Marat A. Mukhamedyarov

Coronary artery bypass surgery provokes alzheimer's disease-like changes in the cerebrospinal fluid

Several biomarkers are used in confirming the diagnosis of cognitive disorders. This study evaluates whether the level of these markers after heart surgery correlates with the development of cognitive dysfunction, which is a frequent complication of cardiac interventions. Concentrations of amyloid-β peptide, tau, and S100β in the cerebro-spinal fluid were assessed, as well as cognitive functions were evaluated before and after coronary artery bypass grafting, utilizing immuno-assays and psychometric tests, respectively. A drastic rise in the level of S100β was observed one week after the surgery, a mark of a severe generalized cerebral injury. The level of amyloid-β peptide significantly decreased, whereas the concentration of tau markedly increased six months postoperatively. Gradual cognitive decline was also present. These findings clearly demonstrate post-surgical cognitive impairment associated with changes in biomarkers similar to that seen in Alzheimers disease, suggesting a unifying pathognomic factor between the two disorders. A holistic approach to coronary heart disease and Alzheimers type dementia is proposed.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF165) and human fibroblast growth factor 2 (FGF2) (pBud-VEGF-FGF2). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models (G93A mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β 3-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF2. Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β 3-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF2, hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF2 on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

Potential role of dental stem cells in the cellular therapy of cerebral ischemia.

Stem cell based therapies for cerebral ischemia (CI) utilize different cell sources including embryonic stem cells (ESCs), neural stem cells (NSCs), umbilical cord blood cells (UCBCs), mesenchymal stem cells (MSCs), and some immortalized cell lines. To date, experimental studies showed that all of these cell sources have been successful to some extent in attenuating the ischemic damage and improving functional recovery after brain injury. Bone marrow derived MSCs seem to be the most widely used and well characterized cell source, which can be also employed for autologous transplantation. Currently, there are two main theories behind the therapeutic effect of stem cell transplantation for treating CIs. The first concept is cell replacement theory in which transplanted stem cells differentiate into progenitor and specialized somatic cells to supersede dying cells. The other hypothesis is based on immuno-modulatory, neuro-protective and neuro-trophic abilities of stem cells which help reducing stroke size and increasing the recovery of behavioral functions. Recent studies focusing on alternative stem cell sources have revealed that dental stem cells (DSCs), including dental pulp stem cells (DPSCs) and dental follicle cells (DFCs) possess properties of MSCs and NSCs. They differentiate into neural linage cells and some other cell types such as osteocytes, adipocytes, chondrocytes, muscle cells and hepatocytes. This review is intended to examine stem cell therapy approaches for CI and emphasize potential use of DSCs as an alternative cell source for the treatment of brain ischemia.

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals.

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.

Modulation of Neurotransmitter Release by Carbon Monoxide at the Frog Neuro-Muscular Junction

Carbon monoxide (CO) is an endogenous gaseous messenger, which regulates numerous physiological functions in a wide variety of tissues. Using extracellular microelectrode recording from frog neuro-muscular preparation the mechanisms of exogenous and endogenous CO action on evoked quantal acetyl-choline (Ach) release were studied. It was shown that CO application increases Ach-release in dose-dependent manner without changes in pre-synaptic Na+ and K+ currents. The effect of exogenous CO on Ach-release was decreased by prior application of guanylate cyclase inhibitor ODQ and prevented by application of a cyclic guanylate monophosphate (cGMP) analog 8Br-cGMP. Pre-treatment of the preparation with adenylate cyclase inhibitor MDL-12330A has completely abolished the effect of CO, whereas elevation of intracellular level of cyclic adenosine monophosphate (cAMP) mimicked and eliminated CO action. Application of cGMP-activated phosphodiesterase-2 inhibitor EHNA did not prevent CO action, whereas inhibition of cGMP-inhibited phosphodiesterase-3 by quazinone has partially blocked the effect of CO. Utilizing immuno-histochemical methods CO-producing enzyme heme-oxygenase-2 (HO-2) was shown to be expressed in skeletal muscle fibers, mostly in sub-sarcolemmal region, karyolemma and sarcoplasmic reticulum. Zn-protoporphirin-IX, the selective HO-2 blocker, has depressed Ach-release, suggesting the tonic activating effect of endogenous CO on pre-synaptic function. These results suggest that facilitatory effect of CO on Ach-release is mediated by elevation of intracellular cAMP level due to activation of adenylate cyclase and decrease of cAMP breakdown. As such, endogenous skeletal muscle-derived CO mediates tonic retrograde up-regulation of neuro-transmitter release at the frog neuro-muscular junction.

