渡部 博貴

BACKGROUND: Microglia play a crucial role in brain homeostasis through phagocytosis of amyloid-β (Aβ) fibrils, a hallmark of Alzheimer disease (AD) pathology. The balance between Aβ production and clearance is critical for AD pathogenesis, with impaired clearance mechanisms potentially contributing to disease progression. G-protein coupled receptor 34 (GPR34), a microglia-enriched Gi/o-coupled receptor, is highly expressed in homeostatic microglia and may regulate phagocytic functions, yet its role in Aβ clearance remains poorly understood. METHODS: Using flow cytometry-based assays, we investigated the effect of a selective GPR34 agonist (M1) on Aβ uptake in mouse primary microglia and human induced pluripotent stem cell-derived microglia. We evaluated uptake specificity across different Aβ species and phagocytic substrates, and measured intracellular cyclic adenosine monophosphate (cAMP) levels to determine the signaling mechanism. We performed in vivo studies using human amyloid precursor protein knock-in mice with intrahippocampal M1 injections. Additionally, we analyzed GPR34 expression in Japanese AD patient brain samples using single-nucleus RNA sequencing and examined age-dependent expression changes across multiple datasets. RESULTS: M1 specifically enhanced uptake of Aβ fibrils through reduction of intracellular cAMP levels, without affecting monomeric or oligomeric Aβ internalization. Gpr34 knockdown experiments confirmed GPR34 as the molecular target of M1. An intrahippocampal injection of M1 significantly increased microglial Aβ uptake in vivo, an effect that required functional TREM2 signaling. GPR34 expression was significantly reduced in microglia from AD patients and showed age-dependent decline in both humans and mice. CONCLUSIONS: Our findings identify GPR34 as a promising therapeutic target for enhancing microglial Aβ clearance and highlight the potential of GPR34 agonists for AD treatment. The age-dependent decline in GPR34 expression may contribute to reduced Aβ clearance efficiency in aging brains, exacerbating amyloid accumulation. Pharmacological activation of GPR34 represents a novel strategy to counteract impaired Aβ clearance in both aging and AD brains, potentially modifying disease progression through enhancement of microglial phagocytic function.

Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC), a rare and complex neurological disorder, is predominantly observed in the Western Pacific islands, including regions of Japan, Guam, and Papua. This enigmatic condition continues to capture medical attention due to affected patients displaying symptoms that parallel those seen in either classical amyotrophic lateral sclerosis (ALS) or Parkinson's disease (PD). Distinctly, postmortem examinations of the brains of affected individuals have shown the presence of α-synuclein aggregates and TDP-43, which are hallmarks of PD and classical ALS, respectively. These observations are further complicated by the detection of phosphorylated tau, accentuating the multifaceted proteinopathic nature of ALS/PDC. The etiological foundations of this disease remain undetermined, and genetic investigations have yet to provide conclusive answers. However, emerging evidence has implicated the contribution of astrocytes, pivotal cells for maintaining brain health, to neurodegenerative onset, and likely to play a significant role in the pathogenesis of ALS/PDC. Leveraging advanced induced pluripotent stem cell technology, our team cultivated multiple astrocyte lines to further investigate the Japanese variant of ALS/PDC (Kii ALS/PDC). CHCHD2 emerged as a significantly dysregulated gene when disease astrocytes were compared to healthy controls. Our analyses also revealed imbalances in the activation of specific pathways: those associated with astrocytic cilium dysfunction, known to be involved in neurodegeneration, and those related to major neurological disorders, including classical ALS and PD. Further in-depth examinations revealed abnormalities in the mitochondrial morphology and metabolic processes of the affected astrocytes. A particularly striking observation was the reduced expression of CHCHD2 in the spinal cord, motor cortex, and oculomotor nuclei of patients with Kii ALS/PDC. In summary, our findings suggest a potential reduction in the support Kii ALS/PDC astrocytes provide to neurons, emphasizing the need to explore the role of CHCHD2 in maintaining mitochondrial health and its implications for the disease.

