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With the objective to improve the amyloid-β (Aβ) targeting immunotherapy, we investigated the safety and efficacy of an oral vaccine with recombinant adeno-associated virus vector carrying a signal sequence and Aβ1-43 cDNA (rAAV/Aβ) in old non-human primates, 12 African green and 10 cynomolgus monkeys. The enteric-dissolving coated capsules containing rAAV/Aβ were orally administered once or twice, then monkeys' conditions were carefully observed with complete blood count and laboratory examinations of the sera. General conditions, food intake, water intake, stool conditions, body weight changes, and menstruation cycles were not significantly altered, and laboratory tests and pathological examinations of the systemic organs were unremarkable. Pathological examinations of the brain showed significant reduction of the amyloid plaque burden and intracellular Aβ without inflammatory or hemorrhagic changes in the brain. However, soluble Aβ and some Aβ oligomers were increased in rAAV-treated monkey brains without changes of the neuronal density and vascular amyloidosis. Thus, this vaccine seems to be safe in general, but we must be cautious about the increase of Aβ oligomers after vaccination. This vaccine may be recommended at a very early stage of Alzheimer's disease when little amyloid is deposited.

Huperzia serrata has been used as a Chinese folk medicine for many years. It contains huperzine A, which has a protective effect against memory deficits in animal models; however, it is unclear if H. serrata extract exerts any effects in Alzheimer’s disease (AD) models. We used H. serrata collected in Japan and determined its huperzine A content using HPLC. We determined its inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. H. serrata extract (30 mg/kg/day) and donepezil (10 mg/kg/day) were orally administrated for 7 days. After repeated administration, we performed the Y-maze and passive avoidance tests. H. serrata extract contained 0.5% huperzine A; H. serrata extract inhibited AChE, but not BuChE. H. serrata extract ameliorated cognitive function in mice. These results indicate that Japanese H. serrata extract ameliorates cognitive function deficits by inhibiting AChE. Therefore, H. serrata extract may be valuable for the prevention or treatment of dementia in AD.

The truncated tau protein is a component of the neurofibrillary tangles found in the brains with tauopathies. However, the molecular mechanisms by which the truncated tau fragment causes neurodegeneration remain unknown. Tau pathology was recently suggested to spread through intercellular propagation, and required the formation of 'prion-like' species. We herein identified a new fragment of the tau protein that consisted of four binding domains and a C-terminal tail (Tau-CTF24), but lacked the N-terminal projection domain, and found that it increased with aging in tauopathy model mice (Tg601). Tau-CTF24-like fragments were also present in human brains with tauopathies. A mass spectroscopic analysis revealed that Tau-CTF24 was cleaved behind R242. The digestion of full-length tau (Tau-FL) by calpain produced Tau-CTF24 in vitro and calpain activity increased in old Tg601. Recombinant Tau-CTF24 accelerated heparin-induced aggregation and lost the ability to promote microtubule assembly. When insoluble tau from diseased brains or aggregated recombinant tau was introduced as seeds into SH-SY5Y cells, a larger amount of insoluble tau was formed in cells overexpressing Tau-CTF24 than in those overexpressing Tau-FL. Furthermore, lysates containing the Tau-CTF24 inclusion propagated to naive tau-expressing cells more efficiently than those containing the Tau-FL inclusion. Immunoblot and confocal microscopic analyses revealed that aggregated Tau-CTF24 bound to cells more rapidly and abundantly than aggregated Tau-FL. Our results suggest that Tau-CTF24 contributes to neurodegeneration by enhancing prion-like propagation as well as deteriorating the mechanisms involved in microtubule function.

The imbalance between ß-amyloid (Aß) generation and clearance plays a fundamental role in the pathogenesis of Alzheimer’s disease (AD). The sporadic form of AD is characterized by an overall impairment in Aß clearance. Immunotherapy targeting Aß clearance is believed to be a promising approach and is under active clinical investigation. Autophagy is a conserved pathway for degrading abnormal protein aggregates and is crucial for Aß clearance. We previously reported that oral vaccination with a recombinant AAV/Aß vaccine increased the clearance of Aß from the brain and improved cognitive ability in AD animal models, while the underlying mechanisms were not well understood. In this study, we first demonstrated that oral vaccination with rAAV/Aß decreased the p62 level and up-regulated the LC3B-II/LC3B-I ratio in APP/PS1 mouse brain, suggesting enhanced autophagy. Further, inhibition of the Akt/mTOR pathway may account for autophagy enhancement. We also found increased anti-Aß antibodies in the sera of APP/PS1 mice with oral vaccination, accompanied by elevation of complement factors C1q and C3 levels in the brain. Our results indicate that autophagy is closely involved in oral vaccination-induced Aß clearance, and modulating the autophagy pathway may be an important strategy for AD prevention and intervention.

Amyloid-β protein (Aβ) accumulates in the neurons of Alzheimer's disease (AD) patients at an early stage of the disease. Recently, we found that Aβ with a toxic turn at positions 22 and 23 accumulates in neurons in AD brain. Here, we studied the accumulation of Aβ, toxic turn Aβ and high-molecular-weight Aβ oligomers in presenilin 1 (PS1) gene-transfected SH-SY5Y cells as well as in the brains of 3xTg-AD mice and AD patients. Immunostaining revealed that accumulation of toxic turn Aβ was promoted in G384A- and I143T-mutant PS1-transfected cells and further enhanced by co-transfection of cells with the Aβ-precursor protein (AβPP) gene. In contrast, accumulation of high-molecular-weight Aβ oligomers was promoted in mutant PS1 cells but attenuated by co-transfection of cells with the AβPP gene. Toxic turn Aβ was detected in the neurons of 3xTg-AD mice aged 2 months, when the mice were cognitively unimpaired. In contrast, high-molecular-weight Aβ oligomers were detected in the neurons of 7-month-old mice, when memory dysfunction is apparent. Furthermore, immunostaining and western blotting for Rab4, Rab6 and GRP78 revealed increased levels of these proteins in mutant PS1 cells and their accumulation in the neurons of 3xTg-AD mice. Remarkably, GRP78 immunoreactivity was increased at 2 months of age. Double-label immunostaining of AD brain revealed an apparent association between toxic turn Aβ and GRP78, an endoplasmic reticulum (ER) stress marker. Intraneuronal accumulation of toxic turn Aβ may be associated with ER stress in the brains of AD model mice and AD patients at an early stage.

