Development of a Novel and Robust Pharmacological Model of Okadaic Acid-induced Alzheimer's Disease in Zebrafish.

Alzheimer's disease (AD) is the leading neurodegenerative disorder affecting the world's elderly population. Most experimental models of AD are transgenic or pharmacological in nature, and do not simulate the entire pathophysiology. In the present study, we developed a pharmacologically induced AD using the zebrafish, a species that can recapitulate most of the phenotypes of the disease. The pharmacological agent being used, okadaic acid (OKA) has also been utilized to study AD in other species. In this model, the immunohistochemistry of phosphorylated glycogen synthase-3α/ß, Aß, p-tau, tau protein, and senile plaque formation in zebrafish brain were all significantly increased with increasing exposure to OKA. These represent the majority of the histological hallmarks of AD pathophysiology. The observed changes were also accompanied by learning and memory deficits which are also important components in AD pathophysiology. Zebrafish disease models are gaining popularity mostly due to their economic cost and relevance to human disease pathophysiology. Current pharmacological methods of inducing AD in zebrafish are not adequately developed and do not represent all the features of the disease. OKA-induced AD in zebrafish can become a cost efficient model to study drug discovery for AD. It may also be used to unravel the molecular mechanisms underlying the complex pathophysiology that leads to AD using relatively economical species.

Obesity and hyperglycemia lead to impaired post-ischemic recovery after permanent ischemia in mice.

OBJECTIVE: Obesity-induced diabetes has increased over the years and has become one of the risk factors for stroke. We investigated the influence of diet-induced obesity and hyperglycemia on permanent distal middle cerebral artery occlusion (pMCAO)-induced ischemic stroke in mice. METHODS: Male C57/Bl6 mice were treated with a high-fat/high-carbohydrate diet [HFCD/obese and hyperglycemia (O/H)] or a normal diet (control) for 3.5 months, subjected to pMCAO, and sacrificed after 7 days. RESULTS: Infarct volume analysis showed no differences between the O/H and control group, whereas neurological deficits were significantly higher in the O/H group compared to the control group. Sirtuin (Sirt1) was overexpressed and NADPH oxidase was reduced in the O/H group. O/H mice had significantly lower expression of Wnt and glycogen synthase kinase 3 α and ß, a key component in the Wnt signaling pathway. Translocation of apoptosis inducing factor (AIF) to the nucleus was observed in both the O/H and control groups, but O/H mice showed a higher expression of AIF in the nucleus. CONCLUSIONS: These data suggest that impaired Wnt signaling and active apoptosis result in reduced post-stroke recovery in obese and hyperglycemic mice.

Heme oxygenase 1-mediated neurogenesis is enhanced by Ginkgo biloba (EGb 761®) after permanent ischemic stroke in mice.

Stroke is the fourth leading cause of death and a major cause of disability in stroke survivors. Studies have underlined the importance of repair mechanisms in the recovery phase of stroke. Neurogenesis in response to brain injury is one of the regeneration processes that, if enhanced, may offer better stroke treatment alternatives. Previously, we have demonstrated antioxidant, neuritogenic, and angiogenic properties of Ginkgo biloba/EGb 761® (EGb 761) in different mouse models of stroke. In the present study, we were interested to study whether EGb 761 could protect mice from permanent middle cerebral artery occlusion (pMCAO) and enhance neurogenesis. EGb 761 pre- and posttreated mice had lower infarct volume and improved motor skills with enhanced proliferation of neuronal stem/progenitor cells (NSPCs) at 24 h and 7 days posttreatment. Netrin-1 and its receptors (DCC and UNC5B) that mediate axonal attraction and repulsion were observed to be overexpressed in NSPCs only, implying that netrin-1 and its receptors might have partly played a role in enhanced neurogenesis. Interestingly, in heme oxygenase 1 knockout mice (HO1(-/-)), neurogenesis was significantly lower than in vehicle-treated mice at day 8. Furthermore, EGb 761 posttreated mice also demonstrated heme oxygenase 1 (HO1)-activated pathway of phosphorylated glycogen synthase kinase 3 α/ß (p-GSK-3 α/ß), collapsin response mediator protein 2 (CRMP-2), semaphorin3A (SEMA3A), and Wnt, suggesting probable signaling pathways involved in proliferation, differentiation, and migration of NSPCs. Together, these results propose that EGb 761 not only has antioxidant, neuritogenic, and angiogenic properties, but can also augment the repair and regeneration mechanisms following stroke.

Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death.

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of the structures of several classes of neuroprotective natural products such as limonoids and cardiac glycosides revealed the presence of a common structural motif which may account for their observed neuroprotective activity. Several natural product mimics that incorporate this shared structural motif were synthesized and were found to possess significant neuroprotective activity. These compounds enhanced cell viability against H(2)O(2) induced oxidative stress or cell death in PC12 neuronal cells. The compounds were also found to enhance and modulate Na(+)/K(+)-ATPase activity of PC12 cells, which may suggest that the observed neuroprotective activity is mediated, at least partly, through interaction with Na(+)/K(+)-ATPase.

A derivative of the CRMP2 binding compound lanthionine ketimine provides neuroprotection in a mouse model of cerebral ischemia.

Lanthionines are novel neurotrophic and neuroprotective small molecules that show promise for the treatment of neurodegenerative diseases. In particular, a recently developed, cell permeable lanthionine derivative known as LKE (lanthionine ketimine 5-ethyl ester) promotes neurite growth at low nanomolar concentrations. LKE also has neuroprotective, anti-apoptotic, and anti-inflammatory properties. Its therapeutic potential in cerebral ischemia and its mechanisms of neurotrophic action remain to be fully elucidated. Here, we hypothesize that the neuroprotective actions of LKE could result from induction or modulation of CRMP2. We found that treating primary cultured mouse neurons with LKE provided significant protection against t-butyl hydroperoxide-induced neuronal death possibly through CRMP2 upregulation. Similarly, in vivo studies showed that LKE pre and/or post-treatment protects mice against permanent distal middle cerebral artery occlusion (p-MCAO) as evidenced by lower stroke lesions and improved functional outcomes in terms of rotarod, grip strength and neurologic deficit scores in treated groups. Protein expression levels of CRMP2 were higher in brain cortices of LKE pretreated mice, suggesting that LKE's neuroprotective activity may be CRMP2 dependent. Lower activity of cleaved PARP-1 and higher activity of SIRT-1 was also observed in LKE treated group suggesting its anti-apoptotic properties. Our results suggest that LKE has potential as a therapeutic intervention in cerebral ischemia and that part of its protective mechanism may be attributed to CRMP2 mediated action and PARP-1/SIRT-1 modulation.

Calcium alginate nanoparticles synthesized through a novel interfacial cross-linking method as a potential protein drug delivery system.

The goal of this research work was to develop a novel technique to synthesize calcium alginate nanoparticles using pharmaceutically relevant microemulsions. Stable microemulsion-based reactors were prepared using aqueous sodium alginate, aqueous calcium chloride, dioctyl sodium sulfosuccinate (DOSS), and isopropyl myristate. The reactor microemulsions were characterized via conductivity and dynamic light scattering (DLS) experiments. The conductivity data indicated composition- and reagent-dependent variations in electrical conductivity when the aqueous phase containing reagents were present at or above a Wo (Wo = [DOSS]/[water]) value of 14. The reactor microemulsions were of approximately 6 nm sized droplets. When the reactor microemulsions were mixed and sonicated for 1 h approximately, 350-nm-sized calcium alginate nanoparticles were produced, as indicated by DLS measurements. The particles were isolated and characterized via low-vacuum scanning electron microscopy. The electron micrographs corroborate the DLS results. The nanoparticles were evaluated as a drug delivery system by incorporating bovine serum albumin (BSA) and performing in vitro release and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies. The BSA release profile was characterized by an initial burst release followed by a sustained-release phase. SDS-PAGE studies indicated that the incorporated protein did not suffer covalent aggregation or degradation via fragmentation.

Preconditioning with Ginkgo biloba (EGb 761®) provides neuroprotection through HO1 and CRMP2.

