Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

Fibroblast growth factor receptor modulators employing diamines with reduced phospholipidosis-inducing potential.

SUN13837 (1), a fibroblast growth factor receptor modulator, has been an attractive candidate for treating neurodegenerative diseases. However, one of its metabolites, N-benzyl-4-(methylamino)piperidine (BMP), turned out to possess phospholipidosis-inducing potential (PLIP) in vitro. To obtain SUN13837 analogs with reduced phospholipidosis risk, we replaced BMP with other diamines possessing low PLIP. Our effort led to the discovery of compound 6 with increased efficacy. Further structural modifications to reduce hydrogen bond donors afforded 17 with improved brain exposure. Oral administration of 17 at 1 mg/kg once daily for 10 days showed enhanced recovery of coordinated movement in a rat acute stroke model, suggesting that it is a promising follow-up compound for 1 with reduced risk of phospholipidosis.

Systemic treatment with a novel basic fibroblast growth factor mimic small-molecule compound boosts functional recovery after spinal cord injury.

Neurotrophic factors have been regarded having promising potentials for neuronal protection and regeneration, and thus promoting beneficial effects of kinesiological functions. They can be suspected to play important roles in cell/tissue grafting for various neural diseases. The clinical applications of such trophic factors to the central nervous system (CNS), however, have caused problematic side effects on account of the distinctive bioactive properties. In the course of developing synthetic compounds reflecting beneficial properties of basic fibroblast growth factor (bFGF), we conducted screening candidates that stimulate to trigger the intracellular tyrosine phosphorylation of FGF receptor and lead to the subsequent intracellular signaling in neurons. A small synthetic molecule SUN13837 was characterized by mimicking the beneficial properties of bFGF, which have been known as its specific activities when applied to CNS. What is more remarkable is that SUN13837 is eliminated the bioactivity to induce cell proliferation of non-neuronal somatic cells. On the bases of studies of pharmacology, behavior, physiology and histology, the present study reports that SUN13837 is characterized as a promising synthetic compound for treatment of devastating damages onto the rat spinal cord.

Discovery of 1,2,3-triazole-based fibroblast growth factor receptor modulators.

To avoid production of a phospholipidosis-inducing metabolite, we replaced the amide structure of SUN13837 (1) with a 1,2,3-triazole. The resulting 1,2,3-triazole analog of 1 (compound 2) displayed greater neuroprotective activity than 1. Structural modification of 2 yielded compound 10, which showed improved neuroprotective activity and negligible mechanism-based inactivation against CYP3A4. In addition, installation of a methyl group at the 5-position of 1,2,3-triazole of 10 significantly boosted the neuroprotective activity. These 1,2,3-triazole derivatives displayed reduced phospholipidosis risk, sufficient systemic exposure, and high central nervous system penetration, and therefore may be potentially useful agents for the treatment of neurodegenerative diseases.

Synthesis and evaluation of N-(4-benzylphenyl)piperazines as VGF inducers.

A series of compounds was discovered that induce the production of VGF mRNA in SH-SY5Y cells and exhibit cytoprotection under tunicamycin induced endoplasmic reticulum (ER) stress. The aminophenol ring and linker chain of the template SUN N8075 (1) was modified to yield compounds with higher efficacy and lower propensity for adverse effects.

SUN11602, a novel aniline compound, mimics the neuroprotective mechanisms of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) offers some measure of protection against excitotoxic neuronal injuries by upregulating the expression of the calcium-binding protein calbindin-D28k (Calb). The newly synthesized small molecule 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) mimics the neuroprotective effects of bFGF, and thus, we examined how SUN11602 exerts its actions on neurons in toxic conditions of glutamate. In primary cultures of rat cerebrocortical neurons, SUN11602 and bFGF prevented glutamate-induced neuronal death. This neuroprotection, which occurred in association with the augmented phosphorylation of the bFGF receptor-1 (FGFR-1) and the extracellular signal-regulated kinase-1/2 (ERK-1/2), was abolished by pretreatment with PD166866 (a FGFR-1 tyrosine kinase-specific inhibitor) and PD98059 (a mitogen-activated protein kinase [MAPK]/[ERK-1/2] kinase [MEK] inhibitor). In addition, SUN11602 and bFGF increased the levels of CALB1 gene expression in cerebrocortical neurons. Whether this neuroprotection was linked to Calb was investigated with primary cultures of cerebrocortical neurons from homozygous knockout (Calb(-/-)) and wild-type (WT) mice. In WT mice, SUN11602 and bFGF increased the levels of newly synthesized Calb in cerebrocortical neurons and suppressed the glutamate-induced rise in intracellular Ca(2+). This Ca(2+)-capturing ability of Calb allowed the neurons to survive severe toxic conditions of glutamate. In contrast, Calb levels remained unchanged in Calb(-/-) mice after exposure to SUN11602 or bFGF, and due to a loss of function of the gene, these neurons were no longer resistant to toxic conditions of glutamate. These findings indicated that SUN11602 activated a number of cellular molecules (FGFR-1, MEK/ERK intermediates, and Calb) and consequently contributed to intracellular Ca(2+) homeostasis as observed in the case of bFGF.

Abeta-induced BACE-1 cleaves N-terminal sequence of mPGES-2.