Active and passive immunization of Alzheimer model mice with Aβ showed clearance of aggregated amyloid β deposits and improved memory and learning. Although human trial was halted because of autoimmune encephalitis, the trial revealed that immunization with Aβ also deleted amyloid deposits in humans without clinical benefit. On these proof of concept, several clinical trials using monoclonal antibodies are on-going. Although solanezumab which recognizes Aβ monomer turned out ineffective in the primary endpoint, it showed significant beneficial effect in mild AD cases in the secondary outcome. Solanezumab is now on a large scale phase III trial in mild AD cases in the world. If it turns out to be effective, it will be the first disease modifying drug for AD in a few years. However, since monoclonal antibodies are extremely expensive, less expensive and long acting active immunization will be widely accepted. More effective and sophisticated vaccines such as DNA vaccine and recombinant viral vaccines will be utilized in future.

Growing evidence suggests that decreased brain-derived neurotrophic factor (BDNF) levels are associated with Alzheimer's disease (AD) pathogenesis. Therefore, BDNF gene therapy is considered to be a promising therapeutic strategy for treating AD. Sendai virus (SeV) is a type I parainfluenza virus that does not interact with host chromosomes because of its strict cytoplasmic life cycle. Although SeV is nonpathogenic in primates, including humans, its infectivity for neurons is strong. Here we demonstrate that SeV vectors effectively infected neurons, even though they were injected into subcortical white matter. Moreover, SeV vectors significantly induced BDNF expression, ameliorating synaptic degeneration and memory deficits in a transgenic mouse model of AD (Tg2576). This is the first study to demonstrate that viral vector administration in white matter is sufficient to restore cognitive function in vivo. These results also support the feasibility of using SeV vectors for gene therapy targeting the brain. © 2012 Wiley Periodicals, Inc.

Based on the amyloid cascade hypothesis, many reports have indicated that immunotherapy is beneficial for Alzheimer's disease (AD). We developed a mucosal immunotherapy for AD by nasal administration of recombinant Sendai virus vector carrying Aβ1–43 and mouse IL-10 cDNA. Nasal but not intramuscular administration of the vaccine induced good antibody responses to Aβ. When APP transgenic mice (Tg2576) received this vaccine once nasally, the Aβ plaque burden was significantly decreased 8 weeks after without inducing inflammation in the brain. The amount of Aβ measured by ELISA was also reduced in both soluble and insoluble fractions of the brain homogenates, and notably the Aβ oligomer (12-mer) was also apparently decreased. Tg2576 mice showed significant improvement in cognitive functions examined at 3 months after vaccination. Thus, this is an alternative immunotherapy for AD, which has an advantage in non-invasive, safe and relatively long lasting features.

Aging is thought to impair prefrontal cortical (PFC) structure-sensitive cognitive functions and flexibility, such as working memory and reversal learning. A traditional Japanese medicine, yokukansan (YKS), is frequently used to treat age-related neurodegenerative disorders such as Alzheimer's disease in Japan, but its pharmacological properties have not been elucidated. The present study was designed to examine whether YKS improves age-related cognitive deficits using aged rats. YKS was administered to 21-month-old rats for 3 months. The ability to learn initially a reward rule for a T-maze discrimination task (initial learning) was examined in young control (4-month-old), aged control (24-month-old) and YKS-treated aged (24-month-old) rats. Subsequently, working memory and reversal learning were examined in delayed alternation and reversal discrimination T-maze tasks, respectively. Locomotor activity was also measured in new environments. Although performance accuracy in the initial learning procedure did not differ among any experimental groups, accuracy in the delayed alternation task was significantly decreased in aged rats compared to young rats. Aged rats also showed significant decreases in accuracy in the reversal discrimination task. YKS treatment significantly ameliorated the age-related decreases in accuracy in the delayed alternation and reversal discrimination tasks. The ameliorative effects of YKS on impaired delayed alternation performance were reduced by intracranial infusions of a dopamine D1 receptor antagonist, SCH 23390, into the prelimbic cortical region of the PFC, and the YKS effects on impaired reversal learning were done by the infusions into the orbitofrontal cortex (OFC). Locomotor activity did not change in any experimental group. Thus, YKS ameliorated age-related impairments of working memory and reversal learning, which might be mediated by a dopaminergic mechanism in the PFC structure. These investigations provide information important for the treatment of brain dysfunctions in the elderly people.

Based on the amyloid cascade hypothesis, various strategies targeting amyloid beta protein (Abeta) have been invented for prevention and treatment of Alzheimer disease (AD). Active and passive immunizations with Abeta and Abeta antibodies successfully reduced AD pathology and improved cognitive functions in an AD mouse model. However, active immunization with AN-1792, a mixture of Abeta1-42 peptide and adjuvant QS21 induced autoimmune encephalitis in humans. Surprisingly, although AN-1792 cleared senile plaque amyloid, it showed no benefit in humans. It is speculated that AN-1792 failed in deleting more toxic forms of Abeta such as oligomers and intracellular Abeta, suggesting that newly developing vaccines should delete these toxic molecules. Since T cell epitopes exist mainly in the C-terminal portion of Abeta, vaccines using shorter N-terminal peptides are under development. In addition, since T helper 1 (Th1) immune responses activate encephalitogenic T cells and induce continuous inflammation in the central nervous system, vaccines inducing Th2 immune responses seem to be more promising. These are N-terminal short Abeta peptides with Th2 adjuvant or Th2-stimulating molecules, DNA vaccines, recombinant viral vector vaccines, recombinant vegetables and others. Improvement of vaccines will be also achieved by the administration method, because Th2 immune responses are mainly induced by mucosal or trans-cutaneous immunizations. Here I review recent progress in active immunization strategies for AD.

