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To review the recent developments on the effect of chronic high mean arterial blood pressure (MAP) on cerebral blood flow (CBF) autoregulation and supporting the notion that CBF autoregulation impairment has connection with chronic cerebral diseases. Method: A narrative review of all the relevant papers known to the authors was conducted. Results: Our understanding of the connection between cerebral perfusion impairment and chronic high MAP and cerebral disease is rapidly evolving, from cerebral perfusion impairment being the result of cerebral diseases to being the cause of cerebral diseases. We now better understand the intertwined impact of hypertension and Alzheimer's disease (AD) on cerebrovascular sensory elements and recognize cerebrovascular elements that are more vulnerable to these diseases. Conclusion: We conclude with the suggestion that the sensory elements pathology plays important roles in intertwined mechanisms of chronic high MAP and AD that impact cerebral perfusion. [ABSTRACT FROM AUTHOR]

Vascular cognitive impairment and dementia (VCID) is a diagnostic term applied to cognitively impaired individuals with heterogeneous cerebrovascular conditions affecting large and/or small vessels. Individual biomarkers have been identified as instrumental in relating VCID to specific underlying pathologies to better characterize this syndrome. Emerging research to refine panels of biomarkers will increase classification sensitivity and specificity. Refined VCID clustering based on the severity and pathology of vascular injury will permit the development of optimal prevention and treatment strategies. Here, we review recently reported data concerning the diversity of VCID-related pathology and attempts for VCID clustering based on biomarkers obtained from different sets of measurements. We discuss three major sets of biomarkers: 1) neuroimaging biomarkers, 2) neuropsychological performance measures, and 3) biochemical markers in current VCID clustering. Finally, we highlight the effect of blood-brain barrier health on cerebrovascular disease trajectory. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is the deadliest of brain tumors. Standard treatment for GBM is surgery, followed by combined radiation therapy and chemotherapy. Current therapy prolongs survival but does not offer a cure. We report on a novel immunotherapy against GBM, tested in an animal model of C57BL/6 mice injected intra-cranially with a lethal dose of murine GL261 glioma cells.Methods: Ten week-old C57BL/6 mice were anesthetized before injection of 2 × 10(4) GL261 cells in the right cerebral hemisphere and after 3 days half of the mice were administered a single subcutaneous (s.c.) injection of irradiated semi-allogeneic vaccine, while mock-vaccinated mice received a s.c. injection of phosphate-buffered saline (PBS). Tumor engraftment was monitored through bioluminescence imaging (BLI). Length of animal survival was measured by Kaplan-Meier graphs and statistics. At time of sacrifice brain tissue was processed for estimation of tumor size and immunohistochemical studies.Results: Overall survival of vaccinated mice was significantly longer compared to mock-vaccinated mice. Five to ten percent of vaccinated mice survived more than 90 days following the engraftment of GL261 cells in the brain and appeared to be free of disease by BLI. Tumor volume in the brain of vaccinated mice was on average five to ten-fold smaller compared to mock-vaccinated mice. In vaccinated mice, conspicuous microglia infiltrates were observed in tumor tissue sections and activated microglia appeared to form a fence along the perimeter of the tumor cells. The results of these animal studies persuaded the Office of Orphan Products Development of the Food and Drug Administration (FDA) to grant Orphan Drug Designation for treatment of GBM with irradiated, semi-allogeneic vaccines.Conclusions: Our preclinical observations suggest that semi-allogeneic vaccines could be tested clinically on subjects with GBM, as an adjuvant to standard treatment. [ABSTRACT FROM AUTHOR]

Therapeutic agents that improve the memory loss of Alzheimer's disease (AD) may eventually be developed if drug targets are identified that improve memory deficits in appropriate AD animal models. One such target is β-secretase which, in most AD patients, cleaves the wild-type (WT) β-secretase site sequence of the amyloid-β protein precursor (AβPP) to produce neurotoxic amyloid-β (Aβ). Thus, an animal model representing most AD patients for evaluating β-secretase effects on memory deficits is one that expresses human AβPP containing the WT β-secretase site sequence. BACE1 and cathepsin B (CatB) proteases have β-secretase activity, but gene knockout studies have not yet validated that the absence of these proteases improves memory deficits in such an animal model. This study assessed the effects of deleting these protease genes on memory deficits in the AD mouse model expressing human AβPP containing the WT β-secretase site sequence and the London γ-secretase site (AβPPWT/Lon mice). Knockout of the CatB gene in the AβPPWT/Lon mice improved memory deficits and altered the pattern of Aβ-related biomarkers in a manner consistent with CatB having WT β-secretase activity. But deletion of the BACE1 gene had no effect on these parameters in the AβPPWT/Lon mice. These data are the first to show that knockout of a putative β-secretase gene results in improved memory in an AD animal model expressing the WT β-secretase site sequence of AβPP, present in the majority of AD patients. CatB may be an effective drug target for improving memory deficits in most AD patients. [ABSTRACT FROM AUTHOR]

