Hippocampus-specific knockdown of Shati/Nat8l impairs cognitive function and electrophysiological response in mice.

Shati/Nat8l was identified as an upregulated molecule in the nucleus accumbens (NAc) of mice following repeated methamphetamine administration. Region-specific roles of this molecule are associated with psychiatric disorders. In the present study, we examined the importance of Shati/Nat8l in the hippocampus because of its high expression in this region. Mice with a hippocampus-specific knockdown of Shati/Nat8l (hippocampal Shati-cKD) were prepared by the microinjection of adeno-associated virus (AAV) vectors carrying Cre into the hippocampus of Shati/Nat8lflox/flox mice, and their phenotypes were investigated. Drastic reduction in the expression and function of Shati/Nat8l in the hippocampus was observed in Shati-cKD mice. These mice exhibited cognitive dysfunction in behavioral experiments and impaired the electrophysiological response to the stimuli, which elicits long-term potentiation. Shati/Nat8l in the hippocampus is suggested to possibly play an important role in synaptic plasticity to maintain cognitive function. This molecule could be a therapeutic target for hippocampus-related disorders such as dementia.

Neuronal activation of nucleus accumbens by local methamphetamine administration induces cognitive impairment through microglial inflammation in mice.

More than half of methamphetamine (METH) users present with cognitive impairment, making it difficult for them to reintegrate into society. However, the mechanisms of METH-induced cognitive impairment remain unclear. METH causes neuronal hyperactivation in the nucleus accumbens (NAc) by aberrantly releasing dopamine, which triggers dependence. In this study, to clarify the involvement of hyperactivation of NAc in METH-induced cognitive impairment, mice were locally microinjected with METH into NAc (mice with METH (NAc)) and investigated their cognitive phenotype. Mice with METH (NAc) exhibited cognitive dysfunction in behavioral analyses and decreased long-term potentiation in the hippocampus, with NAc activation confirmed by expression of FosB, a neuronal activity marker. In the hippocampus of mice with METH (NAc), activated microglia, but not astroglia, and upregulated microglia-related genes, Il1b and C1qa were observed. Finally, administration of minocycline, a tetracycline antibiotic with suppressive effect on microglial activation, to mice with METH (NAc) ameliorated cognitive impairment and synaptic dysfunction by suppressing the increased expression of Il1b and C1qa in the hippocampus. In conclusion, activation of NAc by injection of METH into NAc elicited cognitive impairment by facilitating immune activation in mice. This study suggests that immunological intervention could be a therapeutic strategy for addiction-related cognitive disturbances.

Insulin resistance induces earlier initiation of cognitive dysfunction mediated by cholinergic deregulation in a mouse model of Alzheimer's disease.

Although insulin resistance increases the risk of Alzheimer's disease (AD), the mechanisms remain unclear, partly because no animal model exhibits the insulin-resistant phenotype without persistent hyperglycemia. Here we established an AD model with whole-body insulin resistance without persistent hyperglycemia (APP/IR-dKI mice) by crossbreeding constitutive knock-in mice with P1195L-mutated insulin receptor (IR-KI mice) and those with mutated amyloid precursor protein (AppNL-G-F mice: APP-KI mice). APP/IR-dKI mice exhibited cognitive impairment at an earlier age than APP-KI mice. Since cholinergic dysfunction is a major characteristic of AD, pharmacological interventions on the cholinergic system were performed to investigate the mechanism. Antagonism to a nicotinic acetylcholine receptor α7 (nAChRα7) suppressed cognitive function and cortical blood flow (CBF) response to cholinergic-regulated peripheral stimulation in APP-KI mice but not APP/IR-dKI mice. Cortical expression of Chrna7, encoding nAChRα7, was downregulated in APP/IR-dKI mice compared with APP-KI. Amyloid ß burden did not differ between APP-KI and APP/IR-dKI mice. Therefore, insulin resistance, not persistent hyperglycemia, induces the earlier onset of cognitive dysfunction and CBF deregulation mediated by nAChRα7 downregulation. Our mouse model will help clarify the association between type 2 diabetes mellitus and AD.

The Role of GABA in the Dorsal Striatum-Raphe Nucleus Circuit Regulating Stress Vulnerability in Male Mice with High Levels of Shati/Nat8l.