Exposure to stress is thought to play an important role in the etiology of depression. Dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis characterized by glucocorticoid negative feedback resistance is frequently observed in human depressives. Additionally, dysfunctions of the dopaminergic and serotonergic systems in the prefrontal cortex (PFC) are thought to be involved in the development of a depressive state. In rats, chronic stress induces a behaviorally depressive state, concomitant with dysregulation of the HPA axis and reductions in dopaminergic and serotonergic transmissions in the PFC. Considering that dysregulation of the HPA axis is associated with relapse and persistency of depression, it is possible that the chronic stress-induced depressive state persists during long-term rest after its exposure. In the present study, we examined this possibility in rats and found that the behaviorally depressive state in the rotarod test, negative feedback resistance in the dexamethasone suppression test, and a decrease in the extracellular concentration of dopamine but not serotonin in the PFC persisted for 3 months following a 4-week stress session. These results suggest that dysregulation of the HPA system and reduced dopaminergic transmission in the PFC underlies persistent behavioral depression following chronic stress.

Glucocorticoid hormones are important in the maintenance of many brain functions, and their receptors distribute abundantly throughout the brain. In patients with several neuropsychiatric disorders such as depression, dysregulation of the glucocorticoid negative feedback system is the consistent observations, which is thought to be caused by reduced glucocorticoid response at the several feedback sites including the brain. In the present study, we examined whether reduced glucocorticoid actions via suppression of circulating glucocorticoids by adrenalectomy (ADX) induced a behavioral depressive state using the rotarod test. We found that ADX impaired the rotarod performance while it did not affect the traction performance and locomotor activity. Moreover, this impairment was significantly reversed by corticosterone replacement treatment and was ameliorated by the infusion of D1 receptor agonist SKF 81297 into the prefrontal cortex (PFC) in a dose-dependent manner. Considering the previous findings that ADX reduces dopaminergic transmission in the PFC, the present results suggest that suppression of circulating glucocorticoids induces a behaviorally depressive state that is caused by a D1 receptor-mediated hypodopaminergic mechanism in the PFC. This finding would help to understand the involvement of the dysregulated feedback system in the pathogenesis of depression.

We previously demonstrated that suppression of glucocorticoid secretion by adrenalectomy (ADX) impaired prefrontal cortex-sensitive working memory, but not reference memory. Since the cholinergic system in the hippocampus is also involved in these memories, we examined the effects of glucocorticoid suppression on cholinergic transmission in the rat hippocampus. A microdialysis study revealed that ADX did not affect the basal acetylcholine release, but enhanced the KCl-evoked response. This enhanced response was reversed by the corticosterone replacement treatment. The extracellular choline concentrations increased under both basal and KCl-stimulated conditions in the ADX rats, and these increases were also reversed by the corticosterone replacement. These results indicate that suppression of glucocorticoid secretion enhances cholinergic transmission in the hippocampus in response to stimuli. It is possible that this enhanced cholinergic transmission may not contribute to the ADX-induced working memory impairment, but it may be involved in maintenance of reference memory.

Anxiety is frequently observed in several neuropsychiatric disorders, and stress is thought to precipitate or exacerbate anxiety. In this study, the anxiolytic action of a herbal medicine, saikokaryukotsuboreito, (SRBT) was examined in normal healthy rats using the elevated plus-maze test. Moreover, the improving effect of SRBT on chronic stress-induced anxiety was also examined. Single administration of SRBT did not have anxiolytic action in normal rats. Repeated administration of SRBT significantly improved chronic stress-induced anxiety. On the other hand, single administration of a typical anxiolytic, diazepam, had anxiolytic action in normal rats but repeated administration did not improve chronic stress-induced anxiety. These results suggest that SRBT does not have anxiolytic activity equivalent to that of diazepam but has potency for improving stress-related anxiety. This finding provides information important for the treatment of anxiety.

Microglia are the main resident immunocompetent and phagocytic cells in the central nervous system (CNS). Activated microglia could play phagocytic roles as well as mediate inflammatory processes in the CNS. Involvement of activated microglia in the pathogenesis has been demonstrated in several neurological diseases including Alzheimer's disease (AD). Juzen-taiho-to (JTT), a traditional herbal medicine, has been reported to have effects on activating immune responses and phagocytosis. So far, little is known about the effects of this Kampo formulation JTT on microglia and in AD. In this report, we studied the effects of JTT on the activation and phagocytic functions of mouse microglia and bone marrow-derived macrophages (BMM). JTT could activate microglia, which was confirmed by the prominent morphological change and increased surface expression of an activation marker CD11b. In addition, JTT was revealed to induce microglial proliferation, and enhance microglial phagocytosis of, without eliciting an excessive production of nitric oxide. Furthermore, when mice were administrated with JTT in vivo, their BMM showed more effective phagocytosis of fibrillar Abeta(1-42). These findings implicate the therapeutic potential of JTT in AD and other neurological diseases accompanied by microglial activation.

Presenilin 1 (PS1) forms the gamma-secretase complex with at least three components: nicastrin, APH-1, and PEN-2. This complex mediates intramembrane cleavage of amyloid precursor protein (APP) to generate beta-amyloid protein (Abeta) as well as other type 1 transmembrane proteins. Although PS1 mutations linked to familial Alzheimer's disease influence these cleavages, their biological consequences have not been fully understood. In this study, we used mRNA differential display analysis to identify a gene, denoted adoplin-1/ORMDL-1, which displays significantly reduced expression in association with PS1 mutations. Adoplin-1 and two highly homologous genes (adoplin-2, -3) constitute a gene family that encodes transmembrane proteins. The mRNA and protein levels of adoplins (particularly adoplin-1, -2) were markedly elevated in PS-deficient fibroblasts, compared to wild-type cells. Moreover, knockdown of the three adoplins by RNA interference affected maturation of nicastrin and its association with PS1. Adoplin knockdown additionally resulted in elevated levels of APP C-terminal fragments and decreased Abeta production, suggestive of reduced gamma-secretase activity. Our data collectively indicate that adoplins are unique molecules with PS-related expression and functions that may play important role(s) in the maturation and activity of the gamma-secretase complex.

