[Mechanism of action and clinical trial results of Lecanemab (Leqembi® 200†|mg, 500†|mg for Intravenous Infusion), a novel treatment for Alzheimer's disease].

Lecanemab is a humanized monoclonal antibody directed against human soluble amyloid-ß aggregates. It was developed for the treatment of early Alzheimer's disease (mild cognitive impairment or mild dementia stage of Alzheimer's disease). Among the amyloid-ß (Aß) involved in Alzheimer's disease, Lecanemab selectively binds to the highly neurotoxic Aß protofibrils, and is thought to reduce Aß protofibrils and amyloid plaques (Aß plaques) in the brain. The efficacy and safety of Lecanemab in early Alzheimer's disease were investigated in an international Phase II placebo-controlled study (Study 201) and an international Phase III placebo-controlled study (Study 301). Both studies included Japanese subjects. Lecanemab was given accelerated approval in the United States in January 2023, followed by traditional approval in July 2023. In Japan, it was approved for "control of the progression of mild cognitive impairment or mild dementia stage of Alzheimer's disease" in September 2023, and was added to the NHI drug price list in December 2023.

Methylcobalamin prevents mutant superoxide dismutase-1-induced motor neuron death in vitro.

Amyotrophic lateral sclerosis (ALS) is an incurable progressive neurodegenerative disorder that causes motor dysfunction. Treatments and drugs that slow progression of ALS have garnered great interest. In the present study, we show that the vitamin B12 analog methylcobalamin (MBL) effectively and dose dependently prevented embryonic stem cell-derived motor neuron death induced by cocultivation with astrocytes expressing mutant human superoxide dismutase-1 (G93A). Moreover, cotreatment of MBL with a conventional ALS drug, riluzole, further enhanced survival of motor neurons in this in-vitro ALS model. Our results show the potential use of MBL as a treatment for ALS and suggest a possible combination therapy strategy with other types of approved ALS drugs.

[Fundamental study of memory impairment and non-cognitive behavioral alterations in APPswe/PS1dE9 mice].

In addition to cognitive decline, Alzheimer's disease patients also exhibit non-cognitive symptoms commonly referred to as behavioral and psychological symptoms of dementia, or BPSD. These symptoms have a serious impact on the quality of life of these patients, as well as that of their caregivers, but there are currently no effective therapies. The amyloid ß-peptide (Aß) is suspected to play a central role in the cascade leading to Alzheimer's disease, but the precise mechanisms are still incompletely known. To assess the influence of Aß pathology on cognitive and non-cognitive behaviors, we examined locomotor activity, motor coordination, and spatial memory in male and female APPswePS1dE9 mice (Alzheimer's disease model, double transgenic mice expressing an amyloid precursor protein with Swedish mutation and a presenilin-1 with deletion of exon 9) at 5 months of age, when the mice had subtle Aß deposits, and again at 9 months of age, when the mice had numerous Aß deposits. Compared to wild-type mice, the male and female APPswe/PS1dE9 mice showed normal motor coordination in the rotarod test at both 5 and 9 months. In the Morris water maze test, male and female APPswe/PS1dE9 mice showed impaired spatial memory at 9 months; however, no such deficits were found at 5 months. In a locomotor activity test, male APPswe/PS1dE9 mice exhibited locomotor hyperactivity at 9 months, while females exhibited locomotor hyperactivity at both 5 and 9 months compared to the control mice. Together, these results indicate that APPswe/PS1dE9 mice developed spatial memory impairment and BPSD-like behavioral alterations resulting from Aß accumulation.

Integrin-associated protein promotes neuronal differentiation of neural stem/progenitor cells.

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

Glycogen synthase kinase-3ß activation mediates rotenone-induced cytotoxicity with the involvement of microtubule destabilization.

Rotenone, a mitochondrial complex I inhibitor, has been used to generate animal and cell culture models of Parkinson's disease. Recent studies suggest that microtubule destabilization causes selective dopaminergic neuronal loss. In this study, we investigated glycogen synthase kinase-3ß (GSK3ß) involvement in rotenone-induced microtubule destabilization. Rotenone-induced cytotoxicity in SH-SY5Y cells was attenuated by the GSK3ß inhibitor SB216763. Tau, a microtubule-associated protein and substrate for GSK3ß, has been implicated in the pathogenesis of tauopathies such as Alzheimer's disease. Rotenone induced an increase in phosphorylated tau, the effect of which was attenuated by concomitant treatment with SB216763. Rotenone treatment also decreased tau expression in the microtubule fraction and increased tau expression in the cytosol fraction. These effects were suppressed by SB216763, which suggests that rotenone reduces the capacity of tau to bind microtubules. Rotenone treatment increased the amount of free tubulin and reduced the amount of polymerized tubulin, indicating that rotenone destabilizes microtubules. Rotenone-induced microtubule destabilization was suppressed by SB216763 and taxol, a microtubule stabilizer. Taxol prevented rotenone-induced cytotoxicity and morphological changes. Taken together, these results suggest that rotenone-induced cytotoxicity is mediated by microtubule destabilization via GSK3ß activation, and that microtubule destabilization is caused by reduction in the binding capacity of tau to microtubules, which is a result of tau phosphorylation via GSK3ß activation.

