T-Type Ca2+ Enhancer SAK3 Activates CaMKII and Proteasome Activities in Lewy Body Dementia Mice Model.

Lewy bodies are pathological characteristics of Lewy body dementia (LBD) and are composed of α-synuclein (α-Syn), which is mostly degraded via the ubiquitin-proteasome system. More importantly, 26S proteasomal activity decreases in the brain of LBD patients. We recently introduced a T-type calcium channel enhancer SAK3 (ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)- 2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate) for Alzheimer's disease therapeutics. SAK3 enhanced the proteasome activity via CaMKII activation in amyloid precursor protein knock-in mice, promoting the degradation of amyloid-ß plaques to improve cognition. At this point, we addressed whether SAK3 promotes the degradation of misfolded α-Syn and the aggregates in α-Syn preformed fibril (PFF)-injected mice. The mice were injected with α-Syn PFF in the dorsal striatum, and SAK3 (0.5 or 1.0 mg/kg) was administered orally for three months, either immediately or during the last month after injection. SAK3 significantly inhibited the accumulation of fibrilized phosphorylated-α-Syn in the substantia nigra. Accordingly, SAK3 significantly recovered mesencephalic dopamine neurons from cell death. Decreased α-Syn accumulation was closely associated with increased proteasome activity. Elevated CaMKII/Rpt-6 signaling possibly mediates the enhanced proteasome activity after SAK3 administration in the cortex and hippocampus. CaMKII/Rpt-6 activation also accounted for improved memory and cognition in α-Syn PFF-injected mice. These findings indicate that CaMKII/Rpt-6-dependent proteasomal activation by SAK3 recovers from α-Syn pathology in LBD.

T-type Ca2+ channel enhancer SAK3 administration improves the BPSD-like behaviors in AppNL-G-F/NL-G-F knock-in mice.

Alzheimer's disease (AD) accounts for the majority of dementia among the elderly. In addition to cognitive impairment, behavioral and psychological symptoms (BPSD) such as depression tendency and increased aggression impose a great burden on the patient. However, there is still no rational therapeutic drug for BPSD. Recently, we developed a novel AD therapeutic candidate, SAK3, and demonstrated that it improved cognitive dysfunction in AppNL-G-F/NL-G-F knock-in (NL-G-F) mice. In this study, we investigated whether acute SAK3 administration improved BPSD in addition to cognitive improvement. Acute SAK3 administration improved BPSD, including anxiolytic and depressive-like behaviors, and ameliorated aggressive behaviors. Furthermore, continuous SAK3 administration improved anxiolytic and depressive-like behaviors. Intriguingly, the anti-anxiolytic and cognitive improvement lasted two weeks after the withdrawal of SAK3, whereas the anti-depressive action did not. Taken together, SAK3 had comprehensive beneficial effects on BPSD behavior.

Single Administration of the T-Type Calcium Channel Enhancer SAK3 Reduces Oxidative Stress and Improves Cognition in Olfactory Bulbectomized Mice.

Alzheimer's disease (AD), characterized by cognitive impairments, is considered to be one of the most widespread chronic neurodegenerative diseases worldwide. We recently introduced a novel therapeutic agent for AD treatment, the T-type calcium channel enhancer ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)-2H-spiro[cyclopentane-1,3-imidazo[1,2-a]pyridin]-2-ene-3-carboxylate (SAK3). SAK3 enhances calcium/calmodulin-dependent protein kinase II and proteasome activity, thereby promoting amyloid beta degradation in mice with AD. However, the antioxidative effects of SAK3 remain unclear. We investigated the antioxidative effects of SAK3 in olfactory bulbectomized mice (OBX mice), compared with the effects of donepezil as a positive control. As previously reported, single oral administration of both SAK3 (0.5 mg/kg, p.o.) and donepezil (1.0 mg/kg, p.o.) significantly improved cognitive and depressive behaviors in OBX mice. Single oral SAK3 administration markedly reduced 4-hydroxy-2-nonenal and nitrotyrosine protein levels in the hippocampus of OBX mice, which persisted until 1 week after administration. These effects are similar to those observed with donepezil therapy. Increased protein levels of oxidative stress markers were observed in the microglial cells, which were significantly rescued by SAK3 and donepezil. SAK3 could ameliorate oxidative stress in OBX mice, like donepezil, suggesting that the antioxidative effects of SAK3 and donepezil are among the neuroprotective mechanisms in AD pathogenesis.