Depression is a frequent and serious illness, and stress is considered the main risk factor for its onset. First-line antidepressants increase serotonin (5-hydroxytryptamine; 5-HT) levels in the brain. We previously reported that an N-acetyltransferase, Shati/Nat8l, is upregulated in the dorsal striatum (dSTR) of stress-susceptible mice exposed to repeated social defeat stress (RSDS) and that dSTR Shati/Nat8l overexpression in mice (dSTR-Shati OE) induces stress vulnerability and local reduction in 5-HT content. Male mice were used in this study, and we found that dSTR 5-HT content decreased in stress-susceptible but not in resilient mice. Moreover, vulnerability to stress in dSTR-Shati OE mice was suppressed by the activation of serotonergic neurons projecting from the dorsal raphe nucleus (dRN) to the dSTR, followed by upregulation of 5-HT content in the dSTR using designer receptors exclusively activated by designer drugs (DREADD). We evaluated the role of GABA in modulating the serotonergic system in the dRN. Stress-susceptible after RSDS and dSTR-Shati OE mice exhibited an increase in dRN GABA content. Furthermore, dRN GABA content was correlated with stress sensitivity. We found that the blockade of GABA signaling in the dRN suppressed stress susceptibility in dSTR-Shati OE mice. In conclusion, we propose that dSTR 5-HT and dRN GABA, controlled by striatal Shati/Nat8l via the dSTR-dRN neuronal circuitry, critically regulate stress sensitivity. Our study provides insights into the neural processes that underlie stress and suggests that dSTR Shati/Nat8l could be a novel therapeutic target for drugs against depression, allowing direct control of the dRN serotonergic system.

Detection of Dietary Chalcone and Flavonoid Metabolites in Mice Using UPLC-MS/MS and Their Modulatory Effects on Amyloid ß Aggregation.

Amyloid ß-protein (Aß42) aggregates have been demonstrated to induce cognitive decline and neurodegeneration in Alzheimer's disease (AD). Thus, functional food ingredients that inhibit Aß42 aggregation are valuable for AD prevention. Although several food ingredients have been studied for their anti-aggregation activity, information on their bioavailability in the brain, incorporated forms, and relevance to AD etiology is limited. Here, we first detected the sulfate- and glucuronic-acid-conjugated forms of green perilla-derived chalcone (1) and taxifolin (2), which inhibit Aß42 aggregation, in the brain, small intestine, and plasma of mice (1 and 2 were administered orally) using ultra-performance liquid chromatography-tandem mass spectrometry. We observed that the conjugated metabolites (sulfate (4) and glucuronide (5)) of 1 prevented the fibrillization and oligomerization of Aß42. These findings imply that the conjugated metabolites of 1 can prove beneficial for AD treatment.

DNA methylation status of SHATI/NAT8L promoter in the blood of cigarette smokers.

AIMS: Cigarette smoking is a preventable risk factor for various diseases such as cancer, ischemic stroke, cardiac stroke, and chronic obstructive pulmonary disease. Smoking cessation is of great importance not only for individual smokers but also for social health. Regarding current cessation therapies, the effectiveness of nicotine replacement is limited, and the cost of varenicline medication is considerable. Thus, a method for screening smokers who are responsive to cessation therapy based on the therapeutic effectiveness is required. Peripheral biomarkers reflecting smoking dependence status are necessary to establish a method for achieving effective cessation therapy. METHODS: Methylation status of smokers' blood DNA was evaluated focusing on SHATI/NAT8L, an addiction-related gene. Eight CpG sites in SHATI/NAT8L were quantified by pyrosequencing. RESULTS: There was no difference in the methylation status of this gene between smokers (n = 129) and non-smokers (n = 129) at all CpG sites. No correlations between the methylation status of SHATI/NAT8L and indicators of smoking dependence were found. CONCLUSIONS: Although the present study found no significance in the DNA methylation of SHATI/NAT8L among smokers, the exploration of predictable peripheral biomarkers for the effectiveness of smoking cessation therapy is required.

Sleep disturbance after cessation of cannabis administration in mice.

Cannabis withdrawal syndrome (CWS) in humans is characterized by various somatic symptoms, including sleep disturbances. In the present study, we investigated sleep alterations in mice after the cessation of arachidonylcyclopropylamide (ACPA), a cannabinoid type 1 receptor agonist, administration. ACPA-administered mice (ACPA mice) displayed an increased number of rearings after the cessation of ACPA administration compared to saline-administered mice (Saline mice). Moreover, the number of rubbings was also decreased in ACPA mice compared with those of the control mice. Electroencephalography (EEG) and electromyography (EMG) were measured for 3 days after the cessation of ACPA administration. During ACPA administration, there was no difference in the relative amounts of total sleep and wake time between ACPA and Saline mice. However, ACPA-induced withdrawal decreased total sleep time during the light period in ACPA mice after ACPA cessation. These results suggest that ACPA cessation induces sleep disturbances in the mouse model of CWS.