Immunotherapy with Abeta is expected to bring great improvement for Alzheimer disease (AD). However, clinical trials have been suspended because of meningoencephalitics, which accompanied lymphocytic infiltration. We have developed an oral vaccine for AD with a recombinant adeno-associated viral vector carrying Abeta cDNA (AAV/Abeta). The vaccine reduces the amount of Abeta deposited without lymphocytic infiltration in APP transgenic (Tg2576) mice. In the present study, Tg2576 mice showed progressive cognitive impairments in the novel object recognition test, Y-maze test, water maze test, and contextual conditioned fear learning test. A single oral administration of AAV/Abeta to Tg2576 mice at the age of 10 months alleviated progressive cognitive impairment with decreased Abeta deposition, insoluble Abeta, soluble Abeta oligomer (Abeta*56), microglial attraction, and synaptic degeneration induced in the brain regions at the age of 13 months. A histological analysis with hematoxylin and eosin and an immunohistochemical analysis with antibodies against CD3, CD4, CD8, and CD19 suggested there was no lymphocytic infiltration or microhemorrhage in the brain of AAV/Abeta-vaccinated Tg2576 mice at 13 months of age. Taken together, these results suggest that immunotherapy with AAV/Abeta is a safe and effective treatment for AD.

Disruption of the hypothalamo-pituitary-adrenal (HPA) axis characterized by dysfunction of the glucocorticoid negative feedback system is frequently observed in human depressives and is thought to involve a reduction in glucocorticoid receptor (GR) function in the feedback sites including the brain. Recently, we found that chronic stress in rats induces similar HPA disruption that is caused by abolishment of feedback ability in the prefrontal cortex (PFC) and hippocampus, which involves decreased cytosolic GRs or increased nuclear GRs, respectively. Also, we found that saikokaryukotsuboreito (SRBT), a herbal medicine, prevents the chronic stress-induced HPA disruption. We therefore examined here the effects of this drug on the chronic stress-induced changes in GRs in the PFC and hippocampus. Chronic stress was induced in rats by water immersion and restraint (2 h/day) for 4 weeks. SRBT significantly prevented decreased cytosolic GRs in the PFC and increased nuclear GRs in the hippocampus in the chronically stressed rats. Moreover, SRBT significantly prevented the abolishment of feedback ability in both regions. These results suggest that the beneficial effects of SRBT on the GR level are involved in its ameliorating actions on the HPA disruption. This finding provides information important for the prevention and treatment of depression.

Presenilin-2 (PS2) is a gene of unknown function linked with some forms of familial Alzheimer's disease. To investigate the biological role of PS2 in neurons, we overexpressed PS2 in primary cortical neurons using recombinant adenoviral vectors. Western blot and immunohistochemical analyses showed enhanced expression of PS2 proteins in infected neurons after infection of recombinant adenoviruses containing the human wild-type or mutant PS2 gene. Neuronal survival was decreased by approximately 30% in cultures infected with adenovirus expressing either wild-type or mutant PS2, as compared with those infected with adenovirus expressing the LacZ gene. Fragmented nuclei were frequently observed in dying neurons. These data suggest that apoptotic death of cultured cortical neurons is enhanced by PS2 overexpression.

The mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis (EAE) was studied in SJL/J mice. In vivo experiments showed that NK cell depletion by anti-NK1.1 monoclonal antibody treatment enhanced EAE in mice. To investigate the mechanism, we cultured proteolipid protein (PLP)136-150 peptide-specific, encephalitogenic T cell lines, which were used as the NK cell target. Our results show that NK cells exert a direct cytotoxic effect on autoantigen-specific, encephalitogenic T cells. Furthermore, cytotoxicity to PLP-specific, encephalitogenic T line cells was enhanced by using enriched NK cells as effector cells. However, the cytotoxic effect of NK cells to ovalbumin-specific T line cells and ConA-stimulated T cells could also be detected with a lesser efficiency. Our studies indicate that NK cells play a regulatory role in EAE through killing of syngeneic T cells which include myelin antigen-specific, encephalitogenic T cells, and thus ameliorate EAE.

The amyloid beta-protein (Abeta) ending at 42 plays a pivotal role in Alzheimer's disease (AD). We have reported previously that intracellular Abeta42 is associated with neuronal apoptosis in vitro and in vivo. Here, we show that intracellular Abeta42 directly activated the p53 promoter, resulting in p53-dependent apoptosis, and that intracellular Abeta40 had a similar but lesser effect. Moreover, oxidative DNA damage induced nuclear localization of Abeta42 with p53 mRNA elevation in guinea-pig primary neurons. Also, p53 expression was elevated in brain of sporadic AD and transgenic mice carrying mutant familial AD genes. Remarkably, accumulation of both Abeta42 and p53 was found in some degenerating-shape neurons in both transgenic mice and human AD cases. Thus, the intracellular Abeta42/p53 pathway may be directly relevant to neuronal loss in AD. Although neurotoxicity of extracellular Abeta is well known and synaptic/mitochondrial dysfunction by intracellular Abeta42 has recently been suggested, intracellular Abeta42 may cause p53-dependent neuronal apoptosis through activation of the p53 promoter; thus demonstrating an alternative pathogenesis in AD.

Objective: The effect of chemical sympathectomy on experimental autoimmune encephalomyelitis (EAE) was studied in interferon-γ (IFN-γ) knockout C57BL/6 mice. Methods: Mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide. Sympathectomy was achieved by subcutaneous administration of 6-hydroxydopamine. Results: We showed previously that wild-type mice developed a mild form of EAE with complete recovery. Sympathectomy caused more serious disease and mice did not recover completely, indicating that the sympathetic nervous system (SNS) downmodulates the process of EAE. The clinical signs of disease were more serious in untreated IFN-γ–/– mice than in wild-type animals. Unexpectedly, sympathectomy resulted in suppression of active EAE in IFN-γ–/– mice, implying that control of actively induced EAE by the SNS depends on INF-γ and the integrity of the cytokine network. We also induced EAE by passive transfer of MOG35–55-reactive lymph node cells, and this disease was aggravated by sympathectomy in both wild-type and knockout animals. Conclusions: Our study supports the idea that the integrity of the whole cytokine network is necessary to maintain normal nervous-immune system interactions.