Anti-Epileptic Effects of FABP3 Ligand MF1 through the Benzodiazepine Recognition Site of the GABAA Receptor.

Recently, we developed the fatty acid-binding protein 3 (FABP3) ligand MF1 (4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid) as a therapeutic candidate for α-synucleinopathies. MF1 shows affinity towards γ-aminobutyric acid type-A (GABAA) receptor, but its effect on the receptor remains unclear. Here, we investigate the pharmacological properties of MF1 on the GABAA receptor overexpressed in Neuro2A cells. While MF1 (1-100 µm) alone failed to evoke GABA currents, MF1 (1 µm) promoted GABA currents during GABA exposure (1 and 10 µm). MF1-promoted GABA currents were blocked by flumazenil (10 µm) treatment, suggesting that MF1 enhances receptor function via the benzodiazepine recognition site. Acute and chronic administration of MF1 (0.1, 0.3 and 1.0 mg/kg, p.o.) significantly attenuated status epilepticus (SE) and the mortality rate in pilocarpine (PILO: 300 mg/kg, i.p.)-treated mice, similar to diazepam (DZP: 5.0 mg/kg, i.p.). The anti-epileptic effects of DZP (5.0 mg/kg, i.p.) and MF1 (0.3 mg/kg, p.o.) were completely abolished by flumazenil (25 mg/kg, i.p.) treatment. Pentylenetetrazol (PTZ: 90 mg/kg, i.p.)-induced seizures in mice were suppressed by DZP (5.0 mg/kg, i.p.), but not MF1. Collectively, this suggests that MF1 is a mild enhancer of the GABAA receptor and exercises anti-epileptic effects through the receptor's benzodiazepine recognition site in PILO-induced SE models.

SAK3 Administration Improves Spine Abnormalities and Cognitive Deficits in AppNL-G-F/NL-G-F Knock-in Mice by Increasing Proteasome Activity through CaMKII/Rpt6 Signaling.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by neuropathological hallmarks consisting of accumulation of extracellular amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles (NFT). Recently, we have identified a new AD therapeutic candidate, ethyl-8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo [1,2-a] pyridin]-2-ene-3-carboxylate (SAK3), which ameliorates the AD-like pathology in AppNL-F/NL-F knock-in mice. However, the detailed mechanism underlying the therapeutic effects of SAK3 remains unclear. In this study, we found that SAK3 administration improved the reduced proteasome activity through the activation of CaMKII/Rpt6 signaling in AppNL-F/NL-F knock-in (NL-G-F) mice. Moreover, spine abnormalities observed in NL-G-F mice were significantly reversed by SAK3 administration. Along with this, cognitive impairments found in NL-G-F mice were markedly ameliorated by SAK3. In summary, our data suggest that SAK3 administration increases the activity of the proteasome via activation of the CaMKII/Rpt6 signaling pathway, contributing to improvements in spine abnormalities and cognitive deficits in NL-G-F mice. Overall, our findings suggest that SAK3 might be a new attractive drug candidate, representing a new mechanism for the treatment of AD pathology.