Functional and molecular interactions between Rac1 and FE65.

FE65 is reported to act as an adaptor protein with several protein-interaction domains, including one WW domain and two phosphotyrosine interaction/binding domains. Through these binding domains, FE65 was considered to recruit various binding partners together to form functional complexes in a certain cellular compartment. In this study, we demonstrated that Rac1, a member of the Rho family GTPases, bound with FE65. We also elucidated that Rac1 inhibitor significantly suppressed FE65 expression, and Rac1 small interfering RNA transduction significantly decreased FE65 expression. FE65 small interfering RNA, however, did not influence Rac1 expression and its activity. Taken together, our results reveal that Rac1 interacts with FE65, and Rac1 activity regulates FE65 expression.

Design, synthesis, evaluation and QSAR analysis of N(1)-substituted norcymserine derivatives as selective butyrylcholinesterase inhibitors.

We synthesized a series of N(1)-substituted norcymserine derivatives 7a-p and evaluated their anti-cholinesterase activities. In vitro evaluation showed that the pyridinylethyl derivatives 7m-o and the piperidinylethyl derivative 7p improved the anti-butyrylcholinesterase activity by approximately threefold compared to N(1)-phenethylnorcymserine (PEC, 2). A quantitative structure-activity relationship (QSAR) study indicated that logS might be a key feature of the improved compounds.

Design, synthesis and evaluation of carbamate-modified (-)-N(1)-phenethylnorphysostigmine derivatives as selective butyrylcholinesterase inhibitors.

We synthesized carbamate-modified (-)-N(1)-phenethylnorphysostigmine derivatives 3a-u and evaluated their anti-cholinesterase activities. In vitro evaluation showed that cyclohexylmethylcarbamate derivative 3u potently and selectively inhibits butyrylcholinesterase.

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.

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes.

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer's disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.

Mechanisms of chronic nicotine treatment-induced enhancement of the sensitivity of cortical neurons to the neuroprotective effect of donepezil in cortical neurons.

We have previously shown that chronic donepezil treatment induces nicotinic acetylcholine receptor up-regulation and enhances the sensitivity of the neurons to the neuroprotective effect of donepezil. Further analyses revealed that the nicotinic receptor is involved in this enhancement. In this study, we examined whether nicotinic receptor stimulation is sufficient to make neurons more sensitive to donepezil. We treated primary cultures of rat cortical neurons with nicotine and confirmed that chronic nicotine treatment induced nicotinic receptor up-regulation and made the neurons more sensitive to the neuroprotective effects of donepezil. Analyses with receptor antagonists and kinase inhibitors revealed that the effects of chronic nicotine treatment are mediated by nicotinic receptors and their downstream effectors including phosphatidylinositol 3-kinase. In contrast to chronic donepezil treatment that enhanced the level of nicotine-induced Ca(2+) influx, chronic nicotine treatment did not significantly alter the level of Ca(2+) influx.

Basic fibroblast growth factor promotes the generation of microtubule-associated protein 2-positive cells from microglia.

We recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the role of fibroblast growth factor (FGF) signaling in the molecular mechanism underlying the generation of microglia-derived MAP2-positive and GFAP-positive cells. Real-time quantitative PCR analyses demonstrated that mRNA levels of a family of three FGF receptors, Fgfr1-3, were upregulated in microglia treated with 70% fetal bovine serum (FBS). Immunocytochemical analyses demonstrated that basic FGF (bFGF) promoted the generation of microglia-derived MAP2-positive and GFAP-positive cells, and the FGF receptor tyrosine kinase inhibitor SU5402 and the MEK inhibitor PD98059 both inhibited this process. Western blot analyses demonstrated that bFGF increased phosphorylated ERK1/2 levels without altering total ERK1/2 levels. These results suggest that bFGF promotes the generation of microglia-derived MAP2-positive and GFAP-positive cells via FGF receptors and the ERK-MAP kinase pathway.

Non-fibrillar amyloid-beta peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity.

Amyloid-beta peptide (Abeta) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that Abeta has important physiological roles in addition to its pathological roles. We recently demonstrated that Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between Abeta42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar Abeta42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar Abeta42 on glutamate-induced neurotoxicity. Non-fibrillar Abeta42, but not fibrillar Abeta42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar Abeta42 decreased both neurotoxicity and increases in the intracellular Ca(2+) concentration induced by N-methyl-d-aspartate (NMDA), but not by alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

Identification of an antiamyloidogenic substance from mulberry leaves.