Genetic variants in the apolipoprotein E (APOE) gene affect the onset and progression of Alzheimer's disease (AD). The APOE Christchurch (APOE Ch) variant has been identified as the most prominent candidate for preventing the onset and progression of AD. In this study, we generated isogenic APOE3Ch/3Ch human induced pluripotent stem cells (iPSCs) from APOE3/3 healthy control female iPSCs and induced them into astrocytes. RNA expression analysis revealed the inherent resilience of APOE3Ch/3Ch astrocytes to induce a reactive state in response to inflammatory cytokines. Moreover, cytokine treatment changed astrocytic morphology with more complexity in APOE3/3 astrocytes, but not in APOE3Ch/3Ch astrocytes, indicating resilience of the rare variant to a reactive state. Interestingly, we observed robust morphological alterations containing more intricate processes when cocultured with iPSC-derived cortical neurons, in which APOE3Ch/3Ch astrocytes reduced complexity compared with APOE3/3 astrocytes. To assess the impacts of tau propagation effects, we next developed a sophisticated and sensitive assay utilizing cortical neurons derived from human iPSCs, previously generated from donors of both sexes. We showed that APOE3Ch/3Ch astrocytes effectively mitigated tau propagation within iPSC-derived neurons. This study provides important experimental evidence of the characteristic functions exhibited by APOE3Ch/3Ch astrocytes, thereby offering valuable insights for the advancement of novel clinical interventions in AD research.Significance Statement Alzheimer's disease (AD) is a degenerative disease that causes cognitive decline. Familial AD is a severe form caused by mutations in the PSEN1, PSEN2, or APP genes. One carrier of the PSEN1 mutation did not develop dementia. This carrier also had a rare variant of the APOE gene, the Christchurch variant. The APOE Christchurch variant may protect against familial AD. The mechanism of this protection is not fully understood. In the present study, we have successfully demonstrated that the APOE Christchurch variant suppresses the propagation of tau and exhibits a diminished capacity to convert native astrocytes into reactive astrocytes. These significant findings contribute novel insights to the field of the APOE gene and AD research.

BACKGROUND: Microglia are innate immune cells that are the only residential macrophages in the central nervous system. They play vital physiological roles in the adult brain and during development. Microglia are particularly in the spotlight because many genetic risk factors recently identified for neurodegenerative diseases are largely expressed in microglia. Rare polymorphisms in these risk alleles lead to abnormal activity of microglia under traumatic or disease conditions. METHODS: In the present study, to investigate the multifaceted functions of human microglia, we established a novel robust protocol to generate microglia from human induced pluripotent stem cells (hiPSCs) using a combination of cytokines and small chemicals essential for microglia ontogeny. Moreover, we highly enhanced the microglial differentiation efficiency by forcing the expression of PU.1, a crucial transcription factor for microglial development, during posterior mesoderm differentiation. RESULTS: By our novel method, we demonstrated the generation of a greater number of hiPSC-derived microglia (hiMGLs, approximately 120-folds) than the prior methods (at most 40-folds). Over 90% of the hiMGLs expressed microglia-specific markers, such as CX3CR1 and IBA-1. Whole-transcriptome analysis revealed that these hiMGLs are similar to human primary microglia but differ from monocytes/macrophages. Furthermore, the specific physiological functions of microglia were confirmed through indices of lipopolysaccharide responsiveness, phagocytotic ability, and inflammasome formation. By co-culturing these hiMGLs with mouse primary neurons, we demonstrated that hiMGLs can regulate the activity and maturation of neurons. CONCLUSIONS: In this study, our new simple, rapid, and highly efficient method for generating microglia from hiPSCs will prove useful for future investigations on microglia in both physiological and disease conditions, as well as for drug discovery.

Quantitative assessment of neuropathological changes is essential for the characterization of animal models of neurodegenerative disease. Here, we describe a detailed protocol for the detection and quantification of key neuropathological changes in Alzheimer's mouse models. The protocol covers detailed methods including perfusion, dissection, and paraffinization of the brain, preparation of serial brain sections, immunohistochemical analysis, stereological quantification, and sample coding methods for genotype blind analysis. This protocol may be applied to the analysis of neuropathological changes of other neurological disorders. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020), Kang and Shen (2020), Giaime et al. (2017), Xia et al. (2015), Watanabe et al. (2012, 2014), Wines-Samuelson et al. (2010), and Saura et al. (2004).

Amyloid β (Aβ) peptides are the main component of the characteristic insoluble deposits in brain parenchyma and small blood vessels in the patients afflicted with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). These small peptides are attributed to the pathogenesis of both AD and CAA, suggesting an important index for disease stage and progression. In the brain tissue, Aβs are released mainly from neuronal cells into extracellular space. Here, we describe a step-by-step protocol to measure Aβs secreted from human pluripotent stem cell-derived neuronal cells.

Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers and germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem cell-based therapy, gene-modified animal production, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrate the iPSC derivation from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the fibroblasts temporarily acquired a neural stem cell-like state during the reprogramming. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem cell-based research using various animals in Mammalia.

