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Background The high complexity of neurodegenerative diseases, including Alzheimer's disease (AD), and the lack of effective treatments point to the need for a broader therapeutic approach to target multiple components involved in the disease pathogenesis. Objective To test the efficacy of 'cerebrospinal fluid (CSF) exchange therapy' in AD-mice. This novel therapeutic approach we recently proposed is based on the exchange of the endogenous pathogenic CSF with a new and healthy one by drainage of the endogenous CSF and its continuous replacement with artificial CSF (aCSF) enriched with secretions from human mesenchymal stem cells (MSCs). Methods We treated AD-mice (amyloid-beta injected) with MSC secretions-enriched-aCSF using an intracerebroventricular CSF exchange procedure. Cognitive and histological analysis were performed. Results We show that the MSC secretions enriched CSF exchange therapy improved cognitive performance, paralleled with increased neuronal counts (NeuN positive cells), reduced astrocytic burden (GFAP positive cells), and increased cell proliferation and neurogenesis (Ki67 positive cells and DCX positive cells) in the hippocampus. This beneficial effect was noted on days 5-10 following 3-consecutive daily exchange treatments (3 hours a day). A stronger effect was noted using a more prolonged CSF exchange protocol (3-consecutive daily exchange treatments with 3 additional treatments twice weekly), with cognitive follow-up performed as early as 2-3 days after treatment. Some increase in hippocampal cell proliferation, but no change in the other histological parameters, was noticed when performing CSF exchange therapy using unenriched aCSF relative to untreated AD-mice, yet smaller than with the enriched aCSF treatment. Conclusion These findings point to the therapeutic potential of the CSF exchange therapy using MSC secretions-enriched aCSF in AD, and might be applied to other neurodegenerative and dementia diseases.

Pathogenesis of neurodegenerative diseases involves dysfunction of mitochondria, one of the most important cell organelles in the brain, with its most prominent roles in producing energy and regulating cellular metabolism. Here we investigated the effect of transferring active intact mitochondria as a potential therapy for Alzheimer's disease (AD), in order to correct as many mitochondrial functions as possible, rather than a mono-drug related therapy. For this purpose, AD-mice (amyloid-β intracerebroventricularly injected) were treated intravenously (IV) with fresh human isolated mitochondria. One to two weeks later, a significantly better cognitive performance was noticed in the mitochondria treated AD-mice relative to vehicle treated AD-mice, approaching the performance of non-AD mice. We also detected a significant decrease in neuronal loss and reduced gliosis in the hippocampus of treated mice relative to untreated AD-mice. An amelioration of the mitochondrial dysfunction in brain was noticed by the increase of citrate-synthase and cytochrome c oxidase activities relative to untreated AD-mice, reaching activity levels of non-AD-mice. Increased mitochondrial activity was also detected in the liver of mitochondria treated mice. No treatment-related toxicity was noted. Thus, IV mitochondrial transfer may possibly offer a novel therapeutic approach for AD.

Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune β-cell failure, whereas systemic resistance to insulin is considered the hallmark of insulin-independent type-2 diabetes (T2D). In contrast to this canonical dichotomy, insulin resistance appears to precede the overt diabetic stage of T1D and predict its progression, implying that insulin sensitizers may change the course of T1D. However, previous attempts to ameliorate T1D in animal models or patients by insulin sensitizers have largely failed. Sensitization to insulin by MEthyl-substituted long-chain DICArboxylic acid (MEDICA) analogs in T2D animal models surpasses that of current insulin sensitizers, thus prompting our interest in probing MEDICA in the T1D context. MEDICA efficacy in modulating the course of T1D was verified in streptozotocin (STZ) diabetic rats and autoimmune nonobese diabetic (NOD) mice. MEDICA treatment normalizes overt diabetes in STZ diabetic rats when added on to subtherapeutic insulin, and prevents/delays autoimmune T1D in NOD mice. MEDICA treatment does not improve β-cell insulin content or insulitis score, but its efficacy is accounted for by pronounced total body sensitization to insulin. In conclusion, potent insulin sensitizers may counteract genetic predisposition to autoimmune T1D and amplify subtherapeutic insulin into an effective therapeutic measure for the treatment of overt T1D.