Excess manganese (Mn) in brain can be neurotoxic, implicated in several neurodegenerative disorders such as sporadic Alzheimer's disease (AD). However, little is known about the altered metal environment including elevated Mn in the progressive cognitive impairment of AD. Indeed, whether high Mn is associated with AD risk remains elusive. In the study, we recruited 40 Chinese elders with different cognitive statuses and investigated concentrations of Mn in whole blood and plasma amyloid-β (Aβ) peptides. Surprisingly, there were significant correlations of Mn with Mini-Mental State Examination score and Clinical Dementia Rating Scale score. In addition, plasma Aβ peptides increased with elevated Mn. Further studies both in vitro and in vivo demonstrated dose-related neurotoxicity and increase of Aβ by Mn treatment, which was probably caused by disrupted Aβ degradation. These data suggested that high Mn may be involved in the progress of AD as an essential pathogenic factor.

Microglia are the main immunocompetent and phagocytic cells in Alzheimer's disease (AD). Bone marrow-derived microglia have been demonstrated to be more effective in antigen presentation and phagocytosis than inherent microglia in AD. Thus, microglia have received much attention in the pathogenesis of AD. The herbal medicine Juzen-taiho-to (JTT) has been reported to reduce β-amyloid (Aβ) burden in the mouse brain of an AD model. In this study, we explored the effects of JTT on the migration and differentiation of bone marrow-derived cells in the mouse brain of acutely induced AD. To chase bone marrow-derived cells, we made a chimeric mouse line in C57BL/6 by transplanting fresh bone marrow cells, isolated from the transgenic mice expressing enhanced green fluorescent protein gene. The chimeric mice were orally administrated with JTT or distilled water, and were left untreated or given intrahippocampal injection of fibrillar Aβ 1-42 (fAβ42) or vehicle. In the hippocampus of the vehicle-injected mouse, JTT treatment for 37 days caused a significant increase in the number of microglial cells. In the fAβ42-injected mouse hippocampus, a larger number of bone marrow-derived cells were detected in JTT-treated mice than control mice in the non-neighboring regions of the fAβ42-injected site but not around the injected site. These results suggest that JTT might contribute to the reduction of Aβ burden and the immune surveillance in non-pathological as well as pathological brain regions. The results also implicate the therapeutic potential of JTT in AD.

We previously demonstrated that chronic stress impaired prefrontal cortex-sensitive working memory, but not reference memory. Since the hippocampal cholinergic system is also involved in these memories, we examined the effects of chronic stress on cholinergic transmission in the rat hippocampus. A microdialysis study revealed that the stress did not affect the basal acetylcholine release, but enhanced the KCl-evoked response. These results suggest that cholinergic transmission in the chronically stressed hippocampus does not contribute to working memory impairment, but it may be involved in maintenance of reference memory.

Exposure to chronic stress is thought to play an important role in the etiology of depression. In this disorder, a disrupted negative feedback response to exogenous glucocorticoids on cortisol secretion has been indicated. However, the regulation of glucocorticoid negative feedback by chronic stress is not fully understood. In the present study, we investigated the effects of chronic stress administered by water immersion and restraint (2 h/day) for four weeks on the glucocorticoid feedback in rats. In the acutely (one-time) stressed rats, the basal plasma corticosterone (CORT) level was markedly elevated, remained at high levels for 5 h after the termination of stress, and then decreased. In the chronically stressed rats, the CORT level was initially elevated similarly, but rapidly decreased at 2 h. In the dexamethasone (DEX) suppression test, the peak CORT level in response to stress was not suppressed by DEX in the acutely stressed rats, but was significantly suppressed in the chronically stressed rats. In contrast, the suppressive effects of DEX on the basal CORT secretion in naive rats were attenuated in the chronically stressed rats. In the chronically stressed hippocampus, which plays an important role in the regulation of the glucocorticoid feedback response, the binding of [3H]DEX was decreased and the increased response of activator protein-1 induced by acute stress was abolished. These results suggest that chronic stress induces a hypersuppressive state for induced CORT secretion in response to acute stress, which is caused by partial habituation, coping, and adaptation to the stressor, whereas it induces a hyposuppressive state for the basal CORT secretion, which is caused by glucocorticoid receptor downregulation. These mechanisms may be involved in the stress-induced neural abnormalities observed in depression.

Experimental autoimmune encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease that can be protected against by stimulating regulatory cells. Here we examined whether EAE can be purposefully modulated by stimulating Vα14 NK T cells with the CD1d-restricted ligand α-galactosylceramide (α-GC). EAE induced in wild-type C57BL/6 (B6) mice was not appreciably altered by injection of α-GC. However, EAE induced in IL-4 knockout mice and IFN-γ knockout mice was enhanced or suppressed by α-GC, respectively. This indicates that the IL-4 and IFN-γ triggered by α-GC may play an inhibitory or enhancing role in the regulation of EAE. We next studied whether NK T cells of wild-type mice may switch their Th0-like phenotype toward Th1 or Th2. Notably, in the presence of blocking B7.2 (CD86) mAb, α-GC stimulation could bias the cytokine profile of NK T cells toward Th2, whereas presentation of α-GC by CD40-activated APC induced a Th1 shift of NK T cells. Furthermore, transfer of the α-GC-pulsed APC preparations suppressed or enhanced EAE according to their ability to polarize NK T cells toward Th2 or Th1 in vitro. These results have important implications for understanding the role of NK T cells in autoimmunity and for designing a therapeutic strategy targeting NK T cells.

HIV-1 infection is often complicated by the dysfunction of central nervous system (CNS). Degenerative neuronal changes as well as neuronal loss have been documented in individuals with acquired immunodeficiency syndrome. Feline immunodeficiency virus (FIV) causes similar CNS manifestation and FIV infected cats provide an animal model for human immunodeficiency virus infection in humans. In this study, we examined the brain of FIV-infected cats and controls with immunohistochemical techniques using antibodies to microtubule-associated protein 2 (MAP-2) and glutamic acid decarboxylase (GAD). We found a significant decrease in expression of MAP-2 and GAD in neurons of infected animals compared to controls. In contrast, the expression of neurofilaments and glial fibrillary acidic protein was rather increased. The changes observed in the brain were similar to those seen in humans undergoing the normal aging process as well as those suffering from neurological diseases like Alzheimer's disease and other dementing disorders. These changes in the feline brain give insight into the deleterious effects of FIV on the CNS.