Ginkgo biloba/EGb 761® (EGb 761) is a popular and standardized natural extract used worldwide for the treatment of many ailments. Although EGb 761 is purported to have a plethora of benefits, here, we were interested to study the neuroprotective properties of EGb 761 and its components and determine whether nuclear factor E2 (Nrf2)/heme oxygenase 1 (HO1) induction of the collapsin response mediator protein 2 (CRMP2) pathway contributes to neuroprotection. Mice were pretreated with EGb 761 or one of its constituents (bilobalide, ginkgolide A, ginkgolide B, and terpene free material [TFM]) for 7days and then subjected to transient middle cerebral artery occlusion (tMCAO) and 48 h of reperfusion. All components except TFM significantly reduced infarct volumes and neurologic deficits. Next, we examined the antioxidant and neuritogenic properties of EGb 761 in primary neurons. Compared with vehicle-treated cells, pretreatment with EGb 761 significantly enhanced the survival of neurons exposed to tertiary butylhydroperoxide (t-BuOOH), hydrogen peroxide (H2O2), and N-methyl-D-aspartate (NMDA). Bilobalide and ginkgolide A also protected cells against NMDA-induced excitotoxicity. Immunofluorescence and Western blot analysis showed that EGb 761 pretreatment significantly increased the protein expression levels of Nrf2, HO1, GAPDH, ß-actin, CRMP2, and histone H3 during t-BuOOH-induced oxidative stress. These findings suggest that EGb 761 not only has antioxidant activity but also neuritogenic potential. Demonstrating such effects for possible drug discovery may prove beneficial in stroke and ischemic brain injury.

Developmental programming: differential effects of prenatal testosterone excess on insulin target tissues.

Polycystic ovarian syndrome (PCOS) is the leading cause of infertility in reproductive-aged women with the majority manifesting insulin resistance. To delineate the causes of insulin resistance in women with PCOS, we determined changes in the mRNA expression of insulin receptor (IR) isoforms and members of its signaling pathway in tissues of adult control (n = 7) and prenatal testosterone (T)-treated (n = 6) sheep (100 mg/kg twice a week from d 30-90 of gestation), the reproductive/metabolic characteristics of which are similar to women with PCOS. Findings revealed that prenatal T excess reduced (P < 0.05) expression of IR-B isoform (only isoform detected), insulin receptor substrate-2 (IRS-2), protein kinase B (AKt), peroxisome proliferator-activated receptor-γ (PPARγ), hormone-sensitive lipase (HSL), and mammalian target of rapamycin (mTOR) but increased expression of rapamycin-insensitive companion of mTOR (rictor), and eukaryotic initiation factor 4E (eIF4E) in the liver. Prenatal T excess increased (P < 0.05) the IR-A to IR-B isoform ratio and expression of IRS-1, glycogen synthase kinase-3α and -ß (GSK-3α and -ß), and rictor while reducing ERK1 in muscle. In the adipose tissue, prenatal T excess increased the expression of IRS-2, phosphatidylinositol 3-kinase (PI3K), PPARγ, and mTOR mRNAs. These findings provide evidence that prenatal T excess modulates in a tissue-specific manner the expression levels of several genes involved in mediating insulin action. These changes are consistent with the hypothesis that prenatal T excess disrupts the insulin sensitivity of peripheral tissues, with liver and muscle being insulin resistant and adipose tissue insulin sensitive.

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78.

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations. Activation of ATPase activity of Rep68 by DNA is cooperative with a Hill coefficient of 1.8 +/- 0.2. When examined by gel filtration at 0.5 M NaCl in the absence of DNA, Rep68 self-associates in a concentration-dependent manner. In the presence of DNA containing the binding element, Rep68 (and Rep78) forms protein-DNA complexes that exhibit concentration-dependent self-association in gel filtration analysis. The ATPase activity of the isolated Rep68-DNA and Rep78-DNA complexes is not activated by additional target DNA. Results of sedimentation velocity experiments in the presence of saturating target DNA are consistent with Rep68 forming a hexamer of the protein with two copies of the DNA element. Activation of the ATPase activity of Rep68 is associated with the formation of a protein-DNA oligomer.