Background: Experimental results from studies with inbred mice and their syngeneic tumors indicated that the inoculation of semi-allogeneic cell hybrids (derived from the fusion between syngeneic tumor cells and an allogeneic cell line) protects the animal host from a subsequent lethal challenge with unmodified syngeneic tumor cells. Methods: Semi-allogeneic somatic cell hybrids were generated by the fusion of EL-4 T lymphoma cells (H-2b) and BALB/c-derived renal adenocarcinoma RAG cells (H-2d). Cell hybrids were injected intra-peritoneally (i.p.) in C57BL/6 mice (H-2b) before challenging the mice with a tumorigenic dose of EL-4 cells. Results: Semi-allogeneic tumor cell hybrids could not form a tumor in the animal host because they expressed allogeneic determinants (H-2d) and were rejected as a transplant. However, they conferred protection against a tumorigenic challenge of EL-4 cells compared to control mice that were mock-vaccinated with i.p.-injected phosphate-buffered saline (PBS) and in which EL-4 lymphomas grew rapidly to a large size in the peritoneal cavity. Screening of spleen-derived RNA by means of focused microarray technology revealed up-regulation of genes involved in the Th-1- type immune response and in the activation of dendritic antigen-presenting cells (APC). Conclusion: The results of our studies are entirely consistent with the concept that CD80- and CD86-expressing APC play a central role in mediating the immune protection induced by semiallogeneic vaccines by activating a Th-1 response and instructing T cells responsible for killing autologous tumor cells. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is characterized by the tissue deposition of beta-amyloid peptide (Abeta) in the brain. Recent studies have shown apoproteins (apo) in amyloid plaques and associated with high-density lipoprotein (HDL) particles in the cerebrospinal fluid (CSF). Western blot analysis revealed that serum amyloid A (apoSAA) protein was present in control and AD patients at low levels compared to apoE and apoA-I, however, AD brains showed a significant increase over control values. Analysis of CSF-HDL from control and AD individuals showed that apoA-I, apoE and apoSAA were on the particle. Immuno-cytochemical analysis showed that SAA was detected in senile plaques in AD tissue, but was predominantly localized to neuritic plaques. ApoE staining of AD brain confirmed that most plaques contained the apoprotein, similar to Abeta immunoreactivity, whereas apoA-I expressed little staining of senile plaques. No significant differences were detected in the level of apoSAA when compared to APOE genotype in AD samples, suggesting that interactions with apoE were non-specific. These data imply that the specific interactions of SAA with Abeta in the neuritic plaques may play a role in AD. [ABSTRACT FROM AUTHOR]

Describes the use of biotynilated beta-galactosidase conjugate prepared by the posttranslational modification of a recombinant fusion protein in the development of a heterogeneous binding assay for biotin. Identical dose-response curves obtained by employing two different batches of bioetynilated beta-galactosidase.

Previous studies have shown that amyloid‐β oligomers (AβO) bind with high affinity to cellular prion protein (PrPC). The AβO‐PrPC complex binds to cell‐surface co‐receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen‐1 cells, 67LR is the major co‐receptor involved in the cellular uptake of AβO and AβΟ‐induced cell death. Both pharmacological (dibutyryl‐cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase‐activating polypeptide) cAMP‐elevating agents decreased cell‐surface PrPC and 67LR, thereby attenuating the uptake of AβO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AβO‐induced cytotoxicity by decreasing cell‐surface‐associated PrPC and 67LR. [ABSTRACT FROM AUTHOR]

The Atlantic bottlenose dolphin has been the focus of much attention owing to the considerable impact of environmental stress on its health and the associated implications for human health. Here, we used skin cells from the dolphin to investigate the protective role of the vitamin D pathway against environmental stressors. We previously reported that dolphin skin cells respond to 1,25-dihydroxyvitamin D3 (1,25D3), the bioactive metabolite of vitamin D3, by upregulation of the vitamin D receptor (VDR) and expression of several genes. Methylmercury is a highly bioaccumulative environmental stressor of relevance to the dolphin. We currently report that in dolphin cells sublethal concentrations of methylmercury compromise the ability of 1,25D3 to upregulate VDR, to transactivate a vitamin D-sensitive promoter, and to express specific target genes. These results help elucidate the effects of vitamin D and methylmercury on innate immunity in dolphin skin and potentially in human skin as well, considering similarities in the vitamin D pathway between the two species. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet β-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1 R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine-induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Discusses the role of amyloid beta protein in Alzheimer's disease. Association of genes in familial Alzheimer's disease; Details on the role of amyloid beta protein in inflammation and the disease; Effect of amyloid beta protein accumulation in mouse models.

The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen-1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA, but dependent on Epac. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries. Image 1 • Cyclic-AMP induces a reversible internalization of NgR1 from the cell surface. • Epac (exchange protein directly activated by cAMP) plays a crucial role in this. • NgR1 is redistributed back to the cell surface with a decrease in intracellular cAMP. • Internalization of cell surface NgR1 inhibits Nogo-A-induced growth cone collapse. [ABSTRACT FROM AUTHOR]

Serum amyloid A (SAA) proteins are acute-phase reactant associated with high-density lipoprotein (HDL) particles and increase in the plasma 1000-fold during inflammation. Recent studies have implicated SAAs in innate immunity and various disorders; however, the precise mechanism eludes us. Previous studies have shown SAAs are elevated following stroke and cerebral ischemia, and our studies demonstrated that SAA-deficient mice reduce inflammation and infarct volumes in a mouse stroke model. Our studies demonstrate that SAA increases the cytokine interleukin-1β (IL-1/3), which is mediated by Nod-like receptor protein 3 (NLRP3) inflammasome, cathepsin B, and caspase-1 and may play a role in the pathogenesis of neurological disorders. SAA induced the expression of NLRP3, which mediated IL-β induction in murine BV-2 cells and both sex primary mouse microglial cells, in a dose- and time-dependent fashion. Inhibition or KO of the NLRP3 in microglia prevented the increase in IL-β. N-acetyl-i-cysteine and mito-TEMPO blocked the induction of IL-β by inhibiting ROS with SAA treatment. In addition, inhibition of cathepsin B with different drugs or microglia from CatBdeficient mice attenuated inflammasome activation. Our studies suggest that the impact of SAA on inflammasome stimulation is mediated in part by the receptor for advanced glycation endproducts and Toll-like receptor proteins 2 and 4. SAA induced inflammatory cytokines and an M1 phenotype in the microglial cells while downregulating anti-inflammation M2 phenotype. These studies suggest that brain injury to can elicit a systemic inflammatory response mediated through SAA that contributes to the pathological outcomes. [ABSTRACT FROM AUTHOR]