Lysine-targeting inhibition of amyloid ß oligomerization by a green perilla-derived metastable chalcone in vitro and in vivo.

Oligomers of amyloid ß (Aß) represent an early aggregative form that causes neurotoxicity in the pathogenesis of Alzheimer's disease (AD). Thus, preventing Aß aggregation is important for preventing AD. Despite intensive studies on dietary compounds with anti-aggregation properties, some identified compounds are susceptible to autoxidation and/or hydration upon incubation in water, leaving unanswered issues regarding which active structures in metastable compounds are actually responsible for the inhibition of Aß aggregation. In this study, we observed the site-specific inhibition of 42-mer Aß (Aß42) oligomerization by the green perilla-derived chalcone 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC), which was converted to its decomposed flavonoids (dDDC, 1-3) via nucleophilic aromatic substitution with water molecules. DDC suppressed Aß42 fibrillization and slowed the transformation of the ß-sheet structure, which is rich in Aß42 aggregates. To validate the contribution of dDDC to the inhibitory effects of DDC on Aß42 aggregation, we synthesized 1-3 and identified 3, a catechol-type flavonoid, as one of the active forms of DDC. 1H-15N SOFAST-HMQC NMR revealed that 1-3 as well as DDC could interact with residues between His13 and Leu17, which were near the intermolecular ß-sheet (Gln15-Ala21). The nucleation in Aß42 aggregates involves the rate-limiting formation of low-molecular-weight oligomers. The formation of a Schiff base with dDDC at Lys16 and Lys28 in the dimer through autoxidation of dDDC was associated with the suppression of Aß42 nucleation. Of note, in two AD mouse models using immunoaffinity purification-mass spectrometry, adduct formation between dDDC and brain Aß was observed in a similar manner as reported in vitro. The present findings unraveled the lysine-targeting inhibitory mechanism of metastable dietary ingredients regarding Aß oligomerization.

APP Knock-In Mice Produce E22P-Aß Exhibiting an Alzheimer's Disease-like Phenotype with Dysregulation of Hypoxia-Inducible Factor Expression.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that requires further pathological elucidation to establish effective treatment strategies. We previously showed that amyloid ß (Aß) toxic conformer with a turn at positions 22-23 is essential for forming highly toxic oligomers. In the present study, we evaluated phenotypic changes with aging in AD model AppNL-P-F/NL-P-F (NL-P-F) mice with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aß, a mimic of toxic conformer utilizing the knock-in technique. Furthermore, the role of the toxic conformer in AD pathology was investigated. NL-P-F mice produced soluble toxic conformers from an early age. They showed impaired synaptic plasticity, glial cell activation, and cognitive decline, followed by the accumulation of Aß plaques and tau hyperphosphorylation. In addition, the protein expression of hypoxia-inducible factor (HIF)-1α was increased, and gene expression of HIF-3α was decreased in NL-P-F mice. HIF dysregulation due to the production of soluble toxic conformers may be involved in AD pathology in NL-P-F mice. This study could reveal the role of a highly toxic Aß on AD pathogenesis, thereby contributing to the development of a novel therapeutic strategy targeting the toxic conformer.

Cannabinoid Type 1 Receptors in the Basolateral Amygdala Regulate ACPA-Induced Place Preference and Anxiolytic-Like Behaviors.

The number of cannabis users is increasing in the world. However, the mechanisms involved in the psychiatric effects and addiction formation remain unclear. Medical treatments against cannabis addiction have not yet been established. Δ9-Tetrahydrocannabinol (THC), the main active substance in cannabis, binds and affects cannabinoid type 1 receptors (CB1R) in the brain. The mice were intraperitoneally (i.p.) administered arachidonylcyclopropylamide (ACPA), a CB1R-selective agonist, and then two behavioral experiments on anxiety and addiction were performed. Administration of ACPA caused anxiolytic-like behavior in the elevated plus maze test. In addition, ACPA increased place preference in a conditioned place preference (CPP) test. The basolateral amygdala (BLA), which is the focus of this study, is involved in anxiety-like behavior and reward and is reported to express high levels of CB1R. We aimed to reveal the role of CB1R in BLA for ACPA-induced behavior. AM251, a CB1R selective antagonist, was administered intra-BLA before i.p. administration of ACPA. Intra-BLA administration of AM251 inhibited ACPA-induced anxiolytic-like behavior and place preference. These results suggest that CB1R in the BLA contributes to behavior disorders caused by the acute or chronic use of cannabis.