Symptomatic Improvement, Increased Life-Span and Sustained Cell Homing in Amyotrophic Lateral Sclerosis After Transplantation of Human Umbilical Cord Blood Cells Genetically Modified with Adeno-Viral Vectors Expressing a Neuro-Protective Factor and a Neural Cell Adhesion Molecule

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function. Human umbilical cord blood cells (hUCBCs) transduced with adeno-viral vectors encoding human VEGF, GDNF and/or NCAM genes were transplanted into transgenic ALS mice models. Significant improvement in behavioral performance (open-field and grip-strength tests), as well as increased life-span was observed in rodents treated with NCAM-VEGF or NCAM-GDNF co-transfected cells. Active trans-gene expression was found in the spinal cord of ALS mice 10 weeks after delivering genetically modified hUCBCs, and cells were detectable even 5 months following transplantation. Our gene-cell therapy model yielded prominent symptomatic control and prolonged life-time in ALS. Incredible survivability of xeno-transpanted cells was also observed without any immune-suppression. These results suggest that engineered hUCBCs may offer effective gene-cell therapy in ALS.

Disrupted Serotonergic and Sympathoadrenal Systems in Patients with Chronic Heart Failure May Serve as New Therapeutic Targets and Novel Biomarkers to Assess Severity, Progression and Response to Treatment

Background: It is well established that the serotonergic system (SS) plays important roles in the pathogenesis of cardiovascular diseases. However, the impact of serotonin and its inter-relation with the sympathoadrenal system (SAS) in chronic heart failure (CHF) is poorly understood. Methods: Utilizing high-performance liquid chromatography with electrochemical detection, we determined blood plasma levels of serotonin (5-hydroxy-triptamine, [5-HT]p), 5- hydroxy-indole-acetic acid ([5-HIAA]p), epinephrine ([E]p), norepinephrine ([NE]p), 3,4-dihydroxy-L-phenyl-alanine ([DOPA]p), dopamine ([DA]p) and the platelet concentration of serotonin ([5-HT]pt) in CHF patients with different morphofunctional alterations of myocardium. The morphofunctional alterations included diastolic dysfunction (DD), diastolic dysfunction with left ventricular hypertrophy (DD&LVH), and diastolic and systolic dysfunction (D&SD). Results: All CHF groups showed significant rises of [5-HT]p and [5-HT]pt. DD&LVH and D&SD individuals also had increased [5-HIAA]p. Levels of SAS blood biomarkers were also significantly changed. The correlation between SS and SAS was increased in CHF and corresponded with disease severity. Conclusions: These results clearly demonstrate that in CHF patients significant changes in SS and SAS occur, which are thought to relate to the morphofunctional alterations of myocardium. The observed changes in the levels of these biomarkers may serve as potential surrogates to monitor severity of disease, to evaluate response to drug treatment, and as a rational basis for new therapeutic approaches.

