SUN11602-induced hyperexpression of calbindin D-28k is pivotal for the survival of hippocampal neurons under neurotoxic conditions.

Basic fibroblast growth factor (FGF-2/bFGF) possesses neuroprotective activity and promotes cell proliferation. In this study, the novel synthetic compound 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) exhibited neuroprotective activities similar to those of FGF-2 without promoting cell proliferation. In primary cultures of hippocampal neurons, stimulation with SUN11602 or FGF-2 increased calbindin D-28k (CalB) gene expression and prevented glutamate-induced neuronal death. These effects were abolished by pretreatment with PD166866 (FGF receptor 1 [FGFR1] tyrosine kinase-specific inhibitor). This indicated that FGFR1 activation and increased CalB expression were involved in SUN11602-mediated neuroprotection. However, receptor-binding assays revealed that unlike FGF-2, SUN11602 did not alter the binding of (125)I-labeled FGF-2 to FGFR1. To investigate the possible proliferative activity of SUN11602, we utilized BHK21 and SKN cells expressing endogenous FGFR1. FGF-2 promoted cell proliferation whereas SUN11602 did not. In in vivo studies, wild-type (WT) and CalB-deficient (CalB(-/-)) mice were injected with aggregated Aß1-40 and ibotenate (NMDA receptor agonist) to severely damage the hippocampal tissue. Treatment with SUN11602 (orally) or FGF-2 (intraparenchymally) at the midpoint of Aß1-40 and ibotenate injections prevented the hippocampal damage in WT mice, however this effect was abolished in CalB(-/-) mice. Thus, SUN11602 exerted protective effects on hippocampal neurons through activation of FGFR1 and increased CalB expression. Moreover, the neuroprotective effects of SUN11602 depended upon the various biological activities of FGF-2.

SUN11602 has basic fibroblast growth factor-like activity and attenuates neuronal damage and cognitive deficits in a rat model of Alzheimer's disease induced by amyloid ß and excitatory amino acids.

Basic fibroblast growth factor (bFGF/FGF-2) is known to possess neuroprotective and neurite outgrowth activity properties. In this study, the effects of a novel synthetic compound that mimics the neuroprotective properties of bFGF - SUN11602 - were examined in vitro and in vivo. SUN11602 promoted neurite outgrowth of primarily cultured rat hippocampal neurons. For the in vivo study, an Alzheimer's disease (AD) model with severe damage to the hippocampal tissue was constructed by injecting the hippocampi of rats with aggregated Aß1-40, followed 48 h later by an injection of ibotenate [an agonist for N-methyl-d-aspartate (NMDA) receptor]. Oral administration of SUN11602 at the midpoint of Aß1-40 and ibotenate injections attenuated short-term memory impairment in the Y-maze test, as well as spatial learning deficits in the water maze task. In addition, the SUN11602 treatment inhibited the increase of peripheral-type benzodiazepine-binding sites (PTBBS), which are a marker for gliosis. A negative correlation was found between PTBBS numbers and learning capacity in the water maze task. These results suggest that SUN111602 improved memory and learning deficits in the hippocampally lesioned rats by preventing neuronal death and/or promotion of neurite outgrowth. Taken together, these results indicate that SUN11602, a bFGF-like compound with neuroprotective and neurite outgrowth activity, may be beneficial for the treatment of progressive neurodegenerative diseases such as AD.

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.

Effect of bFGF on neuronal damage induced by sequential treatment of amyloid ß and excitatory amino acid in vitro and in vivo.

Effects of basic fibroblast growth factor (bFGF), a potent neurotrophin, on neuronal damage induced by sequential treatment of amyloid ß (Aß) peptide and excitatory amino acid were examined in vitro and in vivo. Treatment of rat primary cortical neurons with glutamate (10µM, 30µM) resulted in neuronal damage, and pretreatment of the neurons with Aß(25-35) (1.0µM) at 48h before glutamate stimulation augmented the susceptibility of the cells to the glutamate-induced neurotoxicity. Application of bFGF (0.3, 1, 3ng/ml) and MK-801 (1, 3, 10, 30nM) to the culture at 24h before glutamate stimulation markedly decreased the neuronal damage elicited by Aß(25-35) and glutamate. In a rat model of Alzheimer's disease, in which aggregated Aß(1-40) (4µg/1µl) was injected into the hippocampus, followed by an injection of ibotenate (an NMDA receptor agonist, 0.3µg/0.5µl) into the same sites at 48h later, significant neuronal damage and learning deficit was induced. Administration of bFGF (25ng/1µl) into the hippocampus at 24h before ibotenate inhibited the neuronal damage and demonstrated a trend of attenuating spatial learning deficits. These results suggest that bFGF might be a useful agent for treatment of Alzheimer's disease in which Aß peptide and glutamate would be involved as causative substances.

Cognitive dysfunction induced by sequential injection of amyloid-beta and ibotenate into the bilateral hippocampus; protection by memantine and MK-801.

Aggregated 40-residue amyloid-beta peptide (beta40, 4 microg/microl), and 2 days later, ibotenate (NMDA receptor agonist, 0.3 microg/0.5 microl), were bilaterally injected into the hippocampus of rats. Five to six weeks after the beta40 injection, the rats showed learning deficits in the Morris water maze task and neuronal damage in the hippocampus, although the injection of beta40 or ibotenate alone did not result in cognitive deficits and hippocampal damage. Memantine (10, 20 mg/kg/day s.c. infusion for 6 weeks starting 24 h before the beta40 injection) significantly prevented learning deficits as measured for 4 days from 5 weeks after the beta40 injection, while a lower dose of memantine (5 mg/kg/day) and MK-801 (0.312, 0.624 mg/kg/day) did not have inhibitory effects on the learning deficits. The neuronal damage in the hippocampus, assessed as an elevation of the levels of the peripheral-type benzodiazepine-binding site (a gliosis marker for neuronal damage) produced by sequential intra-hippocampal injections of beta40 and ibotenate, at 6 weeks (39 days) after the beta40 injection, was significantly attenuated by memantine (10, 20 mg/kg/day) and MK-801 (0.624 mg/kg/day). These protective effects were also confirmed by histochemical examination (Cresyl violet staining of brain slices). In naive rats, MK-801 produced a significant learning impairment in the water maze task at a dose of 0.624 mg/kg/day, while memantine (20 mg/kg/day s.c. infusion) did not, although the beta40 plus ibotenate-induced hippocampal damage was lessened by both treatments. These results suggest that memantine and MK-801 exert protective effects on progressive neuronal damage, but that only memantine prevents memory impairment in hippocampal-lesioned rats, and that memantine may be a beneficial agent for the treatment of progressive cognitive dysfunction including Alzheimer's disease-type dementia.

Mitochondrial peroxiredoxin-3 protects hippocampal neurons from excitotoxic injury in vivo.

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium-buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin-3 (Prx-3), to investigate the mechanism by which toxic properties of ROS were up-regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx-3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx-3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx-3 completely inhibited protein nitration and markedly reduced gliosis, a post-neuronal cell death event. Since mitochondrial Prx-3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx-3 up-regulation might be a useful novel approach for the management of neurodegenerative diseases.