The effect of chemical sympathectomy induced with 6-hydroxydopamine (OHDA) on experimental autoimmune encephalomyelitis (EAE) was studied in wild type and IL-4 −/− C57BL/6 (B6) mice. When actively sensitized with myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide, control B6 mice developed a mild form of EAE with full recovery. The sympathectomized mice developed paralysis with higher maximum disease score and did not recover completely, indicating that the sympathetic nervous system (SNS) down-modulates the process of EAE. Unexpectedly, however, sympathectomy resulted in suppression of EAE in IL-4 −/− mice, implying that control of actively induced EAE by the SNS depends on the genetic background of mice. We also induced EAE by passive transfer of MOG 35–55 -reactive lymph node cells, and this disease was augmented by sympathectomy in both wild type and knockout animals. Further experiments showed that changes in T cell populations and the activity of antigen presenting cells might be responsible for the altered immune response and clinical course after sympathetic ablation. Our studies indicate that the absence of a single cytokine can severely alter nervous–immune system interactions.

We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology.

We screened proteins for interaction with presenilin (PS) 1, and cloned the full-length cDNA of human delta-catenin, which encoded 1225 amino acids. Yeast two-hybrid assay, GST binding assay and immunoprecipitation demonstrated that delta-catenin interacted with a hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1, but not with that of PS-2. These results suggest that PS1 and PS2 partly differ in function. PS1 loop fragment containing the pathogenic mutation retained the binding ability. We also found another armadillo-protein, p0071, interacted with PS1.

We report the cDNA sequence of human Fe65L2. The human Fe65L2 encoded 486 amino acids; the deduced amino acid sequence was shorter by 18 amino acids than the rat protein and had 86% identity to the rat protein Three protein-protein interaction domains, a WW and two PID/PTB elements, were conserved among the Fe65 protein family. Human Fe65L2 mRNA was expressed in various tissues; a transcript of about 2.2 kb was mainly expressed in the brain. A splicing variant lacking two amino acids in the first PID/PTB element was detected. We also confirmed that the carboxyl-terminal region of PID/PTB of the Fe65L2 interacted with the intracellular domain of the Alzheimer's beta-amyloid precursor protein (APP) and APP-like proteins.

We have previously demonstrated that interleukin 3 (IL-3) has a neurotrophic effect on central cholinergic neurons and have demonstrated the presence of IL-3 receptor (IL-3R)beta subunits in septal cholinergic neurons by reverse-transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry. In order to confirm that the expressed IL-3R is functional, we conducted experiments to show an alpha subunit of IL-3R. The alpha subunit was clearly demonstrated by RT-PCR in the central cholinergic neuronal hybrid cell line SN6, but not in its mother cell line N18TG2, and the expression was slightly upregulated after IL-3 treatment. Choline acetyltransferase and vesicular acetylcholine transporter mRNAs were significantly increased in SN6 after treatment with IL-3. Immunohistochemically, IL-3R alpha-positive cells were mainly present in the medial septal and basal forebrain region, and the stained cells were similar to choline acetyltransferase-positive cells in shape and distribution. The IL-3R alpha-positive cells slightly increased two days after fimbria-fornix transection and decreased seven days after. These findings suggest that functional IL-3 receptors are expressed in the central cholinergic neurons and contribute to some physiological roles such as the differentiation and maintenance of these neurons.

In this report, we establish a regulatory role of natural killer (NK) cells in experimental autoimmune encephalomyelitis (EAE), a prototype T helper cell type 1 (Th1)-mediated disease. Active sensitization of C57BL/6 (B6) mice with the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide induces a mild form of monophasic EAE. When mice were deprived of NK cells by antibody treatment before immunization, they developed a more serious form of EAE associated with relapse. Aggravation of EAE by NK cell deletion was also seen in β2-microglobulin−/− (β2m−/−) mice, indicating that NK cells can play a regulatory role in a manner independent of CD8+ T cells or NK1.1+ T cells (NK–T cells). The disease enhancement was associated with augmentation of T cell proliferation and production of Th1 cytokines in response to MOG35-55. EAE passively induced by the MOG35-55-specific T cell line was also enhanced by NK cell deletion in B6, β2m−/−, and recombination activation gene 2 (RAG-2)−/− mice, indicating that the regulation by NK cells can be independent of T, B, or NK–T cells. We further showed that NK cells inhibit T cell proliferation triggered by antigen or cytokine stimulation. Taken together, we conclude that NK cells are an important regulator for EAE in both induction and effector phases.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes are associated with early-onset autosomal dominant familial Alzheimer's disease, and the gene products are endoproteolytically processed to yield N-terminal fragments (NTF) and C-terminal fragments (CTF). We have studied the cleavage site of the PS2 protein in stably transfected human neuroblastoma cells. The 23 kD PS2-CTF was isolated by a combination of anion exchange chromatography and affinity chromatography and directly sequenced. The N-terminus of the PS2-CTF started at residue 307, which indicated that the cleavage occurs between Lys306 and Leu307 in the proximal portion of the large hydrophilic loop. This site is close to the cleavage positions observed in the PS1 protein.

Previous studies have shown a high prevalence of obstructive sleep apnea (OSA) among patients with Alzheimer's disease (AD). However, it is poorly assessed whether chronic intermittent hypoxia (CIH), which is a characteristic of OSA, affects the pathophysiology of AD. We aimed to investigate the direct effect of intermittent hypoxia (IH) in pathophysiology of AD in vivo and in vitro. In vivo, 15 male triple transgenic AD mice were exposed to either CIH or normoxia (5% O2 and 21% O2 every 10 min, 8 h/day for 4 weeks). Amyloid-β (Aβ) profile, cognitive brain function, and brain pathology were evaluated. In vitro, human neuroblastoma SH-SY5Y cells stably expressing wild-type amyloid-β protein precursor were exposed to either IH (8 cycles of 1% O2 for 10 min followed by 21% O2 for 20 min) or normoxia. The Aβ profile in the conditioned medium was analyzed. CIH significantly increased levels of Aβ42 but not Aβ40 in the brains of mice without the increase in hypoxia-inducible factor 1, alpha subunit (HIF-1α) expression. Furthermore, CIH significantly increased intracellular Aβ in the brain cortex. There were no significant changes in cognitive function. IH significantly increased levels of Aβ42 in the medium of SH-SY5Y cells without the increase in the HIF-1α expression. CIH directly and selectively increased levels of Aβ42 in the AD model. Our results suggest that OSA would aggravate AD. Early detection and intervention of OSA in AD may help to alleviate the progression of the disease.