Knockdown of Piccolo in the Nucleus Accumbens Suppresses Methamphetamine-Induced Hyperlocomotion and Conditioned Place Preference in Mice.

Methamphetamine (METH), the most widely distributed psychostimulant, aberrantly activates the reward system in the brain to induce addictive behaviors. The presynaptic protein "Piccolo", encoded by Pclo, was identified as a METH-responsive protein with enhanced expression in the nucleus accumbens (NAc) in mice. Although the physiological and pathological significance of Piccolo has been identified in dopaminergic signaling, its role in METH-induced behavioral abnormalities and the underlying mechanisms remain unclear. To clarify such functions, mice with Piccolo knockdown in the NAc (NAc-miPiccolo mice) by local injection of an adeno-associated virus vector carrying miRNA targeting Pclo were generated and investigated. NAc-miPiccolo mice exhibited suppressed hyperlocomotion, sensitization, and conditioned place preference behavior induced by systemic administration of METH. The excessive release of dopamine in the NAc was reduced in NAc-miPiccolo mice at baseline and in response to METH. These results suggest that Piccolo in the NAc is involved in METH-induced behavioral alterations and is a candidate therapeutic target for the treatment of drug addiction.

Shati/Nat8l Overexpression Improves Cognitive Decline by Upregulating Neuronal Trophic Factor in Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a type of dementia characterized by the deposition of amyloid ß, a causative protein of AD, in the brain. Shati/Nat8l, identified as a psychiatric disease related molecule, is a responsive enzyme of N-acetylaspartate (NAA) synthesis. In the hippocampi of AD patients and model mice, the NAA content and Shati/Nat8l expression were reported to be reduced. Having recently clarified the involvement of Shati/Nat8l in cognitive function, we examined the recovery effect of the hippocampal overexpression of Shati/Nat8l in AD model mice (5XFAD). Shati/Nat8l overexpression suppressed cognitive dysfunction without affecting the Aß burden or number of NeuN-positive neurons. In addition, brain-derived neurotrophic factor mRNA was upregulated by Shati/Nat8l overexpression in 5XFAD mice. These results suggest that Shati/Nat8l overexpression prevents cognitive dysfunction in 5XFAD mice, indicating that Shati/Nat8l could be a therapeutic target for AD.

N-Acetyl Transferase, Shati/Nat8l, in the Dorsal Hippocampus Suppresses Aging-induced Impairment of Cognitive Function in Mice.

As the elderly population rapidly increases worldwide, the onset of cognitive dysfunction is expected to increase. Although neuronal plasticity, neurogenesis, and mitochondrial dysfunction have been reported to be involved in cognitive function, the detailed mechanism of cognitive impairment accompanied by aging is poorly understood as there are many confounding factors associated with aging. Therefore, effective treatments for aging have not yet been developed, and the establishment of therapeutic strategies has not progressed accordingly. We have previously found a decline of cognitive function in the developmental stage in mice who lack the expression of Shati/Nat8l, an N-acetyl transferase However, the contribution of Shati/Nat8l to cognitive impairment in aged mice has not yet been investigated. In this study, we aimed to investigate the role of Shati/Nat8l in cognitive function during aging. We observed a reduction in Shati/Nat8l mRNA expression in the dorsal hippocampus of mice as a result of their aging. Moreover, the cognitive dysfunction observed in aged mice was reversed by Shati/Nat8l overexpression in the dorsal hippocampus. Shati/Nat8l overexpression in the dorsal hippocampus of mice did not alter the expression of neurotrophic factors or mitochondrial function-related genes, including Bdnf or Pgc-1α, which are suggested to be downstream genes of Shati/Nat8l. Decreased N-acetyl aspartate (NAA) in aged mice was upregulated by Shati/Nat8l overexpression, suggesting that the Shati/Nat8l-NAA pathway determines cognitive function with aging. Taken together, Shati/Nat8l and NAA in the dorsal hippocampus may be novel targets for the treatment of cognitive impairment.

Aptamers targeting amyloidogenic proteins and their emerging role in neurodegenerative diseases.