Oral glutathione administration inhibits the oxidative stress and the inflammatory responses in AppNL-G-F/NL-G-F knock-in mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by the presence of extracellular amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles. Reduced antioxidants and increased oxidative stress and inflammation are responsible for the pathological features characteristic of an AD brain. We observed decreased levels of the reduced form of glutathione (GSH), the most abundant brain antioxidant, and decreased GSH/glutathione disulfide (GSSG) ratios in AppNL-G-F/NL-G-F knock-in (NL-G-F) mouse brains. Repeated oral GSH administration for 3 weeks dose-dependently increased GSH levels and restored the GSH/GSSH ratio. Consistent with the restoration of GSH levels, the levels of 4-hydroxy-2-nonenal (4-HNE), a marker of oxidative stress, were significantly decreased in the hippocampus of NL-G-F mice. Additionally, inflammatory responses, such as microgliosis and increased mRNA expression of inflammatory cytokines, were also inhibited. Moreover, behavioral deficits including cognitive decline, depressive-like behaviors, and anxiety-related behaviors observed in NL-G-F mice were significantly improved by oral and chronic GSH administration. Taken together, our data suggest that oral GSH administration is an attractive therapeutic strategy to reduce the excessive oxidative stress and inflammatory responses in the AD brain.

Novel fatty acid-binding protein 3 ligand inhibits dopaminergic neuronal death and improves motor and cognitive impairments in Parkinson's disease model mice.

The main symptom of Parkinson's disease (PD) is motor dysfunction and remarkably approximately 30-40% of PD patients exhibit cognitive impairments. Recently, we have developed MF8, a heart-type fatty acid-binding protein (FABP3)-specific ligand, which can inhibit α-synuclein (α-syn) oligomerization induced by arachidonic acid in FABP3 overexpressing neuro2A cells. The present study aimed to determine whether MF8 attenuates dopaminergic neuronal death and motor and cognitive impairments in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. MF8 can penetrate the blood-brain barrier and its peak brain concentration (21.5 ± 2.1 nM) was achieved 6 h after the oral administration (1.0 mg/kg). We also compared its effects and pharmacological action with those of L-DOPA (3,4-dihydroxy-l-phenylalanine). PD model mice were developed by administering MPTP (25 mg/kg, i.p.) once a day for five consecutive days. Twenty-four hours after the final MPTP injection, mice were administered MF8 (0.3, 1.0 mg/kg, p.o.) or L-DOPA (25 mg/kg, i.p.) once a day for 28 consecutive days and subjected to behavioral and histochemical studies. MF8 (1.0 mg/kg, p.o.), but not L-DOPA, inhibited the dopaminergic neuronal death in the ventral tegmental area and the substantia nigra pars compacta region of the MPTP-treated mice. MF8 also improved both, motor and cognitive functions, while L-DOPA ameliorated only motor dysfunction. Taken together, our results showed that MF8 attenuated the MPTP-induced dopaminergic neuronal death associated with PD pathology. We present MF8 as a novel disease-modifying therapeutic molecule for PD, which acts via a mechanism different from that of L-DOPA.

Combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient brain ischemia.

Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient brain ischemia.

Correction: T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice.

[This corrects the article DOI: 10.1371/journal.pone.0206986.].

Alzheimer's disease therapeutic candidate SAK3 is an enhancer of T-type calcium channels.

Low-threshold Ca2+ spikes are mediated by T-type Ca2+ channels, which have electrophysiological properties of fast inactivation and slow deactivation kinetics. A low membrane potential of approximately -60 mV is sufficient to trigger channel opening. We recently introduced a novel T-type Ca2+ channel enhancer that improves cognition and inhibits amyloid beta aggregation in an Alzheimer's disease (AD) mouse model. The enhancer stimulates ACh release, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, and neurogenesis in the hippocampus. Then, we discuss how T-type Ca2+ channel enhancer improves cognition and impaired neurogenesis and how CaMKII signaling in neurodegenerative diseases reduces amyloid beta aggregation. We provide a perspective of the potential AD therapies to target CaMKII signaling. In this context, we overview our attempts leading to the development of a T-type Ca2+ channel enhancer as cognitive enhancer, the action of which has been associated with CaMKII and presumably proteasome activity.

T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice.

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer's disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55-0396 (1 µM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-ß-erythroidine (100 µM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.

Neuroprotective effects of protein tyrosine phosphatase 1B inhibitor on cerebral ischemia/reperfusion in mice.