Background: Amyotrophic lateral sclerosis (ALS) is currently an incurable disease without highly effective pharmacological treatments. The peptide drug GM604 (GM6 or Alirinetide) was developed as a candidate ALS therapy, which has demonstrated safety and good drug-like properties with a favorable pharmacokinetic profile. GM6 is hypothesized to bolster neuron survival through the multi-target regulation of developmental pathways, but mechanisms of action are not fully understood. Methods: This study used RNA-seq to evaluate transcriptome responses in SH-SY5Y neuroblastoma cells following GM6 treatment (6, 24 and 48 h). Results: We identified 2867 protein-coding genes with expression significantly altered by GM6 (FDR < 0.10). Early (6 h) responses included up-regulation of Notch and hedgehog signaling components, with increased expression of developmental genes mediating neurogenesis and axon growth. Prolonged GM6 treatment (24 and 48 h) altered the expression of genes contributing to cell adhesion and the extracellular matrix. GM6 further down-regulated the expression of genes associated with mitochondria, inflammatory responses, mRNA processing and chromatin organization. GM6-increased genes were located near GC-rich motifs interacting with C2H2 zinc finger transcription factors, whereas GM6-decreased genes were located near AT-rich motifs associated with helix-turn-helix homeodomain factors. Such motifs interacted with a diverse network of transcription factors encoded by GM6-regulated genes (STAT3, HOXD11, HES7, GLI1). We identified 77 ALS-associated genes with expression significantly altered by GM6 treatment (FDR < 0.10), which were known to function in neurogenesis, axon guidance and the intrinsic apoptosis pathway. Conclusions: Our findings support the hypothesis that GM6 acts through developmental-stage pathways to influence neuron survival. Gene expression responses were consistent with neurotrophic effects, ECM modulation, and activation of the Notch and hedgehog neurodevelopmental pathways. This multifaceted mechanism of action is unique among existing ALS drug candidates and may be applicable to multiple neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Neurological disorders represent major health concerns in terms of comorbidity and mortality worldwide. Despite a tremendous increase in our understanding of the pathophysiological processes involved in disease progression and prevention, the accumulated knowledge so far resulted in relatively moderate translational benefits in terms of therapeutic interventions and enhanced clinical outcomes. Aiming at specific neural molecular pathways, different strategies have been geared to target the development and progression of such disorders. The kallikrein-kinin system (KKS) is among the most delineated candidate systems due to its ubiquitous roles mediating several of the pathophysiological features of these neurological disorders as well as being implicated in regulating various brain functions. Several experimental KKS models revealed that the inhibition or stimulation of the two receptors of the KKS system (B1R and B2R) can exhibit neuroprotective and/or adverse pathological outcomes. This updated review provides background details of the KKS components and their functions in different neurological disorders including temporal lobe epilepsy, traumatic brain injury, stroke, spinal cord injury, Alzheimer’s disease, multiple sclerosis and glioma. Finally, this work will highlight the putative roles of the KKS components as potential neurotherapeutic targets and provide future perspectives on the possibility of translating these findings into potential clinical biomarkers in neurological disease. [ABSTRACT FROM AUTHOR]

Background: Cerebral ionic homeostasis impairment, especially Ca2+, has been observed in Alzheimer's disease (AD) and also with hypertension. Hypertension and AD both have been implicated in impaired cerebral autoregulation. However, the relationship between the ionic homeostasis impairment inADand hypertension and cerebral blood flow(CBF) autoregulation is not clear. Objective: To test the hypothesis that a high-salt diet regimen influences the accumulation of amyloid-β (Aβ) and CBF, exacerbates cognitive decline, and increases the propensity to AD. Methods: Double transgenic mice harboring the amyloid-β protein precursor (APPswe), and presenilin-1 (PSEN1) along with control littermates, 2 months of age at initiation of special diet, were divided into 4 groups: Group A, APP/PS1 and Group B, controls fed a high-sodium (4.00%) chow diet for 3 months; Group C, APP/PS1 and Group D, controls fed a low-sodium (0.08%) regular chow diet for 3 months. Mean arterial blood pressure (MAP) and CBF were measured noninvasively using the tail MAP measurement device and magnetic resonance imaging, respectively. Aβ plaques numbers in the cortex and hippocampus of APP/PS1 were quantified. Results: In contrary to controls, APP/PS1 mice fed a high-salt diet did not showmarkedly elevated mean systolic and diastolic blood pressure (134±4.8 compared with 162±2.8 mmHg, and 114±5.0 compared with 137±20 mmHg, p < 0.0001). However, a high-salt diet increased CBF in both APP/PS1 and controls and did not alter the cerebral tissue integrity. Aβ plaques were significantly reduced in the cortex and hippocampus of mice fed a high-salt diet. Conclusion: These data suggest that a high-salt diet differently affects MAP and CBF in APP/PS1 mice and controls. [ABSTRACT FROM AUTHOR]

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints. [ABSTRACT FROM AUTHOR]

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD+-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke. [ABSTRACT FROM AUTHOR]

Background: Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion.Methods: We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke.Results: C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients.Conclusions: Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke. [ABSTRACT FROM AUTHOR]

