Andrius Baskys

Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents

The neurotransmitter glutamate can have both excitotoxic and protective effects on neurons. The excitotoxic effects have been intensively studied, whereas the protective effects, including the involvement of metabotropic glutamate receptors (mGluRs), remain unclear. In the present study, we tested the protective effects of the group-I-mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) on organotypic hippocampal slice cultures exposed to excitotoxic concentrations of N-methyl-D-aspartate (NMDA). Effects of DHPG on electrophysiological responses induced by NMDA receptor activation were also recorded. Experiments were performed on organotypic hippocampal slice cultures derived from 7-day-old rats, with cellular uptake of propidium iodide as a marker for neuronal cell death. Slice cultures pretreated with DHPG (10 or 100 microM) for 2 h prior to exposure to 50 microM NMDA for 30 min displayed reduced propidium iodide uptake, compared to cultures exposed to NMDA only. The neuroprotective effect was confirmed by Hoechst 33342 staining, where the appearance of pycnotic nuclei after NMDA treatment was prevented by the DHPG pretreatment. Using caspase-3 activity to monitor the presence of apoptosis, failed to demonstrate this type of cell death in CA1 after NMDA application. The protective effect of DHPG was abolished by the mGluR1 selective antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; 5 or 10 microM), whereas the mGluR5-selective antagonist 2-methyl-6-phenylethynylpyridine (MPEP; 1 microM) had no effect. Voltage-clamping of CA1 pyramidal cells in cultures treated with 10 microM DHPG for 2 h showed a significant depression of NMDA-induced inward currents compared to untreated controls. We conclude that neuroprotection induced by activation of group-I-mGluRs involve mGluR1 and is associated with decreased NMDA-stimulated currents.

Effects of galantamine on working memory and global functioning in patients with mild cognitive impairment: A double-blind placebo-controlled study

Mild cognitive impairment (MCI) causes memory impairment and executive function deficits in those with the condition. There is also some evidence that MCI patients are impaired in their daily functioning. Cholinesterase inhibitors have been widely used for patients with Alzheimers disease (AD), with evidence of improving cognitive function. There is currently no established treatment for MCI, and cholinesterase inhibitors are beginning to be studied in these patients. Galantamine is a cholinesterase inhibitor that also has nicotinic receptor-modulating properties that has been successful in improving AD patients. This study examined the effects of galantamine in patients with MCI in areas of memory, executive functioning, and global functioning. There was a significant improvement in scores on the Functional Activities Questionnaire, which is a measure of global functioning. There were also improvements in the galantamine group on two of six measures in the Cambridge Automated Neuropsychiatric Test Assessment Battery and in immediate free recall on the California Verbal Learning Test.

Activation of Neuroprotective Pathways by Metabotropic Group I Glutamate Receptors: A Potential Target for Drug Discovery?

Stroke neuroprotection trials suggest that pharmacological manipulations of a single neuroprotective mechanism are generally ineffective and that new approaches, possibly involving simultaneous manipulations of multiple mechanisms, need to be sought. To identify optimal components for such a multipronged approach, we studied NMDA receptor activation‐induced cell death in organotypic hippocampal culture preparations as a model of excitotoxicity. Metabotropic group I glutamate receptor (mGluR) activation by their selective agonist, (S)‐3,5‐dihydroxyphenylglycine (DHPG), resulted in concentration‐dependent reduction of nerve cell susceptibility to NMDA‐mediated injury (neuroprotective effect). The neuroprotection was mediated primarily by mGluR1, required phospholipase C activation, was inhibited by cholesterol‐containing methyl‐β‐cyclodextrin treatment, and occluded by antipsychotic quetiapine. It was associated with suppression of NMDA currents and prolongation of GABAA receptor‐mediated currents in DHPG‐treated cultures. cDNA microarray analysis of 1128 brain‐relevant genes revealed that mGluR‐mediated neuroprotection was associated with simultaneous activation of endocytosis, and inactivation of inflammation, cell adhesion, cell death, and transcription‐related genes. Antisense inhibition of Rab5b, a gene coding for a small GTPase associated with endocytosis, significantly reduced the mGluR‐mediated neuroprotection. These findings expand our understanding of the role that mGluRs play in regulation of nerve cell susceptibility to injury and should facilitate the design of novel therapeutic strategies for stroke and other neurodegenerative diseases.