Akt (Protein kinase B, PKB), a serine/threonine kinase, plays a critical role in cell development, growth, and survival. Akt phosphorylation mediates a neuroprotective effect against ischemic injury. Recently, a protein-tyrosine phosphatase-1B (PTP1B) inhibitor (KY-226) was developed to elicit anti-diabetic and anti-obesity effects via enhancement of insulin signaling. Previously, we reported that the nonselective PTP1B inhibitor, sodium orthovanadate, rescued neurons from delayed neuronal death during brain ischemia. In this study, we confirmed the ameliorative effects of KY-226 on ischemia/reperfusion (I/R) injury using a murine model of middle cerebral artery occlusion (MCAO). ICR mice were subjected to MCAO for 2 h followed by reperfusion. Although KY-226 permeability was poor through the blood-brain barrier (BBB) of normal mice, it could penetrate through the BBB of mice after I/R insult. Intraperitoneal KY-226 administration elicited dose-dependent reductions in infarcted brain areas and improved neurological deficits. The neuroprotective effects of KY-266 were obtained when administered within 0.5 h after reperfusion. KY-226 (10 mg/kg) also restored reduced Akt phosphorylation and eNOS phosphorylation (Ser-1177) levels following I/R insult. Moreover, 10 mg/kg of KY-226 improved I/R-induced decreased extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, KY-226 attenuated the generation of reactive oxygen species (ROS) in mouse cortex. These results suggest that KY-226 may act as a novel therapeutic candidate for ischemic stroke. Activation of Akt and ERK possibly underlie the neuroprotective mechanism of KY-226.

The Disease-modifying Drug Candidate, SAK3 Improves Cognitive Impairment and Inhibits Amyloid beta Deposition in App Knock-in Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common form of elderly dementia in the world. At present, acetylcholine inhibitors, such as donepezil, galantamine and rivastigmine, are used for AD therapy, but the therapeutic efficacy is limited. We recently proposed T-type voltage-gated Ca2+ channels' (T-VGCCs) enhancer as a new therapeutic candidate for AD. In the current study, we confirmed the pharmacokinetics of SAK3 in the plasma and brain of mice using ultra performance liquid chromatography-tandem mass spectrometry. We also investigated the effects of SAK3 on the major symptoms of AD, such as cognitive dysfunction and amyloid beta (Aß) accumulation, in AppNL-F knock-in (NL-F) mice, which have been established as an AD model. Chronic SAK3 (0.5 mg/kg/day) oral administration for 3 months from 9 months of age improved cognitive function and inhibited Aß deposition in 12-month-old NL-F mice. Using microarray and real-time PCR analysis, we discovered serum- and glucocorticoid-induced protein kinase 1 (SGK1) as one of possible genes involved in the inhibition of Aß deposition and improvement of cognitive function by SAK3. These results support the idea that T-VGCC enhancer, SAK3 could be a novel candidate for disease-modifying therapeutics for AD.

Reduced CaM Kinase II and CaM Kinase IV Activities Underlie Cognitive Deficits in NCKX2 Heterozygous Mice.

Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1-5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer's disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/- relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/- relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/- mice. Our findings suggest that like APP23 mice, NCKX2+/- mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.

Reduced expression of Na+/Ca2+ exchangers is associated with cognitive deficits seen in Alzheimer's disease model mice.

Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP-KI mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. Moreover, relative to controls, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/- mouse hippocampus but increased in hippocampus of NCX3+/- mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/- mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP-KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/- mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/- mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/- mice. We conclude that memory impairment seen in NCX2+/- and NCX3+/- mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimer's disease model mice.

Pharmacological properties of SAK3, a novel T-type voltage-gated Ca2+ channel enhancer.