Because the nervous system is most vulnerable to toxicants during development, there is a crucial need for a highly sensitive developmental-neurotoxicity-test model to detect potential toxicants at low doses. We developed a lab-on-chip wherein single-neuron axonal pathfinding under geometric guidance was created using soft lithography and laser cell-micropatterning techniques. After coating the surface with L1, an axon-specific member of the Ig family of cell adhesion molecules (CAMs), and optimizing microunit geometric parameters, we introduced low-dose methylmercury, a well-known, environmentally significant neurotoxicant, in the shared medium. Its developmental neurotoxicity was evaluated using a novel axonal pathfinding assay including axonal turning and branching rates at turning points in this model. Compared to the conventional neurite-outgrowth assay, this model's detection threshold for low-dose methylmercury was 10-fold more sensitive at comparable exposure durations. These preliminary results support study of developmental effects of known and potential neurotoxicants on axon pathfinding. This novel assay model would be useful to study neuronal disease mechanisms at the single-cell level. To our knowledge, the potential of methylmercury chloride to cause acute in vitro developmental neurotoxicity (DNT) at such a low dosage has not been reported. This is the first DNT test model with high reproducibility to use single-neuron axonal pathfinding under precise geometric guidance. [ABSTRACT FROM AUTHOR]

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma. [ABSTRACT FROM AUTHOR]

There are currently no effective therapeutic agents for traumatic brain injury (TBI), but drug treatments for TBI can be developed by validation of new drug targets and demonstration that compounds directed to such targets are efficacious in TBI animal models using a clinically relevant route of drug administration. The cysteine protease, cathepsin B, has been implicated in mediating TBI, but it has not been validated by gene knockout (KO) studies. Therefore, this investigation evaluated mice with deletion of the cathepsin B gene receiving controlled cortical impact TBI trauma. Results indicated that KO of the cathepsin B gene resulted in amelioration of TBI, shown by significant improvement in motor dysfunction, reduced brain lesion volume, greater neuronal density in brain, and lack of increased proapoptotic Bax levels. Notably, oral administration of the small-molecule cysteine protease inhibitor, E64d, immediately after TBI resulted in recovery of TBI-mediated motor dysfunction and reduced the increase in cathepsin B activity induced by TBI. E64d outcomes were as effective as cathepsin B gene deletion for improving TBI. E64d treatment was effective even when administered 8 h after injury, indicating a clinically plausible time period for acute therapeutic intervention. These data demonstrate that a cysteine protease inhibitor can be orally efficacious in a TBI animal model when administered at a clinically relevant time point post-trauma, and that E64d-mediated improvement of TBI is primarily the result of inhibition of cathepsin B activity. These results validate cathepsin B as a new TBI therapeutic target. [ABSTRACT FROM AUTHOR]

The preservation of functional neural tissue after spinal cord injury (SCI) is the basis for spontaneous neurological recovery. Some injured patients in the acute phase have more potential for recovery than others. This fact is problematic for the construction of clinical trials because enrollment of subjects with variable recovery potential makes it difficult to detect effects, requires large sample sizes, and risks Type II errors. In addition, the current methods to assess injury and recovery are non-quantitative and not sensitive. It is likely that therapeutic combinations will be necessary to cause substantially improved function after SCI, thus we need highly sensitive techniques to evaluate changes in motor, sensory, autonomic and other functions. We review several emerging neurophysiological techniques with high sensitivity. Quantitative methods to evaluate residual tissue sparing after severe acute SCI have not entered widespread clinical use. This reduces the ability to correlate structural preservation with clinical outcome following SCI resulting in enrollment of subjects with varying patterns of tissue preservation and injury into clinical trials. We propose that the inclusion of additional measures of injury severity, pattern, and individual genetic characteristics may enable stratification in clinical trials to make the testing of therapeutic interventions more effective and efficient. New imaging techniques to assess tract injury and demyelination and methods to quantify tissue injury, inflammatory markers, and neuroglial biochemical changes may improve the evaluation of injury severity, and the correlation with neurological outcome, and measure the effects of treatment more robustly than is currently possible. The ability to test such a multimodality approach will require a high degree of collaboration between clinical and research centers and government research support. When the most informative of these assessments is determined, it may be possible to identify patients with substantial recovery potential, improve selection criteria and conduct more efficient clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin D promotes the differentiation of prostate cancer cells, raising the possibility that vitamin D deficiency over time may contribute to the progression from subclinical prostate cancer to clinical disease. Since low-risk prostate cancers are monitored over time in an effort to determine which progress into clinically important, more aggressive cancers, they provide an excellent model in which to study, over an extended period of time, the effects of enhancing vitamin D status and related changes in tumor progression. This is particularly relevant to African-American men, who exhibit a high prevalence of vitamin D deficiency as well as higher incidence of prostate cancer and higher mortality rates from prostate cancer than Caucasians. Our research team has recently completed an open-label clinical trial aimed at assessing the safety and potential efficacy of vitamin D3 supplementation at 4000 international units (IU) per day for one year in subjects diagnosed with early stage, low-risk prostate cancer. The results of this clinical study suggest that supplementation with vitamin D3 at 4000IU per day may benefit patients with early stage, low-risk prostate cancer on active surveillance, because of the improved outcome (a decreased number of positive cores at repeat biopsy) in more than half of the subjects enrolled in the trial. We also observed that, after one year of supplementation, there was no difference in circulating levels of vitamin D between African-American and Caucasian subjects who completed the study. These clinical results also suggest that robust and sustained vitamin D3 supplementation can reduce prostate cancer-related health disparities in African-American men and that these health disparities are at least in part the result of widespread hypovitaminosis D within the African-American population. This article is part of a Special Issue entitled ‘Vitamin D Workshop’. [Copyright &y& Elsevier]