The complexity of central nervous system (CNS) degenerative/inflammatory diseases and the lack of substantially effective treatments point to the need for a broader therapeutic approach to target multiple components involved in the disease pathogenesis. We suggest a novel approach directed for the elimination of pathogenic agents from the CNS and, in parallel, its enrichment with an array of neuroprotective substances, using a &ldquo, cerebrospinal fluid (CSF) exchange&rdquo, procedure, in which endogenous (pathogenic) CSF is removed and replaced by artificial CSF (aCSF) enriched with secretions of human mesenchymal stem cells (MSCs). MSCs produce a variety of neuroprotective agents and have shown beneficial effects when cells are transplanted in animals and patients with CNS diseases. Our data show that MSCs grown in aCSF secrete neurotrophic factors, anti-inflammatory cytokines, and anti-oxidant agents, moreover, MSC-secretions-enriched-aCSF exerts neuroprotective and immunomodulatory effects in neuronal cell lines and spleen lymphocytes. Treatment of experimental-autoimmune-encephalomyelitis (EAE) mice with this enriched-aCSF using an intracerebroventricular (ICV) CSF exchange procedure (&ldquo, CSF exchange therapy&rdquo, ) caused a significant delay in the onset of EAE and amelioration of the clinical symptoms, paralleled by a reduction in axonal damage and demyelination. These findings point to the therapeutic potential of the CSF exchange therapy using MSC-secretions-enriched-aCSF in inflammatory/degenerative diseases of the CNS.

Calcium (Ca(2+)) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca(2+) concentration in the secretory vesicles was estimated to be in the range of ∼10 to 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca(2+) dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca(2+) signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Ca(v)2.1) Ca(2+) channels. Blocking the p-type Ca(2+) channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca(2+) dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca(2+) indicator fluorescence. The results suggest that the free Ca(2+) content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process.

The Na(+)-Ca(2+) exchanger (NCX) is a major Ca(2+) regulating protein. It is almost ubiquitously expressed. Cyclophilins (Cyps) make up a class of proteins that are involved in protein folding via their peptidyl prolyl cis-trans isomerase (PPIase) and chaperone domains. They are also the cellular receptors of cyclosporin A (CsA). Binding of CsA to cyclophilins inhibits both PPIase and chaperone activities. We have shown that treatment of transfected HEK 293 cells expressing the Na(+)-Ca(2+) exchanger NCX1 with CsA results in downregulation of surface expression and transport activity, without any reduction in the total level of cell NCX1 protein [Kimchi-Sarfaty, C., et al. (2002) J. Biol. Chem. 277 (4), 2505-2510]. In this work, we show that knockdown of cell CypA using targeting siRNA (without any CsA treatment) results in a reduction in the level of NCX1 surface expression, a decrease in the level of Na(+)-dependent Ca(2+) uptake, and no change in the total amount of cell NCX1 protein in NCX1.5-transfected HEK 293 cells and nontransfected H9c2 cells that express NCX1.1 naturally. It also reduced Na(+)-dependent Ca(2+) fluxes measured by changes in Fluo-4 AM fluorescence in single NCX1.5-transfected HEK 293 and single H9c2 cells. Knockdown of CypB had no significant effect on either transport activity, surface expression, NCX1 cell protein expression, or Ca(2+) fluxes. Overexpression of CypA or its R55A mutant, which exhibits a substantially reduced PPIase activity, alleviated the reduction of NCX1 surface expression caused by CsA treatment, suggesting that the PPIase domain was probably not mandatory for NCX1 functional expression. We suggest that CypA plays a role in the functional expression of NCX1 protein.

Although the three mammalian Na(+)-Ca(2+) exchangers share considerable amino acid sequence homology, they exhibit substantial immunosuppressive drug specificity. We have shown that cyclosporin A (CsA) treatment of NCX1-, NCX2-, or NCX3-transfected HEK 293 cells and non-transfected H9c2, L6, and aortic smooth muscle cells, which express NCX1 protein naturally, reduces NCX surface expression and transport activity but has no impact on total cell NCX protein. Similar effect on functional expression of NCX1 protein can be obtained also without CsA treatment by knockdown of cell cyclophilin A (CypA), one of the cellular receptor of CsA. This suggests that CypA has a role in acquisition of function competence of NCX1 protein.Unlike CsA treatment, which affects the functional expression of all three mammalian NCX proteins similarly, FK506 and rapamycin treatment modulates only the functional expression of NCX2 and NCX3 proteins. FK506 reduces NCX2 and NCX3 surface expression and transport activity without affecting cell NCX protein. Rapamycin reduces NCX2 and NCX3 transport activity but has no effect on their surface expression or total cell NCX protein expression suggesting that, although it shares a common receptor FKBP with FK506, its mode of action follows a different pathway.We are showing now that the large cytosolic loop of NCX1, NCX2, and NCX3 is involved in acquisition of immunosuppressive drug specificity: truncation of the large cytosolic loop of NCX1 renders the protein sensitive to FK506. Exchange of the large cytosolic loop of NCX3 with that of NCX1 renders the mutant protein insensitive to FK506.