T-type voltage-gated Ca2+ channels (T-VGCCs) function in the pathophysiology of epilepsy, pain and sleep. However, their role in cognitive function remains unclear. We previously reported that the cognitive enhancer ST101, which stimulates T-VGCCs in rat cortical slices, was a potential Alzheimer's disease therapeutic. Here, we introduce a more potent T-VGCC enhancer, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate), and characterize its pharmacological properties in brain. Based on whole cell patch-clamp analysis, SAK3 (0.01-10 nM) significantly enhanced Cav3.1 currents in neuro2A cells ectopically expressing Cav3.1. SAK3 (0.1-10 nM nM) also enhanced Cav3.3 but not Cav3.2 currents in the transfected cells. Notably, Cav3.1 and Cav3.3 T-VGCCs were localized in cholinergic neurve systems in hippocampus and in the medial septum. Indeed, acute oral administration of SAK3 (0.5 mg/kg, p.o.), but not ST101 (0.5 mg/kg, p.o.) significantly enhanced acetylcholine (ACh) release in the hippocampal CA1 region of naïve mice. Moreover, acute SAK3 (0.5 mg/kg, p.o.) administration significantly enhanced hippocampal ACh levels in olfactory-bulbectomized (OBX) mice, rescuing impaired memory-related behaviors. Treatment of OBX mice with the T-VGCC-specific blocker NNC 55-0396 (12.5 mg/kg, i.p.) antagonized both enhanced ACh release and memory improvements elicited by SAK3 administration. We also observed that SAK3-induced ACh releases were significantly blocked in the hippocampus from Cav3.1 knockout (KO) mice. These findings suggest overall that T-VGCCs play a key role in cognition by enhancing hippocampal ACh release and that the cognitive enhancer SAK3 could be a candidate therapeutic in Alzheimer's disease.

Dehydroepiandrosterone administration improves memory deficits following transient brain ischemia through sigma-1 receptor stimulation.

Dehydroepiandrosterone (DHEA) is the most abundant neurosteroid synthesized de novo in the central nervous system. Oral DHEA administration elicits neuroprotection and cognitive improvement, but mechanisms underlying these functions in cerebral ischemia have remained unclear. Since DHEA is the endogenous ligand for the sigma-1 receptor (σ1R), we determined whether oral DHEA administration prevents neuronal cell death and improves cognition via σ1R stimulation in brain ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. Twenty-four hours after BCCAO ischemia, mice were administered DHEA (15 or 30mg/kg p.o.) daily for 11 consecutive days. Memory deficits following brain ischemia were improved by DHEA administration dose-dependently. Accordingly, DHEA administration significantly prevented neuronal cell death in the hippocampal CA1 region in BCCAO mice. Interestingly, DHEA administration rescued decreases in Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) in the CA1 region. Moreover, DHEA administration significantly ameliorated decreases in adenosine 5'-triphosphate (ATP) levels and decreased σ1R expression levels in CA1 following BCCAO ischemia. Finally, co-treatment of mice with the σ1R antagonist NE-100 (1mg/kg, p.o.) blocked DHEA effects on memory improvement and neuroprotection in ischemic mice. Taken together, DHEA prevents neuronal cell death and activates CaMKII via σ1R stimulation, thereby improving cognitive deficits following brain ischemia.

Stimulation of Sigma-1 Receptor Ameliorates Depressive-like Behaviors in CaMKIV Null Mice.

Sigma-1 receptor (Sig-1R) is a molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to the mitochondria. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice exhibit depressive-like behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Here, we demonstrate that chronic stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for 14 days improves depressive-like behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig-1R, did not alter these behaviors. Reduced numbers of BrdU-positive cells and decreased brain-derived neurotrophic factor (BDNF) mRNA expression and protein kinase B (Akt; Ser-473) phosphorylation seen in the DG of CaMKIV null mice were significantly rescued by chronic Sig-1R stimulation. Interestingly, reduced ATP production observed in the DG of CaMKIV null mice was improved by chronic Sig-1R stimulation. Such stimulation also improved hippocampal long-term potentiation (LTP) induction and maintenance, which are impaired in the DG of CaMKIV null mice. LTP rescue was closely associated with both increases in calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and GluA1 (Ser-831) phosphorylation. Taken together, Sig-1R stimulation by SA4503 or fluvoxamine treatment increased hippocampal neurogenesis, which is closely associated with amelioration of depressive-like behaviors in CaMKIV null mice.