Fibrillar aggregates of amyloid beta-peptides are major constituents of the plaques found in the brains of patients with Alzheimer's disease, and have been implicated in the neurotoxicity of Alzheimer's. We previously reported that the methanol extract of mulberry leaves inhibits the formation of amyloid beta-peptide (1-42)-fibrils in vitro, and protects hippocampal neurons from amyloid beta-peptide (1-42)-induced cell death. In this study, we identified antiamyloidogenic substances, pheophorbide a, kaempferol -3-O-glucoside, and kaempferol -3-O-(6-malonyl) glucoside, from the methanol extract of mulberry leaves. We also compared the antiamyloidogenic activity of pheophorbide a with that of other porphyrin-related compounds.

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes alpha- and gamma-secretase cleavage and subsequently induces CHOP-mediated cell death.

Roles of nicotinic receptors in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation.

Protection of neurons from neuronal damage and cell death in neurodegenerative disease is a major challenge in neuroscience research. Donepezil, galantamine and tacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease, and were believed to be symptomatic drugs whose therapeutic effects are achieved by slowing the hydrolysis of acetylcholine at synaptic termini. However, recent accumulated evidence strongly suggests that these acetylcholinesterase inhibitors also possess neuroprotective properties whose mechanism is independent of acetylcholinesterase inhibition. We have shown that acetylcholinesterase inhibitors protect neurons from glutamate-induced neurotoxicity in the primary culture of rat cortical neurons. It was also found that acetylcholinesterase inhibitor treatment induces up-regulation of nicotinic receptor expression levels, a property which may also have some bearing on their therapeutic effects. We next showed that alpha4 and alpha7-nicotinic receptors play important roles in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation. Our results also demonstrate the important roles of the phosphatidylinositol 3-kinase pathway downstream of nicotinic receptors in protecting neurons from death and up-regulating nicotinic receptors. This review summarizes recent findings on the roles of the nicotinic receptor in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation.

Rac1 inhibition negatively regulates transcriptional activity of the amyloid precursor protein gene.

Rac1, a member of the Rho family GTPases, participates in a variety of cellular functions including lamellipodia formation, actin cytoskeleton organization, cell growth, apoptosis, and neuronal development. Recent studies have implicated Rac1 in cytoskeletal abnormalities, production of reactive oxygen species, and generation of the amyloid beta-peptide (Abeta) observed in Alzheimer's disease. In this study, we examined the relationship between Rac1 and amyloid precursor protein (APP), because the abnormal proteolytic processing of APP is a pathologic feature of Alzheimer's disease. In primary hippocampal neurons, the Rac1-specific inhibitor NSC23766 decreased both Rac1 activity and APP protein levels in a concentration-dependent manner. To elucidate how NSC23766 decreases APP protein levels, we examined the effects of NSC23766 on APP processing, degradation, and biosynthesis. NSC23766 did not increase the levels of the proteolytic products of APP, sAPPalpha, Abeta40, and Abeta42. The proteasome inhibitor lactacystin did not reverse the NSC23766-induced decrease in APP protein levels. NSC23766 did, however, decrease the levels of both APP mRNA and APP protein. Decreased levels of APP mRNA and protein were also observed when HEK293 cells were transfected with an expression vector containing a dominant-negative Rac1 mutant or with siRNA targeting Rac1. By overexpressing progressively deleted fragments of the APP promoter in HEK293 cells, we identified a Rac1 response site at positions -233 to -41 bp in the APP promoter. Taken together, our results suggest that Rac1 regulates transcription of the APP gene in primary hippocampal neurons.

Serofendic acid promotes stellation induced by cAMP and cGMP analogs in cultured cortical astrocytes.

We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.

A role for SOX2 in the generation of microtubule-associated protein 2-positive cells from microglia.

We recently demonstrated that, as a type of multipotential stem cells, microglia give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells. In this study, we investigated the role of SOX2, a high-mobility group DNA binding domain transcription factor, in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analysis demonstrated that expression of SOX2 was upregulated by treatment with 70% fetal bovine serum treatment. Immunocytochemical analyses demonstrated that SOX2 expression was evident in the nuclei of microglia-derived MAP2-positive and GFAP-positive cells, whereas it was not present in the nuclei of microglia. These assays also showed that Sox2 siRNA inhibited the generation of MAP2-positive and GFAP-positive cells from microglia. Interestingly, this activity was also inhibited by Smad4 siRNA, which reduces SOX2 expression. These results indicate that SOX2 upregulation is involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells through SMAD4.