Although the three mammalian Na+–Ca2+ exchangers share considerable amino acid sequence homology, they exhibit substantial immunosuppressive drug specificity. We have shown that cyclosporin A (CsA) treatment of NCX1-, NCX2-, or NCX3-transfected HEK 293 cells and non-transfected H9c2, L6, and aortic smooth muscle cells, which express NCX1 protein naturally, reduces NCX surface expression and transport activity but has no impact on total cell NCX protein. Similar effect on functional expression of NCX1 protein can be obtained also without CsA treatment by knockdown of cell cyclophilin A (CypA), one of the cellular receptor of CsA. This suggests that CypA has a role in acquisition of function competence of NCX1 protein.

Agonist-induced lipolysis of adipose fat is robustly inhibited by insulin or by feedback inhibition by the long-chain fatty acids (LCFA) produced during lipolysis. However, the mode of action of LCFA in suppressing adipose lipolysis is not clear. β,β’-Tetramethyl hexadecanedioic acid (Mββ/ EDICA16) is a synthetic LCFA that is neither esterified into lipids nor β-oxidized, and therefore, it was exploited for suppressing agonist-induced lipolysis in analogy to natural LCFA. Mββ is shown here to suppress isoproterenol-induced lipolysis in the rat in vivo as well as in 3T3-L1 adipocytes. Inhibition of isoproterenol-induced lipolysis is due to decrease in isoproterenol-induced cAMP with concomitant inhibition of the phosphorylation of hormone-sensitive lipase and perilipin by protein kinase A. Suppression of cellular cAMP levels is accounted for by inhibition of the adenylate cyclase due to suppression of Raf1 expression by Mββ-activated AMPK. Suppression of Raf1 is further complemented by induction of components of the unfolded-protein-response by Mββ. Our findings imply genuine inhibition of agonist-induced adipose lipolysis by LCFA, independent of their β-oxidation or reesterification. Mββ suppression of agonist-induced lipolysis and cellular cAMP levels independent of the insulin transduction pathway may indicate that synthetic LCFA could serve as insulin mimetics in the lipolysis context under conditions of insulin resistance.

Renal failure is associated with aortic valve calcification. Using our rat model of uremia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in that disease. We also explored the effects of raloxifene, an estrogen receptor modulator, on valvular calcification. Gene array analysis was performed in aortic valves obtained from three groups of rats ( n = 7 rats/group): calcified valves obtained from rats fed with uremic diet, valves after calcification resolution following diet cessation, and control. In addition, four groups of rats ( n = 10 rats/group) were used to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet that also received daily raloxifene. Evaluation included imaging, histology, and antigen expression analysis. Gene array results showed that the majority of the altered expressed genes were in diet group valves. Most apoptosis-related genes were changed in a proapoptotic direction in calcified valves. Apoptosis and decreases in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of survival pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. In conclusion, downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure.

Aortic valve calcification (AVC) is an inflammatory regulated process. Growth arrest-specific 6 (Gas6) is involved in atherosclerosis and inflammation; in addition the Gla domain of Gas6 directly inhibits mineralization. We sought to assess the role of Gas6 in AVC using our unique animal model of reversible AVC based on a uremia-inducing diet. We also evaluated the possible role of raloxifene (a selective estrogen modulator), which has demonstrated anti inflammatory effects on blood vessels, in the pathogenesis of AVC. Aortic valves were obtained from four groups of rats (n=10 each): calcified valves- from rats fed with the uremia-inducing diet for 7 weeks (group A), valves after calcification resolution following diet cessation (group B), valves from rats fed with the same diet who also received subcutaneous raloxifen (1 mg/kg/day) (group C), and control valves. Valves were examined using multislice computed tomography (MSCT), histology, RT-PCR and western blot. Histological evaluation of calcified valves (group A) revealed positive staining for calcium deposits and osteoblast’s markers (osteopontin, Runx-2). Gene expression analyses of these valves revealed 2.5 times decrease in Gas6 level compared to control valves (p