Disease-specific expression of the serotonin-receptor 5-HT2C in natural killer cells in Alzheimer's dementia

Alzheimers dementia (AD) is a degenerative brain disorder characterized mainly by cholinergic failure, but other neuro-transmitters are also deficient especially at late stages of the disease. Misfolded β-amyloid peptide has been identified as a causative agent, however inflammatory changes also play a pivotal role. Even though the most prominent pathology is seen in the cognitive functions, specific abnormalities of the central nervous system (CNS) are also reflected in the periphery, particularly in the immune responses of the body. The aim of this study was to characterize the dopaminergic and serotonergic systems in AD, which are also markedly disrupted along with the hallmark acetyl-choline dysfunction. Peripheral blood mono-nuclear cells (PBMCs) from demented patients were judged against comparison groups including individuals with late-onset depression (LOD), as well as non-demented and non-depressed subjects. Cellular sub-populations were evaluated by mono-clonal antibodies against various cell surface receptors: CD4/CD8 (T-lymphocytes), CD19 (B-lymphocytes), CD14 (monocytes), and CD56 (natural-killer (NK)-cells). The expressions of dopamine D(3) and D(4), as well as serotonin 5-HT(1A), 5-HT(2A), 5-HT(2B) and 5-HT(2C) were also assessed. There were no significant differences among the study groups with respect to the frequency of the cellular sub-types, however a unique profound increase in 5-HT(2C) receptor exclusively in NK-cells was observed in AD. The disease-specific expression of 5-HT(2C), as well as the NK-cell cyto-toxicity, has been linked with cognitive derangement in dementia. These changes not only corroborate the existence of bi-directional communication between the immune system and the CNS, but also elucidate the role of inflammatory activity in AD pathology, and may serve as potential biomarkers for less invasive and early diagnostic purposes as well.

All-or-Nothing Type Biphasic Cytokine Production of Human Lymphocytes After Exposure to Alzheimer's β-Amyloid Peptide

BACKGROUND Neuro-inflammation, triggered by beta-amyloid peptide, is implicated as one of the primary contributors to Alzheimers disease (AD) pathogenesis, and several cytokines were identified as key instigating factors. METHODS To reveal the inflammatory response of lymphocytes to the neuro-toxic beta-amyloid peptide, we evaluated the release of several cytokines from peripheral blood mononuclear cells with immuno-assays (ELISA). From hyper-acute to chronic effects of beta-amyloid peptide were assessed at a wide range of concentrations. RESULTS The pro-inflammatory interleukin (IL)-1beta, tumor necrosis factor-alpha, monocyte chemotactic protein-1, and Rantes (acronym for regulated on activation, normal T-cell expressed and secreted) as well as the pleiotropic IL-6 showed a biphasic release pattern over time in both low and high doses of amyloid treatment: after an initial increase, their concentration gradually fell to the baseline. The suppressors IL-4 and IL-10 had a sinus-like secretion panel: an acute increase in their levels turned to a depression and later followed by their over-secretion. Interestingly, beta-amyloid below 10(-8) mol/L produced no effect at all, but any molarity above this threshold caused the very same cytokine secretion pattern, the mark of an all-or-nothing response of beta-amyloid peptide. CONCLUSIONS These results delineate a highly organized pro- and anti-inflammatory response of cells to the neuro-toxic peptide. This is the first study to describe how the beta-amyloid-induced inflammatory processes in Alzheimers dementia are regulated.

Retrogradely transported siRNA silences human mutant SOD1 in spinal cord motor neurons.

The transgenic mouse model of familial amyotrophic lateral sclerosis (ALS) expressing human mutant (G93A) copper/zinc superoxide dismutase (SOD1) is an attractive model for studying the therapeutic effects of RNA interference (RNAi) because of the specific silencing of the mutant gene expression. We studied small interfering RNA (siRNA)-mediated down-regulation of human mutant G93A SOD1 gene in lumbar spinal cord of ALS mice. siRNA was applied onto the proximal nerve stump of severed sciatic nerves. One day after surgery the lumbar spinal cords were processed for RT-PCR examination. Treatment with specific siRNA resulted in 48% decrease in human SOD1 mRNA levels in lumbar spinal cord, but had no effect on the abundance of mouse ChAT and SNAP25 mRNAs which were used as randomly selected internal controls, the mark of a specific silencing of SOD1. Our findings demonstrate for the first time that siRNA, targeting mutant human SOD1 mRNA, is taken up by the sciatic nerve, retrogradely transported to the perikarya of motor neurons, and inhibits mutant SOD1 mRNA in G93A transgenic ALS mice.