Human induced pluripotent stem cells (iPSCs) have great potential to elucidate the molecular pathogenesis of neurological/psychiatric diseases. In particular, neurological/psychiatric diseases often display brain region-specific symptoms, and the technology for generating region-specific neural cells from iPSCs has been established for detailed modeling of neurological/psychiatric disease phenotypes in vitro. On the other hand, recent advances in culturing human iPSCs without feeder cells have enabled highly efficient and reproducible neural induction. However, conventional regional control technologies have mainly been developed based on on-feeder iPSCs, and these methods are difficult to apply to feeder-free (ff) iPSC cultures. In this study, we established a novel culture system to generate region-specific neural cells from human ff-iPSCs. This system is the best optimized approach for feeder-free iPSC culture and generates specific neuronal subtypes with high purity and functionality, including forebrain cortical neurons, forebrain interneurons, midbrain dopaminergic neurons, and spinal motor neurons. In addition, the temporal patterning of cortical neuron layer specification in the forebrain was reproduced in our culture system, which enables the generation of layer-specific cortical neurons. Neuronal activity was demonstrated in the present culture system by using multiple electrode array and calcium imaging. Collectively, our ff-iPSC-based culture system would provide a desirable platform for modeling various types of neurological/psychiatric disease phenotypes.

Mutations in the presenilin genes (PS1, PS2) have been linked to the majority of familial Alzheimer's disease (AD). Although great efforts have been made to investigate pathogenic PS mutations, which ultimately cause an increase in the toxic form of β-amyloid (Aβ), the intrinsic physiological functions of PS in human neurons remain to be determined. In this study, to investigate the physiological roles of PS in human neurons, we generated PS1 conditional knockout induced pluripotent stem cells (iPSCs), in which PS1 can be selectively abrogated under Cre transduction with or without additional PS2 knockout. We showed that iPSC-derived neural progenitor cells do not confer a maintenance ability in the absence of both PS1 and PS2, showing the essential role of PS in Notch signaling. We then generated PS-null human cortical neurons, where PS1 was intact until full neuronal differentiation occurred. Aβ40 production was reduced exclusively in human PS1/PS2-null neurons along with a concomitant accumulation of APP-CTFs, whereas Aβ42 was decreased in neurons devoid of PS2 Unlike previous studies in mice, in which APP cleavage is largely attributable to PS1, γ-secretase activity seemed to be comparable between PS1 and PS2. In contrast, cleavage of another substrate, N-cadherin, was impaired only in neurons devoid of PS1 Moreover, PS2/γ-secretase exists largely in late endosomes/lysosomes, as measured by specific antibody against the γ-secretase complex, in which Aβ42 species are supposedly produced. Using this novel stem cell-based platform, we assessed important physiological PS1/PS2 functions in mature human neurons, the dysfunction of which could underlie AD pathogenesis.Significance Statement Presenilins are crucial catalytic subunits of γ-secretase, an intramembranous protease complex, whose mutations underlie AD pathogenesis via the dysregulation of Aβ generation. The γ-secretase complex exhibits heterogeneity via the assembly of PS1 or PS2, but the correlation of γ-secretase heterogeneity with substrate processing remains to be established in human neurons. Here, using a novel iPSC-derived cellular model carrying PS1 and/or PS2 conditional knockout alleles, we uncovered the unique processing of three substrates, Notch, APP and N-cadherin, by PS1 or PS2 in human neural cell contexts. Furthermore, the intrinsic subcellular localization of γ-secretase depends on PS1 or PS2, leading to putative differences in the processing of substrates. This novel platform will help ensure the correlation of γ-secretase/substrates in human neurons.

Induced pluripotent stem cell (iPSC)-based disease modeling has a great potential for uncovering the mechanisms of pathogenesis, especially in the case of neurodegenerative diseases where disease-susceptible cells can usually not be obtained from patients. So far, the iPSC-based modeling of neurodegenerative diseases has mainly focused on neurons because the protocols for generating astrocytes from iPSCs have not been fully established. The growing evidence of astrocytes' contribution to neurodegenerative diseases has underscored the lack of iPSC-derived astrocyte models. In the present study, we established a protocol to efficiently generate iPSC-derived astrocytes (iPasts), which were further characterized by RNA and protein expression profiles as well as functional assays. iPasts exhibited calcium dynamics and glutamate uptake activity comparable to human primary astrocytes. Moreover, when co-cultured with neurons, iPasts enhanced neuronal synaptic maturation. Our protocol can be used for modeling astrocyte-related disease phenotypes in vitro and further exploring the contribution of astrocytes to neurodegenerative diseases.