Understanding regulation of nerve cell death by mGluRs as a method for development of successful neuroprotective strategies

A common cause of nerve cell death often leading to vascular dementia is ischemic stroke. Attempts to develop clinically effective stroke treatment and prevention strategies based on pharmacological manipulations of a single mechanism have not led to clinical success. Analysis of clinical neuroprotection trials suggests that combination treatments may be more effective. To identify optimal components for such treatment, N-methyl-d-aspartate receptor (NMDAR) activation-induced cell death in organotypic hippocampal preparations was studied as a model of neurodegeneration that occurs in association with stroke or vascular dementia. Pharmacological manipulation of metabotropic glutamate receptors mGluR1 and 5 resulted in significant reduction of nerve cell susceptibility to NMDA-induced injury, suggesting that these receptors may function as physiological regulators of neuronal vulnerability. cDNA microarray analysis of over 1000 brain-related genes performed after the neuroprotective activation of group I metabotropic glutamate receptors (mGluRs) revealed a complex pattern of activation and inactivation of seemingly unrelated genes responsible for regulation of neuronal excitability, inflammation, cell death pathways, cell adhesion and transcriptional activation. Combined pharmacological targeting of these processes may provide basis for clinical trials of effective neuroprotective compounds.

Rab-mediated endocytosis: linking neurodegeneration, neuroprotection, and synaptic plasticity?

Rab proteins are small GTPases involved in endocytosis and recycling of cell surface molecules. Recently they have been implicated in the etiopathogenesis of several neurodegenerative disorders including Alzheimers and Lewy body disease. In experiments on organotypic hippocampal cultures, upregulation of Rab protein family member Rab5b after group I metabotropic glutamate receptor (mGluR) stimulation was associated with reduced neuronal vulnerability to excitotoxic injury. This mGluR‐mediated neuroprotection was abolished by antisense‐induced deficiency of Rab5b. Electrophysiological measurements of excitatory synaptic transmission in the Schaffer collateral–CA1 pathway revealed that mGluR activation that induces neuroprotection also induced long‐term depression (LTD) of synaptic transmission. Similar to the neuroprotection, Rab5b deficiency abolished dihydroxyphenylglycine‐induced LTD. Together, these findings support the idea that Rab proteins, and the Rab5b protein in particular, may provide a link between neurodegenerative disease, neuroprotection, and synaptic plasticity, as well as possibly being a useful target for pharmacological interventions.

Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors

Stimulation of group I metabotropic glutamate receptors (mGluRs) has been shown to protect against N-methyl-D-aspartate receptor-mediated cell death, but the underlying cellular mechanism is unknown. Using cDNA microarrays we have now compared gene expressions in organotypic hippocampal slice cultures after neuroprotective activation of group I mGluRs with (S)-3,5-dihydroxyphenylglycine (DHPG; 10 microM, 2 h) with untreated control cultures. Total RNA was extracted from the cultures immediately after the neuroprotective treatment, reverse transcribed to cDNA with incorporation of [32]P-dCTP, and then hybridized to the arrays. Of a total of 1128 genes on the Neuroarray, 33 genes displayed significant changes in expression after DHPG-treatment (six up- and 27 downregulated). These genes have been associated with regulation of synaptic excitation, inflammation, cell adhesion, cell death, and transcription. The small GTPase RAB5B associated with endocytosis emerged as a primary candidate gene for neuroprotection, and its expression was confirmed by Western blot analysis and real time polymerase chain reaction. By providing insight into genes involved in neuroprotection these data may help to identify novel therapeutic targets.

Antisense oligonucleotide against GTPase Rab5b inhibits metabotropic agonist DHPG-induced neuroprotection.