Stroke is one of the major causes of death and disability in the United States. After cerebral ischemia and reperfusion injury, the generation of reactive oxygen species (ROS) and reactive nitrogen species may contribute to the disease process through alterations in the structure of DNA, RNA, proteins, and lipids. We generated various nanoparticles (liposomes, polybutylcyanoacrylate (PBCA), or poly(lactide-co-glycolide) (PLGA)) that contained active superoxide dismutase (SOD) enzyme (4,000 to 20,000 U/kg) in the mouse model of cerebral ischemia and reperfusion injury to determine the impact of these molecules. In addition, the nanoparticles were untagged or tagged with nonselective antibodies or antibodies directed against the N-methyl-D-aspartate (NMDA) receptor 1. The nanoparticles containing SOD protected primary neurons in vitro from oxygen-glucose deprivation (OGD) and limited the extent of apoptosis. The nanoparticles showed protection against ischemia and reperfusion injury when applied after injury with a 50% to 60% reduction in infarct volume, reduced inflammatory markers, and improved behavior in vivo. The targeted nanoparticles not only showed enhanced protection but also showed localization to the CA regions of the hippocampus. Nanoparticles alone were not effective in reducing infarct volume. These studies show that targeted nanoparticles containing protective factors may be viable candidates for the treatment of stroke. [ABSTRACT FROM AUTHOR]

There is mounting evidence indicating an important role for complement in the pathogenesis of cerebral ischemia-reperfusion injury, or ischemic stroke. The role of the alternative complement pathway in ischemic stroke has not been investigated, and there is conflicting data on the role of the terminal pathway. In this study, we show that compared with wild-type mice, mice deficient in the alternative pathway protein factor B or mice treated with the alternative pathway inhibitor CR2-fH have improved outcomes after 60-min middle cerebral artery occlusion and 24-h reperfusion. Factor B-deficient or CR2-fH-treated mice were protected in terms of improved neurologic function and reduced cerebral infarct, demyelination, P-selectin expression, neutrophil infiltration, and microthrombi formation. Mice deficient in both the classical and lectin pathways (Clq/MBL deficient) were also protected from cerebral ischemia-reperfusion injury, and there was no detectable C3d deposition in the ipsilateral brain of these mice. These data demonstrate that the alternative pathway is not alone sufficient to initiate complement activation and indicate that the alternative pathway propagates cerebral injury via amplification of the cascade. Deficiency of C6, a component of the terminal cytolytic membrane attack complex, had no effect on outcome after ischemic stroke, indicating that the membrane attack complex is not involved in mediating injury in this model. We additionally show that the protective effect of factor B deficiency and CR2-fH treatment is sustained in the subacute stage of infarct development, adding to the clinical relevance of these findings. [ABSTRACT FROM AUTHOR]

Abstract: The resin of Boswellia species is a major anti-inflammatory agent that has been used for centuries to treat various conditions including injuries and inflammatory conditions. Incensole acetate (IA), a major constituent of this resin, has been shown to inhibit NF-κB activation and concomitant inflammation, as well as the neurological deficit following head trauma. Here, we show that IA protects against ischemic neuronal damage and reperfusion injury in mice, attenuating the inflammatory nature of ischemic damage. IA given post-ischemia, reduced infarct volumes and improved neurological activities in the mouse model of ischemic injury in a dose dependent fashion. The protection from damage was accompanied by inhibition of TNF-α, IL-1β and TGF-β expression, as well as NF-κB activation following injury. In addition, IA is shown to have a therapeutic window of treatment up to 6h after ischemic injury. Finally, the protective effects of IA were partially mediated by TRPV3 channels as determined by the TRPV3 deficient mice and channel blocker studies. This study suggests that the anti-inflammatory and neuroprotective activities of IA may serve as a novel therapeutic treatment for ischemic and reperfusion injury, and as a tool in the ongoing research of mechanisms for neurological damage. [Copyright &y& Elsevier]

Self-reactive natural Abs initiate injury following ischemia and reperfusion of certain tissues, but their role in ischemic stroke is unknown. We investigated neoepitope expression in the postischemic brain and the role of natural Abs in recognizing these epitopes and mediating complement-dependent injury. A novel IgM mAb recognizing a subset of phospholipids (C2) and a previously characterized anti-annexin IV mAb (B4) were used to reconstitute and characterize injury in Ab-deficient Rag1-/- mice after 60 min of middle cerebral artery occlusion and reperfusion. Reconstitution with C2 or B4 mAb in otherwise protected Rag1-/- mice restored injury to that seen in wild-type (wt) mice, as demonstrated by infarct volume, demyelination, and neurologic scoring. IgM deposition was demonstrated in both wt mice and reconstituted Rag1-/- mice, and IgM colocalized with the complement activation fragment C3d following B4 mAb reconstitution. Further, recombinant annexin IV significantly reduced infarct volumes in wt mice and in Rag1-/- mice administered normal mouse serum, demonstrating that a single Ab reactivity is sufficient to develop cerebral ischemia reperfusion injury in the context of an entire natural Ab repertoire. Finally, C2 and B4 mAbs bound to hypoxic, but not normoxic, human endothelial cells in vitro. Thus, the binding of pathogenic natural IgM to postischemic neoepitopes initiates complement-dependent injury following murine cerebral ischemia and reperfusion, and, based also on previous data investigating IgM reactivity in human serum, there appears to be a similar recognition system in both mouse and man [ABSTRACT FROM AUTHOR]