Neuromyelitis optica (NMO) is associated with antibodies to aquaporin 4 (AQP4). We hypothesized that antibodies to AQP4 can be triggered by exposure to environmental proteins. We compared human AQP4 to plant and bacterial proteins to investigate the occurrence of significantly similar structures and sequences. High similarity to a known epitope for NMO-IgG, AQP4(207-232), was observed for corn ZmTIP4-1. NMO and non-NMO sera were assessed for reactivity to AQP4(207-232) and the corn peptide. NMO patient serum showed reactivity to both peptides as well as to plant tissue. These findings warrant further investigation into the role of the environment in NMO etiology.

Previously, we reported the presence of multiple neurotransmitters in subclones of SN6, a septal cholinergic hybrid cell line. To obtain information concerning the functionality of these transmitters, we measured transmitter contents, activities of transmitter-producing enzymes, and the effect of serum-free culture medium in two different batches (SN6.1.6 and SN6.10.2.2) and two subclones of the SN6 cell line (SN6.2a and SN6.1b). Except for SN6.1b, SN6 cell lines and subclones had basically the same neurotransmitter characteristics. Among the transmitters, only acetylcholine seemed to be functional. Monoamine oxidase was missing and activity of aromatic amino acid decarboxylase was diminished in SN6 cell lines. Even in serum-containing medium, SN6.1b had a more mature morphology than the other cell lines, and it contained choline acetyltransferase and acetylcholine but not tyrosine hydroxylase or catecholamines. Similar characteristics were acquired by the mother cell line in response to serum-free conditions. Thus, SN6.1b is the most mature of these central cholinergic neuronal cell lines, at least with regard to neurotransmitter profiles. ©1995 Wiley-Liss, Inc.

We previously reported that interleukin-3 (IL-3) acts as a neurotrophic factor for cholinergic neurons. However, it has not yet been determined whether the action is derived from the interaction of IL-3 with IL-3 receptors. As the first step to study IL-3 receptors in the central nervous system, we examined the presence and localization of IL-3 receptor-associated antigen (IL-3RAA) in mouse and rat brain. Immunohistochemically, IL-3RAA, which is closely involved both in the IL-3 binding to IL-3 receptors and the tyrosine phosphorylation in the signal transduction for IL-3 in hematopoietic cells, was demonstrated in neurons throughout the brain. This was confirmed in primary cultured neurons and neuronal cell lines by immunocytochemistry and flow cytometry. The staining intensity varied among regions and the most intense immunoreactivity for IL-3RAA was found in large neurons in the magnocellular basal nuclei, pyramidal cells in the cerebral cortex, and neuronal cells in some nuclei of the brainstem. Not only cholinergic cell lines derived from the septal region but also other neuronal cell lines exhibited IL-3RAA immunoreactivity by flow cytometry. Therefore, we conclude that IL-3RAA is present in a wide variety of neurons in the brain including cholinergic neurons of the basal forebrain. Western blot analysis revealed that the candidates for IL-3RAA are 145, 100, and 50 kDa proteins both in neuronal and IL-3-dependent cell lines.

Based on the amyloid cascade hypothesis, immunotherapy targeting amyloid β (Aβ) for Alzheimer's disease (AD) has been developed. It was reported that active immunization using Aβ peptide attenuates amyloid deposits and memory impairment in AD model mice. However, active immunization of patients with AD (AN-1792) was halted due to adverse effects in which a subset of patients developed meningoencephalitis. In order to avoid autoimmune encephalitis, passive immunotherapy using humanized monoclonal antibodies with specificity to Aβ are in clinical trials. We also developed an anti-Aβ monoclonal antibody 3.4A10, which react with AD brain-specific Aβ oligomers. On the other hand, some studies showed that immunotherapy approach targeting tau could attenuate pathology in AD model mouse. Here we introduce a current trend of immunotherapy for AD.

In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

We investigated the effect of insulin-like growth factors II and I (IGFII and IGFI) on septal primary cultures from mouse embryonic day 15 brains. The addition of IGFII to septal cultures enhanced total choline acetyltransferase (ChAT) activity in a dose-dependent manner. Maximal stimulation of ChAT activity was observed at 10 ng/ml IGFII. The effect of IGFII on ChAT activity was completely blocked by anti-IGFII/M-6-P receptor antibodies, whereas the antisera alone had no effect on the enzyme activity. Double-labeled immunohistochemical studies revealed that most ChAT-positive neurons expressed IGFII/M-6-P receptor immunoreactivity. These results indicate that the trophic effect of IGFII results from the direct action of this molecule through the IGFII/M-6-P receptor in septal cholinergic neurons. IGFI also stimulated ChAT activity, but with less potency than IGFII. Antibodies against the IGFII/M-6-P receptor inhibited approximately 50% of the IGFI response, suggesting that the effect of IGFI is mediated in part by the IGFII/M-6-P receptor. Thus, it appears that IGFII and IGFI are potent trophic factors for central cholinergic neurons and could potentially play a significant role in the differentiation, maintenance and regeneration of these neurons.

SUZUKI, A., TAKASHIMA, S., MIZUGUCHI, M., KATO, M., KUNISHITA, T. and TABIRA, T. High Expression on Kunitz-Type Protease Inhibitor-Containing Substances in the Cerebral Vessels of Patients with Down Syndrome. Tohoku J. Exp. Med., 1994, 174 (3), 181-187 - Down syndrome (DS) brains, from 19 gestational weeks to 50 years of age were studied by immunohistochemical methods with a polyclonal antibody against synthetic peptide comprising part of the Kunitz-type protease inhibitor (KPI) domain of Alzheimer disease amyloid precursor protein (APP), residues 301 to 323 of APP 770. In DS, positive KPI immunoreactivity was observed in early infancy and from child to adulthood on the tunica media of the arteries in the leptomeninges, cerebral cortex and white matter, but negative or little in controls. In DS with Alzheimer type dementia, KPI immunoreactivity in the arteries was reduced, but a gross granular reactivity was noted in neurons and glial cells. The high expression of KPI in DS vessels may be one of the predisposing factors to vascular diseases and amyloid deposition associated with DS.

