10); frontotemporal lobar degeneration linked to tau mutations (n=6); frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (n=4); frontotemporal lobar degeneration with motor neuron disease (n=3); Pick disease (n=3); Parkinson disease (n=8); dementia with Lewy bodies, pure form (n=5); and dementia with Lewy bodies, common form (n=15). UCHL1 (ubiquitin carboxyl-terminal hydrolase 1 gene) was analyzed in the frontal cortex of controls and those with Parkinson disease and related synucleinopathies. DNA methylation sites were very reproducible in every case. No differences in the percentage of CpG methylation were found between control and disease samples or among the different pathological entities in any region analyzed. Because small changes in methylation of DNA promoters in vulnerable cells might have not been detected in total homogenates, however, these results should be interpreted with caution, particularly as they relate to chronic degenerative diseases in which small modifications may be sufficient to modulate disease progression.

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinson's disease and other such diseases. We used SH-SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress-induced pathways, and expression of alpha-synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 microM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N-acetylcysteine and ascorbic acid. Dopamine activates the stress-response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases alpha-synuclein expression. Both cell viability and the increase in alpha-synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3-methyladenine. This indicates that oxidative stress, stress-activated kinases, and factors involved in autophagy up-regulate alpha-synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration.

Adenosine receptors are G-protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A(1) and A(2A) receptors were studied in post-mortem human cortex in Alzheimer's disease (AD) and age-matched controls. Total adenosine A(1) receptor number, determined by radioligand binding assay, using [(3)H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A(1)R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A(2A) receptors were observed in AD samples as determined by a binding assay using [(3)H]ZM 241385 as a radioligand and by Western blot. Increased binding and protein expression levels of adenosine receptors were not associated with increased mRNA levels coding A(1) and A(2A) receptors. Finally, increased A(1) and A(2A) receptor-mediated response was observed. These results show up-regulation of adenosine A(1) and A(2A) receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.

The expression of mitogen-activated protein kinases, extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinases, c-Jun N-terminal kinases (SAPK/JNK), and p38 kinases is examined in Parkinson disease (PD), in Dementia with Lewy bodies (DLB), covering common and pure forms, and in age-matched controls. The study is geared to gaining understanding about the involvement of these kinases in the pathogenesis of Lewy bodies (LBs) and associated tau deposits in Alzheimer changes in the common form of DLB. Active, phosphorylation dependent MAPK (MAPK-P) is found as granular cytoplasmic inclusions in a subset of cortical neurons bearing abnormal tau deposits in common forms of DLB. Phosphorylated p-38 (p-38-P) decorates neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in common forms of DLB. Phosphorylated SAPK/JNK (SAPK/JNK-P) expression occurs in cortical neurons with neurofibrillary tangles in the common form of DLB. Lewy bodies (LBs) in the brain stem of PD and DLB are stained with anti-ERK-2 antibodies, but they are not recognized by MAPK-P, SAPK/JNK-P and p-38-P. Yet MAPK-P, p-38-P and SAPK/JNK-P immunoreactivity is found in cytoplasmic granules in the vicinity of LBs or in association with irregular-shaped or diffuse alpha-synuclein deposits in a small percentage of neurons, not containing phosphorylated tau, of the brain stem in PD and DLB. MAPK-P, p-38-P and SAPK-P are not expressed in cortical LBs or in cortical neurons with alpha-synuclein-only inclusions in DLB. MAPK-P, p-38-P and SAPK/JNK-P are not expressed in alpha-synuclein-positive neurites (Lewy neurites) in PD and DLB as revealed by double-labeling immunohistochemistry. These results show that MAPKs are differentially regulated in neurons with alpha-synuclein-related inclusions and in neurons with abnormal tau deposits in DLB. Moreover, different kinase expression in brain stem and cortical LBs suggest a pathogenesis of brain stem and cortical LBs in LB diseases. Finally, no relationship has been observed between MAPK-P, p-38-P and SAPK/JNK-P expression and increased nuclear DNA vulnerability, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation, and active, cleaved caspase-3 expression in neurons and glial cells in the substantia nigra in PD and DLB.

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.

Adenosine A2A receptor (A2AR) is a G-protein-coupled receptor highly expressed in basal ganglia. Its expression levels are severely reduced in Huntington's disease (HD), and several pharmacological therapies have shown its implication in this neurodegenerative disorder. The main goal of this study was to gain insight into the molecular mechanisms that regulate A2AR gene (ADORA2A) expression in HD. Based on previous data reported by our group, we measured the methylcytosine (5mC) and hydroxymethylcytosine (5hmC) content in the 5'UTR region of ADORA2A in the putamen of HD patients and in the striatum of R6/1 and R6/2 mice at late stages of the disease. In this genomic region, 5mC and 5hmC remained unchanged in both mice strains, although low striatal A2AR levels were associated with reduced 5mC levels in 30-week-old R6/1 mice and reduced 5hmC levels in 12-week-old R6/2 mice in exon m2. In order to analyze when this mechanism appears during the progression of the disease, a time course for A2AR protein levels was carried out in R6/1 mice striatum (8, 12, and 20 weeks of age). A2AR levels were reduced from 12 weeks of age onwards, and this downregulation was concomitant with reduced 5hmC levels in the 5'UTR region of ADORA2A. Interestingly, increased 5mC levels and reduced 5hmC were found in the 5'UTR region of ADORA2A in the putamen of HD patients with respect to age-matched controls. Therefore, an altered DNA methylation pattern in ADORA2A seems to play a role in the pathologically decreased A2AR expression levels found in HD.