Therapeutic agents that improve the memory loss of Alzheimer's disease (AD) may eventually be developed if drug targets are identified that improve memory deficits in appropriate AD animal models. One such target is β-secretase which, in most AD patients, cleaves the wild-type (WT) β-secretase site sequence of the amyloid-β protein precursor (AβPP) to produce neurotoxic amyloid-β (Aβ). Thus, an animal model representing most AD patients for evaluating β-secretase effects on memory deficits is one that expresses human AβPP containing the WT β-secretase site sequence. BACE1 and cathepsin B (CatB) proteases have β-secretase activity, but gene knockout studies have not yet validated that the absence of these proteases improves memory deficits in such an animal model. This study assessed the effects of deleting these protease genes on memory deficits in the AD mouse model expressing human AβPP containing the WT β-secretase site sequence and the London γ-secretase site (AβPPWT/Lon mice). Knockout of the CatB gene in the AβPPWT/Lon mice improved memory deficits and altered the pattern of Aβ-related biomarkers in a manner consistent with CatB having WT β-secretase activity. But deletion of the BACE1 gene had no effect on these parameters in the AβPPWT/Lon mice. These data are the first to show that knockout of a putative β-secretase gene results in improved memory in an AD animal model expressing the WT β-secretase site sequence of AβPP, present in the majority of AD patients. CatB may be an effective drug target for improving memory deficits in most AD patients. [ABSTRACT FROM AUTHOR]

In addition to its function in calcium and bone metabolism, vitamin D is neuroprotective and important for mitigating inflammation. Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system, characterized by neuronal loss in many areas of the brain, and the formation of senile (neuritic) plaques, which increase in number and size over time. The goal of this project was to investigate whether vitamin D3 supplementation would affect amyloid plaque formation in amyloid-β protein precursor (AβPP) transgenic mice that spontaneously develop amyloid plaques within 3-4 months of birth. AβPP mice were fed control, vitamin D3-deficient or vitamin D3-enriched diets for five months, starting immediately after weaning. At the end of the study, the animals were subjected to behavioral studies, sacrificed, and examined for bone changes and brain amyloid load, amyloid-β (Aβ) peptide levels, inflammatory changes, and nerve growth factor (NGF) content. The results obtained indicate that a vitamin D3-enriched diet correlates with a decrease in the number of amyloid plaques, a decrease in Aβ peptides, a decrease in inflammation, and an increase in NGF in the brains of AβPP mice. These observations suggest that a vitamin D3-enriched diet may benefit AD patients. [ABSTRACT FROM AUTHOR]

Current evidence suggests that Alzheimer’s disease (AD) is a multi-factorial disease that starts with accumulation of multiple proteins. We have previously proposed that inhibition of c-secretase may impair membrane recycling causing neurodegeneration starting at synapses (Sambamurti K., Suram A., Venugopal C., Prakasam A., Zhou Y., Lahiri D. K. and Greig N. H. A partial failure of membrane protein turnover may cause Alzheimer’s disease: a new hypothesis. Curr. Alzheimer Res., 3, 2006, 81). We also proposedfamilal AD mutations increase Ab42 by inhibiting c-secretase. Herein, we discuss the failure of Eli Lilly’s c-secretase inhibitor, semagacestat, in clinical trials in the light of our hypothesis, which extends the problem beyond toxicity of Ab aggregates. We elaborate that c-secretase inhibitors lead to accumulation of amyloid precursor protein C-terminal fragments that can later be processed by c-secretase to yields bursts of Ab to facilitate aggregation. Although we do not exclude a role for toxic Ab aggregates, inhibition of c-secretase can affect numerous substrates other than amyloid precursor protein to affect multiple pathways and the combined accumulation of multiple peptides in the membrane may impair its function and turnover. Taken together, protein processing and turnover pathways play an important role in maintaining cellular homeostasis and unless we clearly see consistent diseaserelated increase in their levels or activity, we need to focus on preserving their function rather than inhibiting them for treatment of AD and similar diseases. [ABSTRACT FROM AUTHOR]

Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1-6/CerS1-6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C16:0-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C16:0-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca2+-loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca2+ accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca2+ uptake or decreasing Ca2+-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca2+ homeostasis and calpain, which appears to be important in OL apoptosis during brain development. [ABSTRACT FROM AUTHOR]

Transcranial laser therapy (TLT) was tested for efficacy in a mouse model of Alzheimer's disease (AD) using a near-infrared energy laser system. TLT is thought to stimulate ATP production, increase mitochondrial activity, and help maintain neuronal function. Studies were performed to determine the effect of TLT in an amyloid-β protein precursor (AβPP) transgenic mouse model. TLT was administered 3 times/week at various doses, starting at 3 months of age, and was compared to a control group (no laser treatment). Treatment was continued for a total of six months. Animals were examined for amyloid load, inflammatory markers, brain amyloid-β (Aβ) levels, plasma Aβ levels, cerebrospinal fluid Aβ levels, soluble AβPP (sAβPP) levels, and behavioral changes. The numbers of Aβ plaques were significantly reduced in the brain with administration of TLT in a dose-dependent fashion. Administration of TLT was associated with a dose-dependent reduction in amyloid load. All TLT doses mitigated the behavioral effects seen with advanced amyloid deposition and reduce the expression of inflammatory markers in the AβPP transgenic mice. All TLT doses produced an increase in sAβPPα and a decrease in CTFβ levels consistent with inhibition of the β-secretase activity. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos suggesting an overall improvement in neurological function. These studies suggest that TLT is a potential candidate for treatment of AD. [ABSTRACT FROM AUTHOR]