BACKGROUND: Several genetic alterations, including point mutations and copy number variations in NLGN genes, have been associated with psychiatric disorders, such as autism spectrum disorder (ASD) and X-linked mental retardation (XLMR). NLGN genes encode neuroligin (NL) proteins, which are adhesion molecules that are important for proper synaptic formation and maturation. Previously, we and others found that the expression level of murine NL1 is regulated by proteolytic processing in a synaptic activity-dependent manner. METHODS: In this study, we analyzed the effects of missense variants associated with ASD and XLMR on the metabolism and function of NL4X, a protein which is encoded by the NLGN4X gene and is expressed only in humans, using cultured cells, primary neurons from rodents, and human induced pluripotent stem cell-derived neurons. RESULTS: NL4X was found to undergo proteolytic processing in human neuronal cells. Almost all NL4X variants caused a substantial decrease in the levels of mature NL4X and its synaptogenic activity in a heterologous culture system. Intriguingly, the L593F variant of NL4X accelerated the proteolysis of mature NL4X proteins located on the cell surface. In contrast, other variants decreased the cell-surface trafficking of NL4X. Notably, protease inhibitors as well as chemical chaperones rescued the expression of mature NL4X. LIMITATIONS: Our study did not reveal whether these dysfunctional phenotypes occurred in individuals carrying NLGN4X variant. Moreover, though these pathological mechanisms could be exploited as potential drug targets for ASD, it remains unclear whether these compounds would have beneficial effects on ASD model animals and patients. CONCLUSIONS: These data suggest that reduced amounts of the functional NL4X protein on the cell surface is a common mechanism by which point mutants of the NL4X protein cause psychiatric disorders, although different molecular mechanisms are thought to be involved. Furthermore, these results highlight that the precision medicine approach based on genetic and cell biological analyses is important for the development of therapeutics for psychiatric disorders.

Amyloid precursor protein (APP) is associated with both familial and sporadic forms of Alzheimer's disease. Despite its importance, the role of APP family in neuronal function and survival remains unclear because of perinatal lethality exhibited by knockout mice lacking all three APP family members. Here we report that selective inactivation of APP family members in excitatory neurons of the postnatal forebrain results in neither cortical neurodegeneration nor increases in apoptosis and gliosis up to ∼2 years of age. However, hippocampal synaptic plasticity, learning, and memory are impaired in these mutant mice. Furthermore, hippocampal neurons lacking APP family exhibit hyperexcitability, as evidenced by increased neuronal spiking in response to depolarizing current injections, whereas blockade of Kv7 channels mimics and largely occludes the effects of APP family inactivation. These findings demonstrate that APP family is not required for neuronal survival and suggest that APP family may regulate neuronal excitability through Kv7 channels.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex (ALS/PDC) is a unique endemic neurodegenerative disease, with high-incidence foci in Kii Peninsula, Japan. To gather new insights into the pathological mechanisms underlying Kii ALS/PDC, we performed transcriptome analyses of patient brains. We prepared frozen brains from three individuals without neurodegenerative diseases, three patients with Alzheimer's disease, and 21 patients with Kii ALS/PDC, and then acquired microarray data from cerebral gray and white matter tissues. Microarray results revealed that expression levels of genes associated with heat shock proteins, DNA binding/damage, and senescence were significantly altered in patients with ALS/PDC compared with healthy individuals. The RNA expression pattern observed for ALS-type brains was similar to that of PDC-type brains. Additionally, pathway and network analyses indicated that the molecular mechanism underlying ALS/PDC may be associated with oxidative phosphorylation of mitochondria, ribosomes, and the synaptic vesicle cycle; in particular, upstream regulators of these mechanisms may be found in synapses and during synaptic trafficking. Furthermore, phenotypic differences between ALS-type and PDC-type were observed, based on HLA haplotypes. In conclusion, determining the relationship between stress-responsive proteins, synaptic dysfunction, and the pathogenesis of ALS/PDC in the Kii peninsula may provide new understanding of this mysterious disease.

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.

Mutations in the microtubule-associated protein tau (MAPT) gene are known to cause familial frontotemporal dementia (FTD). The R406W tau mutation is a unique missense mutation whose patients have been reported to exhibit Alzheimer's disease (AD)-like phenotypes rather than the more typical FTD phenotypes. In this study, we established patient-derived induced pluripotent stem cell (iPSC) models to investigate the disease pathology induced by the R406W mutation. We generated iPSCs from patients and established isogenic lines using CRISPR/Cas9. The iPSCs were induced into cerebral organoids, which were dissociated into cortical neurons with high purity. In this neuronal culture, the mutant tau protein exhibited reduced phosphorylation levels and was increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized and the axons of the patient-derived neurons displayed morphological and functional abnormalities, which were rescued by microtubule stabilization. The findings of our study provide mechanistic insight into tau pathology and a potential for therapeutic intervention.