Marta Barrachina

Dopamine induces autophagic cell death and α-synuclein increase in human neuroblastoma SH-SY5Y cells

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinsons 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 α‐synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 μM 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 α‐synuclein expression. Both cell viability and the increase in α‐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 α‐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.

MPP+ increases α-synuclein expression and ERK/MAP-kinase phosphorylation in human neuroblastoma SH-SY5Y cells

Abstract α-Synuclein is a brain presynaptic protein that is linked to familiar early onset Parkinson’s disease and it is also a major component of Lewy bodies in sporadic Parkinson’s disease and other neurodegenerative disorders. α-Synuclein expression increases in substantia nigra of both MPTP-treated rodents and non-human primates, used as animal models of parkinsonism. Here we describe an increase in α-synuclein expression in a human neuroblastoma cell line, SH-SY5Y, caused by 5–100 μM MPP + , the active metabolite of MPTP, which induces apoptosis in SH-SY5Y cells after a 4-day treatment. We also analysed the activation of the MAPK family, which is involved in several cellular responses to toxins and stressing conditions. Parallel to the increase in α-synuclein expression we observed activation of MEK1,2 and ERK/MAPK but not of SAPK/JNK or p38 kinase. The inhibition of the ERK/MAPK pathway with U0126, however, did not affect the increase in α-synuclein. The highest increase in α-synuclein (more than threefold) in 4-day cultures was found in adherent cells treated with low concentrations of MPP + (5 μM). Inhibition of ERK/MAPK reduced the damage caused by MPP + . We suggest that α-synuclein increase and ERK/MAPK activation have a prominent role in the cell mechanisms of rescue and damage, respectively, after MPP + -treatment.

DNA Methylation of Alzheimer Disease and Tauopathy-Related Genes in Postmortem Brain

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.

Brain banks: benefits, limitations and cautions concerning the use of post-mortem brain tissue for molecular studies

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.

Constitutive Dyrk1A is abnormally expressed in Alzheimer disease, Down syndrome, Pick disease, and related transgenic models

DYRK1A, dual-specificity tyrosine-regulated kinase 1A, maps to human chromosome 21 within the Down syndrome (DS) critical region. Dyrk1 phosphorylates the human microtubule-associated protein tau at Thr212 in vitro, a residue that is phosphorylated in fetal tau and hyper-phosphorylated in Alzheimer disease (AD) and tauopathies, including Pick disease (PiD). Furthermore, phosphorylation of Thr212 primes tau for phosphorylation by glycogen synthase kinase 3 (GSK-3). The present study examines Dyrk1A in the cerebral cortex of sporadic AD, adult DS with associated AD, and PiD. Increased Dyrk1A immunoreactivity has been found in the cytoplasm and nuclei of scattered neurons of the neocortex, entorhinal cortex, and hippocampus in AD, DS, and PiD. Dyrk1A is found in sarkosyl-insoluble fractions which are enriched in phosphorylated tau in AD brains, thus suggesting a possible association of Dyrk1A with neurofibrillary tangle pathology. Yet, no clear relationship has been observed between tau phosphorylation at Thr212, and GSK-3 and Dyrk1A expression in diseased brains. Transgenic mice bearing a triple tau mutation (G272V, P301L, and R406W) and expressing hyper-phosphoyrylated tau in neurons of the entorhinal cortex, hippocampus, and cerebral neocortex show increased expression of Dyrk1A in individual neurons in the same regions. However, transgenic mice over-expressing Dyrk1A do not show increased phosphorylation of tau at Thr212, thus suggesting that Dyrk1A over-expression does not trigger per se hyper-phosphorylation of tau at Thr212 in vivo. The present observations indicate modifications in the expression of constitutive Dyrk1A in the cytoplasm and nuclei of neurons in various neurodegenerative diseases associated with tau phosphorylation.

Up-regulation of adenosine receptors in the frontal cortex in Alzheimer's disease.

Adenosine receptors are G‐protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A1 and A2A receptors were studied in post‐mortem human cortex in Alzheimers disease (AD) and age‐matched controls. Total adenosine A1 receptor number, determined by radioligand binding assay, using [3H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A1R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A2A receptors were observed in AD samples as determined by a binding assay using [3H]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 A1 and A2A receptors. Finally, increased A1 and A2A receptor‐mediated response was observed. These results show up‐regulation of adenosine A1 and A2A receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.

Abnormal α-synuclein interactions with rab3a and rabphilin in diffuse Lewy body disease

The present study examines alpha-synuclein interactions with rab3a and rabphilin by antibody arrays, immunoprecipitation and pull-down methods in the entorhinal cortex of control cases and in diffuse Lewy body disease (LBD) cases. Alpha-synuclein immunoprecipitation revealed alpha-synuclein binding to rabphilin in control but not in LB cases. Immunoprecipitation with rab3a disclosed rab3a binding to rabphilin in control but not in LB cases. Moreover, rab3a interacted with high molecular weight (66 kDa) alpha-synuclein only in LB cases, in agreement with parallel studies using antibody arrays. Results were compared with pull-down assays using His(6)/Flag-tagged rab3, rab5 and rab8, and anti-Flag immunoblotting. Weak bands of 17 kDa, corresponding to alpha-synuclein, were obtained in LB and, less intensely, in control cases. In addition, alpha-synuclein-immunoreactive bands of high molecular weight (36 kDa) were seen only in LB cases after pull-down assays with rab3a, rab5 or rab8. These findings corroborate previous observations showing rab3a-rabphilin interactions in control brains, and add substantial information regarding decreased binding of rab3a to rabphilin and increased binding of rab3a to alpha-synuclein aggregates in LB cases. Since, alpha-synuclein, rab3a and rabphilin participate in the docking and fusion of synaptic vesicles, it can be suggested that exocytosis of neurotransmitters may be impaired in LB diseases.

CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP- cleaved products of caspase activation following middle cerebral artery occlusion in the rat

Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue.

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinsons disease, dementia with Lewy bodies pure form and common form, and Alzheimers disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.

RESEARCH ARTICLE: Up-regulation of Adenosine Receptors in the Frontal Cortex in Alzheimer's Disease

Adenosine receptors are G‐protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A1 and A2A receptors were studied in post‐mortem human cortex in Alzheimers disease (AD) and age‐matched controls. Total adenosine A1 receptor number, determined by radioligand binding assay, using [3H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A1R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A2A receptors were observed in AD samples as determined by a binding assay using [3H]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 A1 and A2A receptors. Finally, increased A1 and A2A receptor‐mediated response was observed. These results show up‐regulation of adenosine A1 and A2A receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.