(S)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR1 and 5) agonist reduced NMDA-mediated membrane currents, NMDA-induced cell death and up-regulated Rab5b, a small GTPase involved in endocytosis [M. Blaabjerg, A. Baskys, J. Zimmer and M. P. Vawter, Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors, Molec. Brain Res. 117 (2003) 196-205; M. Blaabjerg, L. Fang, J. Zimmer and A. Baskys, Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents, Experimental Neurol. 183 (2003) 573-580.]. To examine the role of Rab5b on DHPG-mediated neuroprotection in organotypic hippocampal cultures, we developed antisense oligonucleotide targeted to suppress Rab5b translation. Treatment of cultures with the antisense (24 h) but not scrambled sequence oligonucleotide suppressed DHPG-induced increase in Rab5b expression and significantly disrupted DHPG-induced protection against NMDA toxicity in a concentration-dependent manner (0.01-10 nM). Antisense but not scrambled oligonucleotide treatment reduced NMDA toxicity (to 74.4+/-5.9% of control) and this effect could be blocked by protein kinase C inhibitor staurosporine (0.2 microM) or with the protease inhibitor leupeptin (100 microM). Application of osmotic shock followed by K(+) depletion to disrupt endocytosis abolished the protective effect of DHPG. These data suggest that neuroprotection by DHPG against NMDA-mediated injury may involve facilitation of NMDA receptor endocytosis likely stimulated by DHPG-induced increase in Rab5b synthesis.

Neuroprotective effects of extracellular glutamate are absent in hippocampal organotypic cultures treated with the amyloid peptide Aβ25–35☆

Hippocampal cells are particularly vulnerable in Alzheimers disease but the cause of cell death is unknown. Amyloid toxicity has been implicated in hippocampal cell death, but its specific mechanisms are poorly understood. We used confocal microscopy to examine the effects of the amyloid peptide fragment 25-35 (Abeta(25-35)) on cell death in organotypic hippocampal slice cultures. Addition of glutamate to the culture medium significantly improved nerve cell survival in cultures subjected to consecutive medium exchanges. This effect was lost if cultures were treated with the amyloid peptide fragment Abeta(25-35) but not the inactive peptide 35-25. These data suggest that one of the mechanisms responsible for amyloid toxicity may be inhibition of the survival promoting effects of extracellular glutamate.

PITX3 polymorphism is not associated with Parkinson's disease in a Chinese population

Several studies have indicated that three PITX3 single nucleotide polymorphisms (SNPs), rs2281983, rs4919621 and rs3758549, are likely to be associated with Parkinsons disease (PD) in Caucasians. Some studies also suggested an age-of-onset effect. We recently reported that allele and genotype frequencies did not differ between late-onset PD (LOPD) patients and controls for all three SNPs. To extend the analysis to early-onset PD (EOPD) patients, and to test whether an age-of-onset effect exists in Chinese, we genotyped these SNPs in 290 Chinese EOPD patients using a ligase detection reaction (LDR). For all three SNPs, allele and genotype frequencies did not differ between total PD patients and controls, between LOPD patients and controls, between EOPD patients and controls, or between LOPD and EOPD patients. Our results suggest that these PITX3 SNPs do not contribute to the risk of developing PD in EOPD or LOPD in Chinese.

Calpastatin gene (CAST) is not associated with late onset sporadic Parkinson's disease in the Han Chinese population.

Recent studies point to an association between the late-onset sporadic Parkinson’s disease (PD) and single nucleotide polymorphisms (SNPs) rs1559085 and rs27852 in Ca2+-dependent protease calpain inhibitor calpastatin (CAST) gene. This finding is of interest since loss of CAST activity could result in over activated calpain, potentially leading to Ca2+ dysregulation and loss of substantia nigra neurons in PD. We explored the association between CAST SNPs and late-onset sporadic PD in the Han Chinese population. The study included 615 evaluable patients (363 male, 252 female) with PD and 636 neurologically healthy controls (380 male, 256 female) matched for age, gender, ethnicity, and area of residence. PD cases were identified from the PD cohort of the Chinese National Consortium on Neurodegenerative Diseases (www.chinapd.cn). A total of 24 tag-SNPs were genotyped capturing 95% of the genetic variation across the CAST gene. There was no association found between any of the polymorphisms and PD in all models tested (co-dominant, dominant-effect and recessive-effect). Similarly, none of the common haplotypes was associated with a risk for PD. Our data do not support a significant association between the CAST gene polymorphisms and late onset sporadic PD in the Han Chinese population.