Aptamers are oligonucleotides selected from large pools of random sequences based on their affinity for bioactive molecules and are used in similar ways to antibodies. Aptamers provide several advantages over antibodies, including their small size, facile, large-scale chemical synthesis, high stability, and low immunogenicity. Amyloidogenic proteins, whose aggregation is relevant to neurodegenerative diseases, such as Alzheimer's, Parkinson's, and prion diseases, are among the most challenging targets for aptamer development due to their conformational instability and heterogeneity, the same characteristics that make drug development against amyloidogenic proteins difficult. Recently, chemical tethering of aptagens (equivalent to antigens) and advances in high-throughput sequencing-based analysis have been used to overcome some of these challenges. In addition, internalization technologies using fusion to cellular receptors and extracellular vesicles have facilitated central nervous system (CNS) aptamer delivery. In view of the development of these techniques and resources, here we review antiamyloid aptamers, highlighting preclinical application to CNS therapy.

Schizophrenia-Like Behavioral Impairments in Mice with Suppressed Expression of Piccolo in the Medial Prefrontal Cortex.

Piccolo, a presynaptic cytomatrix protein, plays a role in synaptic vesicle trafficking in the presynaptic active zone. Certain single-nucleotide polymorphisms of the Piccolo-encoding gene PCLO are reported to be associated with mental disorders. However, a few studies have evaluated the relationship between Piccolo dysfunction and psychotic symptoms. Therefore, we investigated the neurophysiological and behavioral phenotypes in mice with Piccolo suppression in the medial prefrontal cortex (mPFC). Downregulation of Piccolo in the mPFC reduced regional synaptic proteins, accompanied with electrophysiological impairments. The Piccolo-suppressed mice showed an enhanced locomotor activity, impaired auditory prepulse inhibition, and cognitive dysfunction. These abnormal behaviors were partially ameliorated by the antipsychotic drug risperidone. Piccolo-suppressed mice received mild social defeat stress showed additional behavioral despair. Furthermore, the responses of these mice to extracellular glutamate and dopamine levels induced by the optical activation of mPFC projection in the dorsal striatum (dSTR) were inhibited. Similarly, the Piccolo-suppressed mice showed decreased depolarization-evoked glutamate and -aminobutyric acid elevations and increased depolarization-evoked dopamine elevation in the dSTR. These suggest that Piccolo regulates neurotransmission at the synaptic terminal of the projection site. Reduced neuronal connectivity in the mPFC-dSTR pathway via suppression of Piccolo in the mPFC may induce behavioral impairments observed in schizophrenia.

New Insights Regarding Diagnosis and Medication for Schizophrenia Based on Neuronal Synapse-Microglia Interaction.

Schizophrenia is a common psychiatric disorder that usually develops during adolescence and young adulthood. Since genetic and environmental factors are involved in the disease, the molecular status of the pathology of schizophrenia differs across patients. Recent genetic studies have focused on the association between schizophrenia and the immune system, especially microglia-synapse interactions. Microglia physiologically eliminate unnecessary synapses during the developmental period. The overactivation of synaptic pruning by microglia is involved in the pathology of brain disease. This paper focuses on the synaptic pruning function and its molecular machinery and introduces the hypothesis that excessive synaptic pruning plays a role in the development of schizophrenia. Finally, we suggest a strategy for diagnosis and medication based on modulation of the interaction between microglia and synapses. This review provides updated information on the involvement of the immune system in schizophrenia and proposes novel insights regarding diagnostic and therapeutic strategies for this disease.

[Development of a Novel Alzheimer's Disease Model Based on the Theory of the Toxic-conformer of Amyloid ß].

Development of therapeutics for Alzheimer's disease (AD) is an urgent research task. Amyloid ß (Aß) is one of the causative proteins of AD. Irie et al. identified a toxic conformer among the various structures of 42-mer Aß (Aß42). This conformer, which possesses a turn structure at the positions Glu22-Asp23, exhibits rapid oligomerization and potent neurotoxicity. By the generation of conformationally-specific antibodies against this toxic conformer of Aß, elevation of the toxic conformer in the AD brain was strongly suggested. To investigate the pathogenic role of the toxic conformer in AD, passive immunization experiments against conventional AD model mice were conducted. Specific antibody administration improved the behavioral abnormalities observed in AD model mice without affecting senile plaque pathology. Next, knock-in mice exclusively producing the toxic conformer of Aß were generated. These mice exhibited cognitive dysfunction and oligomerization of Aß, which preceded the onset of the plaque deposition. Taken together, the toxic conformer of Aß is confirmed to be involved in the pathogenesis of AD, and our knock-in mice could be useful in analyzing the Aß oligomer-related pathology of AD.