In addition to its function in calcium and bone metabolism, vitamin D is neuroprotective and important for mitigating inflammation. Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system, characterized by neuronal loss in many areas of the brain, and the formation of senile (neuritic) plaques, which increase in number and size over time. The goal of this project was to investigate whether vitamin D3 supplementation would affect amyloid plaque formation in amyloid-β protein precursor (AβPP) transgenic mice that spontaneously develop amyloid plaques within 3-4 months of birth. AβPP mice were fed control, vitamin D3-deficient or vitamin D3-enriched diets for five months, starting immediately after weaning. At the end of the study, the animals were subjected to behavioral studies, sacrificed, and examined for bone changes and brain amyloid load, amyloid-β (Aβ) peptide levels, inflammatory changes, and nerve growth factor (NGF) content. The results obtained indicate that a vitamin D3-enriched diet correlates with a decrease in the number of amyloid plaques, a decrease in Aβ peptides, a decrease in inflammation, and an increase in NGF in the brains of AβPP mice. These observations suggest that a vitamin D3-enriched diet may benefit AD patients. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca2+/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca2+-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke. [ABSTRACT FROM AUTHOR]

Background: Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimer's disease. Most therapeutic development studies have focused on toxicity directly at the synapse. Methodology/Principal Findings: Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Ab (1-42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods. Conclusions/Significance: The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment. [ABSTRACT FROM AUTHOR]

Abstract: The Atlantic bottlenose dolphin has attracted attention due to the evident impact that environmental stressors have taken on its health. In order to better understand the mechanisms linking environmental health with dolphin health, we have established cell cultures from dolphin skin as in vitro tools for molecular evaluations. The vitamin D3 pathway is one mechanism of interest because of its well established chemopreventative and immunomodulatory properties in terrestrial mammals. On the other hand, little is known of the physiological role of this molecule in aquatic animals. 1,25-dihydroxyvitamin D3 (1,25D3), the bioactive and hormonal form of vitamin D3, exerts its biological function by binding to the vitamin D receptor (VDR), a ligand-activated regulator of gene transcription. Therefore, we investigated the transcriptomic changes induced by 1,25D3 administration in dolphin skin cells. Identification of specific genes activated by 1,25D3 has provided clues to the physiological function of the vitamin D3 pathway in the dolphin. We found that exposure of the cells to 1,25D3 upregulated transactivation of a vitamin D-sensitive promoter. cDNA microarray analysis, using a novel dolphin array, identified specific gene targets within this pathway, and real-time PCR (qPCR) confirmed the enhanced expression of select genes of interest. These transcriptional changes correlated with an increase in VDR levels. This is the first report of the presence and activation of the vitamin D3 pathway in a marine mammal, and our experimental results demonstrate a number of similarities to terrestrial animals. Conservation of this pathway in the Atlantic bottlenose dolphin is consistent with the importance of nonclassic functions of vitamin D3, such as its role in innate immunity, similar to what has been demonstrated in other mammals. [Copyright &y& Elsevier]

Abstract: Although the majority of human illnesses occur during adulthood, most of the available in vitro disease models are based upon cells obtained from embryonic/fetal tissues because of the difficulties involved with culturing adult cells. Development of adult mouse neuronal cultures has a special significance because of the abundance of transgenic disease models that use this species. In this study a novel cell culture method has been developed that supports the long-term survival and physiological regeneration of adult mouse hippocampal cells in a serum-free defined environment. In this well-defined, controlled system, adult mouse hippocampal cells survived for up to 21 days in culture. The cultured cells exhibited typical hippocampal neuronal morphology and electrophysiological properties after recovery from the trauma of dissociation, and stained positive for the expected neuronal markers. This system has great potential as an investigative tool for in vitro studies of adult diseases, the aging brain or transgenic models of age-associated disorders. [Copyright &y& Elsevier]

Abstract: Motoneuronotrophic factor (MNTF) is an endogenous neurotrophin that is highly specific for the human nervous system, and some of the observed effects of MNTF include motoneuron differentiation, maintenance, survival, and reinnervation of target muscles and organs. MNTF is a neuro-signaling molecule that binds to specific receptors. Using In Silico Analysis, one of the active sites of MNTF was identified as an analog of six amino acids (GM6). The effect of chemically synthesized GM6 on ischemic stroke was studied in the middle cerebral artery occlusion (MCAo) mouse model. Mice were subjected to 1 hur of ischemia followed by 24 h of reperfusion. Mice were injected intravenously with a bolus of GM6, at various doses (1 and 5 mg/kg) immediately after the start of reperfusion and examined for changes in physiological parameters, neurological deficits and infarct volume. GM6 was able to penetrate the blood brain barrier, and at both 1 and 5 mg/kg showed a significant protection from infarct damage, which translated to improvement of neurological deficits. Administration of GM6 demonstrated no changes in HR, BP, pO2, pCO2, or pH. A significant increase over the control group in CBF after reperfusion was observed with GM6 administration, which helped to mitigate the ischemic effect caused by the blockage of blood flow. The time window of treatment was assessed at various times following cerebral ischemia with GM6 demonstrating a significant protective effect up to 6–12 h post ischemia. In addition, GM6 increased neurogenesis, and decreased apoptosis and inflammation in the mouse brain following cerebral ischemic injury. These data suggest that GM6 is neuroprotective to the brain following IV injection in the mouse model of MCAo. [Copyright &y& Elsevier]