Whether or not the oral intake of metals such as aluminium (Al) and zinc (Zn) is a risk for Alzheimer's disease (AD) has been a matter of controversy. Lack of AD pathology in patients with Al encephalopathy indicates Al does not cause AD. On the other hand, some epidemiological studies have suggested high Al increases the occurrence of AD. Our purpose is to test if high Al in drinking water is a risk factor for AD. We administered Al and Zn in drinking water to Tg2576, a transgenic mouse model for amyloid β-protein (Aβ) deposition with the Aβ precursor protein (AβPP) mutations (K670N/M671L), and Tg2576/tau(P301L), a model for Aβ and tau deposition. Deionized water was given to the control Tg2576 and Tg2576/tau. After administration for 4-10 months of approximately 100 mg/kg body weight Al or Zn per day, we were not able to find by quantitative immunohistochemical analyses differences in the deposition of Aβ and tau between the treated and untreated groups. Nor did the Al or Zn treatment affect the amount of soluble Aβ and Aβ*56, an Aβ oligomer, measured by ELISA or immunoblot. The oral intake of excess Al or Zn does not accelerate AD pathology in the transgenic mouse models for Aβ and tau accumulation. Such results do not seem to support the notion that excessive oral intake of Al or Zn is a risk factor for AD.

Immunotherapy targeting amyloidbeta (Abeta) for Alzheimer's disease (AD) has been developed based on the amyloid cascade hypothesis. It was shown that active immunization using Abeta peptide attenuates amyloid deposits and memory impairment in AD model mice. However, active immunization of patients with AD (AN1792) was halted because 6 % of patients developed meningoencephalitis. Follow-up studies demonstrated that AN1792 could not improve cognitive functions, although senile plaques were cleared. These results suggest that active immunization is a valid strategy and bring us a new perspective. It is speculated that the next generation immunotherapy should activate humoral immunity but not cellular immunity. Recently, there are a lot of immunization strategies for AD. Here we introduce a current trend of immunotherapy for AD.

Radioiodinated erythropoietin (Epo) was bound specifically to the cells of two non-erythroid clonal lines, PC12 and SN6, which expressed neuronal characteristics. The binding was time-, cell number-, and dose-dependent and was reversible. Although the cloned Epo receptor from PC12 cells (derived from rat adrenal medulla) was identical to that from rat erythroid cells, significant differences in the ligand binding properties between two cell lineages were found; 1) PC12 cells had a single class of binding sites with very low affinity (Kd = 16 nM), whereas erythroid cells had two classes of binding sites with different affinities (Kd = 95 pM for high affinity sites and 1.9 nM for low affinity sites), and 2) cross-linking experiments revealed one cross-linked product of 105 kDa for PC12 cells and two products of 140 and 120 kDa for erythroid cells. Taken together with additional results, the presence of a putative accessory protein(s) that may alter the ligand binding affinity through interaction with Epo receptor is discussed. The binding of Epo to PC12 cells caused a rapid increase in the cytosolic concentration of free calcium. The presence of EGTA had no effect on the Epo binding but completely inhibited the calcium increase, indicating that Epo stimulated the calcium influx from outside of the cells. The addition of Epo to the culture media of PC12 cells elevated the intracellular concentrations of monoamines.

We analyzed the effect of several growth factors and cytokines on the expression of amyloid β protein precursor (APP) mRNAs in cultured mouse neuronal and glial cells. In neuronal cultures from embryonic day-15 brain, Northern blotting revealed that APP mRNAs increased by 1.3 to 2.6-fold when treated with nerve growth factor, basic fibroblast growth factor, interleukin 1, interleukin 2, interleukin 3, interleukin 6 or granulocyte-macrophage colony-stimulating factor but not with tumor necrosis factor α. An S1 nuclease protection assay revealed that the enhanced APP mRNA in neuronal cultures was exclusively APP695 mRNA. On the other hand, astrocyte-enriched cultures prepared from postnatal day-2 brain did not show any significant alteration among these factors. We conclude that certain growth factors and cytokines could enhance APP 695 mRNA expression in neurons in vitro.

We studied the in vitro neurite outgrowth activity of fibronectin, laminin, heparan sulfate proteoglycan, type I collagen, type IV collagen, and type VIII collagen in cholinergic neuronal cell lines and primary cultured neurons. All these substances had high neurite promoting activity on primary cultured neurons from embryonic mouse brain. However, collagens had no such an effect on primary cultured neurons from postnatal brain. When neuronal cell lines were used, collagens and other extracellular matrix substances were equally and highly effective on cells originated from embryonic brain, but collagens were less effective on cells from postnatal brain. These findings suggest that postnatal neurons lose the neuritic responsiveness to collagens earlier than that of other ECM.

Currently, only donepezil is available for the treatment of Alzheimer disease (AD) in Japan. Clinical trials of galantamine, rivastigmine, and memantine have been completed in Japan, and patients are awaiting government approval for the use of these drugs. The herbal medicine yokukansan was found to be effective for behavioral and psychological symptoms of dementia (BPSD) in patients, and juzentaihoto was found to reduce AD pathology in a mouse model. In addition, muscarinic and nicotinic acetylcholine receptor agonists, serotonergic agonists, other drugs are being developed. These medicines have little effect on the improvement of cognitive functions. The anti-histamine dimebolin was expected to have a significant effect on the improvement of cognitive functions, but unfortunately, it was rejected during phase III clinical trials. Disease modifying drugs such as alpha-secretase activators, beta- and gamma-secretase inhibitors or modulators, inhibitors of Abeta and tau aggregation, enhancers of Abeta degradation, immunotherapies to remove Abeta oligomers and fibrils, and neurotrophic factors are being developed. Some of these drugs are in phase III clinical trials and are expected to be available for clinical use in the near future.