We previously indicated that amyloid beta (Abeta) augments protein levels of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) through oxidative stress. In this study, we revealed that BACE-1 is involved in the cleavage of membrane-bound prostaglandin E2 synthase-2 (mPGES-2) in its N-terminal portion, which, in turn, enhanced the generation of prostaglandin E2 (PGE2). PGE2 results in increased Abeta production, initiating a cell-injuring cycle. Using rat primary cortical neurons, a 48 h treatment with Abeta 1-42 (5 microM) resulted in the enhanced extracellular PGE2 levels up to about 1 ng/mL, which was attenuated by treatment with a BACE-1 inhibitor (200 nM). A synthetic peptide sequence of 20-amino acids that included the cleavage site of mPGES-2 (HTARWHL RAQDLHERS AAQLSLSS) was cleaved by recombinant BACE-1, confirmed using reverse-phase high-performance liquid chromatography. Cleaved or activated mPGES-2 augments the generation of PGE2. In addition, mPGES-2 was determined to be colocalized with BACE-1 and cyclooxygenase-2 in the perinuclear region in cells after exposure to Abeta. Exposure of neurons to PGE2 led to cell death, and Abeta production was enhanced by PGE2 (1 ng/mL, 48 h). Collectively, these results suggest that Abeta might cause neuroinflammation that aggravates Alzheimer's disease pathogenesis.

Epigallocatechin-3-gallate and curcumin suppress amyloid beta-induced beta-site APP cleaving enzyme-1 upregulation.

Beta-site APP cleaving enzyme-1 (BACE-1), is a rate-limiting enzyme for beta amyloid production. Beta amyloid induces the production of radical oxygen species and neuronal injury. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease. Recent studies suggest that oxidative stress induces BACE-1 protein upregulation in neuronal cells. Here, we demonstrate that naturally occurring compounds (-)-epigallocatechin-3-gallate and curcumin suppress beta amyloid-induced BACE-1 upregulation. Exposure of beta amyloid 1-42 to neuronal culture increased BACE-1 protein levels. (-)-Epigallocatechin-3-gallate or curcumin significantly attenuated beta amyloid-induced radical oxygen species production and beta-sheet structure formation. These two compounds have novel pharmacological effects that may be beneficial for Alzheimer's disease treatment.

Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features.

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.

Three distinct neuroprotective functions of myricetin against glutamate-induced neuronal cell death: involvement of direct inhibition of caspase-3.

The excitatory neurotransmitter glutamate can accumulate in the brain and is thought to be involved in the etiology of many neurodegenerative disorders, including ischemia and Alzheimer disease. Therefore, it is important to search for compounds that reduce glutamate neurotoxicity. This glutamate-mediated excitotoxicity is caused by intracellular Ca2+ overload via the N-methyl-D-aspartate receptor NMDAR), reactive oxygen species (ROS) generation, and caspase-3 activation. Here we show that the natural flavonoid myricetin inhibited glutamate-induced excitotoxicity and protected neurons by multiple, distinct pathways. First, myricetin affected modulation of the NMDAR by phosphorylation, causing a subsequent reduction in glutamate-induced intracellular Ca2+ overload. Second, myricetin inhibited the ROS production caused by glutamate. Finally, glutamate-induced activation of caspase-3 was reduced by myricetin treatment. Moreover, myricetin directly interacted with the active site of caspase-3 via three hydrogen bonds and inhibited its activity. We conclude that myricetin inhibited glutamate-induced neuronal toxicity by multiple biochemical pathways. These results show that myricetin is a potent antineurodegenerative compound and may contribute to the discovery of a drug with which to combat neurodegeneration.

Multifunction of myricetin on A beta: neuroprotection via a conformational change of A beta and reduction of A beta via the interference of secretases.

Myricetin (3,3',4',5,5',7-hexahydroxyflavone) is classified as a flavonoid with strong antioxidant effects. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease (AD). To elucidate whether myricetin could counter the progress of AD, we examined the effects of myricetin on neurotoxicity induced by beta-amyloid (A beta), a component of senile plaques in the AD brain. We found that cultured rat primary cortical neurons treated for 48 hr with A beta1-42 (1 microM) induced significant neuronal injury. Conformationally altered A beta1-42 caused apoptotic changes, such as nuclear fragmentation, as shown by DAPI staining. Pre- and simultaneous administration of myricetin and A beta1-42 reduced A beta neurotoxicity in a concentration-dependent manner. By using circular dichroism spectroscopy and a thioflavin T binding assay, we show that myricetin (10 microM, 48 hr) prevented structural changes in A beta1-42 from a random coil to a beta-sheet-rich structure. A beta1-42-induced apoptotic changes and caspase-3 activation were reduced by myricetin treatment. Furthermore, we determined that administration of myricetin significantly decreased A beta1-40 and A beta1-42 levels in culture media. These effects were based on two mechanisms: the activation and up-regulation of alpha-secretase (ADAM10) protein levels as indicated by fluorescence resonance energy transfer (FRET) assay and immunoblot analysis and the direct binding and inhibition of beta-secretase (BACE-1) indicated by cell-free FRET assays. Evidently, myricetin has multiple functions to counter the progress of AD by the reduction of A beta production and the detoxification of A beta through a structural change.

Distinct mechanisms underlie distinct polyphenol-induced neuroprotection.

Glutamate excitotoxicity is mediated by intracellular Ca(2+) overload, caspase-3 activation, and ROS generation. Here, we show that curcumin, tannic acid (TA) and (+)-catechin hydrate (CA) all inhibited glutamate-induced excitotoxicity. Curcumin inhibited PKC activity, and subsequent phosphorylation of NR1 of the NMDA receptor. As a result, glutamate-mediated Ca(2+) influx was reduced. TA attenuated glutamate-mediated Ca(2+) influx only when simultaneously administered, directly interfering with Ca(2+). Both curcumin and TA inhibited glutamate-induced caspase-3 activation. Although Ca(2+) influx was not attenuated by CA, caspase-3 was reduced by direct inhibition of the enzyme. All polyphenols reduced glutamate-induced generation of ROS.