Integrins govern cellular adhesion and transmit signals leading to activation of intracellular signaling pathways aimed to prevent apoptosis. Herein we report that attachment of oligodendrocytes (OLs) to fibronectin via αvβ3 integrin receptors rendered the cells more resistant to apoptosis than the cells attached to laminin via α6β1 integrins. Investigation of molecular mechanisms involved in αvβ3integrin-mediated cell survival revealed that ligation of the integrin with fibronectin results in higher expression of activated Lyn kinase. Both in OLs and in the mouse brain, Lyn selectively associates with αvβ3 integrin, not with αvβ5 integrin, leading to suppression of acid sphingomyelinase activity and preventing ceramide-mediated apoptosis. In OLs, knockdown of Lyn with small interfering RNA resulted in OL apoptosis with concomitant accumulation of C16-ceramide due to activation of acid sphingomyelinase (ASMase) and sphingomyelin hydrolysis. Knocking down ASMase partially protected OLs from apoptosis. In the brain, ischemia/reperfusion (IR) triggered rearrangements in the αvβ3 integrin-Lyn kinase complex leading to disruption of Lyn kinase-mediated suppression of ASMase activity. Thus, co-immunoprecipitation studies revealed an increased association of αvβ3 integrin-Lyn kinase complex with ionotropic glutamate receptor subunits, GluR2 and GluR4, after cerebral IR. Sphingolipid analysis of the brain demonstrated significant accumulation of ceramide and sphingomyelin hydrolysis. The data suggest a novel mechanism for regulation of ASMase activity during cell adhesion in which Lyn acts as a key upstream kinase that may play a critical role in cerebral IR injury. [ABSTRACT FROM AUTHOR]

Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer’s disease-like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer’s disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)-deficient mice were fed a high fat/cholesterol diet for an 8-week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR−/− mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat-fed C57BL/6 mice and LDLR−/− mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor-α, interleukin-1β and -6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. β-site APP cleaving enzyme 1 in both high fat/cholesterol-fed C57BL/6 and LDLR−/− mice accompanied by an increased generation of C-terminal fragments of APP. Although ELISA for beta-amyloid failed to record significant changes in the non-transgenic mice, a threefold increase in beta-amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild-type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non-transgenic mouse model. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have demonstrated that tumor necrosis factor-alpha (TNF-α) is one of the most important mediators in spinal cord injury (SCI). However, the role of TNF-α in this process is still under debate due to conflicting evidence. Here, we utilized TNF-α transgenic (tg) rats and wild-type (wt) littermates to further investigate the role of TNF-α in SCI. We observed that, in the acute phase post-SCI (≤3 days), TNF-α tg rats showed higher expression of TNF-α protein and more apoptotic cells in the spinal cord than wt rats, while in the chronic period (≥7 days), TNF-α tg rats exhibited persistent baseline level of TNF-α protein, better tissue healing, and more activated astrocytes in the border of the lesion than wt rats. These data further demonstrate that TNF-α plays a dual role in SCI and its role probably depends on when it is released after SCI and on which cellular population it acts on. [Copyright &y& Elsevier]

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, studies have suggested that accumulation of amyloid beta (Aβ) peptide in the brain plays a key role in the development of Alzheimer''s disease (AD). The steady-state level of Aβ peptide in the brain is determined by the rate of production from amyloid precursor protein (APP) via β- and γ-secretases and degradation by the activity of several enzymes. Neprilysin (NEP) appears to be the most potent Aβ peptide-degrading enzyme in the brain. Decreasing the activity of NEP (due to genetic mutations, age or diseases that alter the expression or activity of NEP) may lead to accumulation of the neurotoxic Aβ peptide in the brain; in turn this leads to neuronal loss. We investigated the efficacy of lentivirus-mediated over-expression of NEP to protect neuronal cells from Aβ peptide in vitro. Incubation of hippocampal neuronal cells (HT22) over-expressing NEP with the monomeric from of Aβ peptide decreases the toxicity of Aβ peptide on the neuronal cells, as measured through cell viability. We conclude that over-expression of NEP by a gene therapy approach in areas vulnerable to Aβ peptide aggregation in AD brain may protect the neurons from the toxicity effects of Aβ peptide and this promises a great potential target for altering the development of AD. [Copyright &y& Elsevier]

Abstract: A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at www.marinegenomics.org). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues. [Copyright &y& Elsevier]

Individuals with Down''s syndrome (DS) develop the pathological hallmarks of Alzheimer''s (AD) disease at an early age, subsequently followed by memory decline and dementia. We have utilized an animal model for DS, mice with segmental trisomy of chromosome 16 (Ts65Dn), to study biological events linked to memory loss. Previous studies demonstrated a cognitive decline and loss of cholinergic markers after 6–8 months of age. In the current study, we found increased levels of amyloid precursor protein (APP) in the striatum by 6–8 months of age, and in the hippocampus and parietal cortex by 13–16 months of age in Ts65Dn but not in normosomic mice. Additionally, Ts65Dn mice exhibited alterations in nerve growth factor (NGF) levels in the basal forebrain and hippocampus. Ts65Dn mice demonstrated a significant decline in NGF levels in the basal forebrain with age, as well as a reduction in hippocampal NGF by 13–16 months of age. These findings demonstrate that elevated APP and decreased NGF levels in limbic areas correlate with the progressive memory decline and cholinergic degeneration seen in middle-aged trisomic mice. [Copyright &y& Elsevier]

Presents a study that tested a lentiviral vector expressing human neprilysin (Lenti-Nep) in transgenic mouse models of amyloidosis. Information on neprilysin; Materials and methods; Expression of active neprilysin from Lenti-Nep-transduced cells.

Examines the role of estrogen receptors in mediating neuroprotective actions of estradiol. Potential biological roles of each estrogen receptor subtype in the injured brain; Effects of estradiol in stroke injury; Effect of the absence of estrogen receptor alpha on estradiol-mediated protection against total brain injury.