Impairment of cognitive function induced by Shati/Nat8l overexpression in the prefrontal cortex of mice.

A novel N-acetyltransferase, Shati/Nat8l, was identified in the brains of mice exposed to methamphetamine. Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC) was found to attenuate methamphetamine-induced dependence. The mPFC is a brain region that plays an important role in cognitive function. However, the effect of Shati/Nat8l on cognition and memory has not yet been clarified. To understand the role of Shati/Nat8l in memory, we generated C57BL/6J mice with overexpressed Shati/Nat8l in the mPFC and performed memory-related experiments, including novel object-location and object-in-context tests. Furthermore, we used quantitative immunohistochemistry to assess the presynaptic and postsynaptic proteins, synaptophysin and postsynaptic density protein (PSD)-95, respectively. Shati/Nat8l overexpression in the mPFC impaired both novel object-location and object-in-context memory. Moreover, Shati/Nat8l overexpression in the mPFC reduced PSD-95 levels, but not synaptophysin levels in the mPFC. These results demonstrated that Shati/Nat8l overexpression in the mPFC is involved in location and contextual memory, and can affect the excitatory postsynaptic protein, PSD-95.

Detection of Amyloid ß Oligomers with RNA Aptamers in AppNL-G-F/NL-G-F Mice: A Model of Arctic Alzheimer's Disease.

RNA aptamers have garnered attention for diagnostic applications due to their ability to recognize diverse targets. Oligomers of 42-mer amyloid ß-protein (Aß42), whose accumulation is relevant to the pathology of Alzheimer's disease (AD), are among the most difficult molecules for aptamer recognition because they are prone to aggregate in heterogeneous forms. In addition to designing haptens for in vitro selection of aptamers, the difficulties involved in determining their effect on Aß42 oligomerization impede aptamer research. We previously developed three RNA aptamers (E22P-AbD4, -AbD31, and -AbD43) with high affinity for protofibrils (PFs) derived from a toxic Aß42 dimer. Notably, these aptamers recognized diffuse staining, which likely originated from PFs or higher-order oligomers with curvilinear structures in a knock-in AppNL-G-F/NL-G-F mouse, carrying the Arctic mutation that preferentially induced the formation of PFs, in addition to a PS2Tg2576 mouse. To determine which oligomeric sizes were mainly altered by the aptamer, ion mobility-mass spectrometry (IM-MS) was carried out. One aptamer, E22P-AbD43, formed adducts with the Aß42 monomer and dimer, leading to suppression of further oligomerization. These findings support the utility of these aptamers as diagnostics for AD.

Synthetic and Biophysical Studies on the Toxic Conformer in Amyloid ß with the E22Δ Mutation in Alzheimer Pathology.

The toxic conformer of the 40- or 42-mer-amyloid ß-proteins (Aß) (Aß40, Aß42) with a turn at positions 22 and 23 plays a role in oligomer formation, leading to neurotoxicity as part of the pathogenesis of Alzheimer's disease (AD). A deletion mutant at Glu22 (E22Δ) of Aß, known as an Osaka mutation, accelerates oligomerization. Although E22Δ-Aß has not been found to be toxic to cultured neuronal cells and is instead synaptotoxic in long-term potentiation, there is no information on the toxic conformer of E22Δ-Aß in AD. The site-directed spin labeling study of E22Δ-Aß40 by continuous wave-electron spin resonance (CW-ESR) spectroscopy in part showed the spatial proximity between positions 10 and 35, which are characteristic of the toxic conformation of Aß, indicating the existence of a toxic conformer of Aß with the E22Δ mutation. To obtain structural insight, E22Δ-Aß42 substitutes with proline (F20P, A21P, D23P, and V24P), in which proline is known as a turn inducer but is a ß-sheet breaker, were synthesized. An enzyme immunoassay using the 24B3 antibody recognizing toxic conformer of Aß was carried out. 24B3 reacted with these substitutes of E22Δ-Aß42 as well as E22Δ-Aß42 in a similar manner to WT-Aß42. Notably, only A21P-E22Δ-Aß42 exhibited strong neurotoxicity in rat primary neurons after 8 days of incubation, with potent high-order oligomerization compared with E22Δ-Aß42. These results suggest that E22Δ-Aß42 could enhance neurotoxicity by generating a toxic oligomer conformation with a turn near position 21.