Modeling APOE ε4 familial Alzheimer's disease in directly converted 3D brain organoids.

Brain organoids have become a valuable tool for studying human brain development, disease modeling, and drug testing. However, generating brain organoids with mature neurons is time-intensive and often incomplete, limiting their utility in studying age-related neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report the generation of 3D brain organoids from human fibroblasts through direct reprogramming, with simplicity, efficiency, and reduced variability. We also demonstrate that induced brain organoids from APOE ε4 AD patient fibroblasts capture some disease-specific features and pathologies associated with APOE ε4 AD. Moreover, APOE ε4-induced brain organoids with mutant APP overexpression faithfully recapitulate the acceleration of AD-related pathologies, providing a more physiologically relevant and patient-specific model of familial AD. Importantly, transcriptome analysis reveals that gene sets specific to APOE ε4 patient-induced brain organoids are highly similar to those of APOE ε4 post-mortem AD brains. Overall, induced brain organoids from direct reprogramming offer a promising approach for more efficient and controlled studies of neurodegenerative disease modeling.

Single-cell RNA-sequencing identifies disease-associated oligodendrocytes in male APP NL-G-F and 5XFAD mice.

Alzheimer's disease (AD) is associated with progressive neuronal degeneration as amyloid-beta (Aß) and tau proteins accumulate in the brain. Glial cells were recently reported to play an important role in the development of AD. However, little is known about the role of oligodendrocytes in AD pathogenesis. Here, we describe a disease-associated subpopulation of oligodendrocytes that is present during progression of AD-like pathology in the male AppNL-G-F and male 5xFAD AD mouse brains and in postmortem AD human brains using single-cell RNA sequencing analysis. Aberrant Erk1/2 signaling was found to be associated with the activation of disease-associated oligodendrocytes (DAOs) in male AppNL-G-F mouse brains. Notably, inhibition of Erk1/2 signaling in DAOs rescued impaired axonal myelination and ameliorated Aß-associated pathologies and cognitive decline in the male AppNL-G-F AD mouse model.

APOE ε4-dependent effects on the early amyloid pathology in induced neurons of patients with Alzheimer's disease.

BACKGROUND: The ε4 allele of apolipoprotein E (APOE ε4) is the strongest known genetic risk factor for late-onset Alzheimer's disease (AD), associated with amyloid pathogenesis. However, it is not clear how APOE ε4 accelerates amyloid-beta (Aß) deposition during the seeding stage of amyloid development in AD patient neurons. METHODS: AD patient induced neurons (iNs) with an APOE ε4 inducible system were prepared from skin fibroblasts of AD patients. Transcriptome analysis was performed using RNA isolated from the AD patient iNs expressing APOE ε4 at amyloid-seeding and amyloid-aggregation stages. Knockdown of IGFBP3 was applied in the iNs to investigate the role of IGFBP3 in the APOE ε4-mediated amyloidosis. RESULTS: We optimized amyloid seeding stage in the iNs of AD patients that transiently expressed APOE ε4. Remarkably, we demonstrated that Aß  pathology was aggravated by the induction of APOE ε4 gene expression at the amyloid early-seeding stage in the iNs of AD patients. Moreover, transcriptome analysis in the early-seeding stage revealed that IGFBP3 was functionally important in the molecular pathology of APOE ε4-associated AD. CONCLUSIONS: Our findings suggest that the presence of APOE ε4 at the early Aß-seeding stage in patient iNs is critical for aggravation of sporadic AD pathology. These results provide insights into the importance of APOE ε4 expression for the progression and pathogenesis of sporadic AD.

Dormant state of quiescent neural stem cells links Shank3 mutation to autism development.

Autism spectrum disorders (ASDs) are common neurodevelopmental disorders characterized by deficits in social interactions and communication, restricted interests, and repetitive behaviors. Despite extensive study, the molecular targets that control ASD development remain largely unclear. Here, we report that the dormancy of quiescent neural stem cells (qNSCs) is a therapeutic target for controlling the development of ASD phenotypes driven by Shank3 deficiency. Using single-cell RNA sequencing (scRNA-seq) and transposase accessible chromatin profiling (ATAC-seq), we find that abnormal epigenetic features including H3K4me3 accumulation due to up-regulation of Kmt2a levels lead to increased dormancy of qNSCs in the absence of Shank3. This result in decreased active neurogenesis in the Shank3 deficient mouse brain. Remarkably, pharmacological and molecular inhibition of qNSC dormancy restored adult neurogenesis and ameliorated the social deficits observed in Shank3-deficient mice. Moreover, we confirmed restored human qNSC activity rescues abnormal neurogenesis and autism-like phenotypes in SHANK3-targeted human NSCs. Taken together, our results offer a novel strategy to control qNSC activity as a potential therapeutic target for the development of autism.

Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI promotes neuronal rejuvenation in aged mice.

An increasing number of studies have indicated that alterations in gut microbiota affect brain function, including cognition and memory ability, via the gut-brain axis. In this study, we aimed to determine the protective effect of Bifidobacterium bifidum BGN4 (B. bifidum BGN4) and Bifidobacterium longum BORI (B. longum BORI) on age-related brain damage in mice. We found that administration of B. bifidum BGN4 and B. longum BORI effectively elevates brain-derived neurotrophic factor expression which was mediated by increased histone 3 lysine 9 trimethylation. Furthermore, administration of probiotic supplementation reversed the DNA damage and apoptotic response in aged mice and also improved the age-related cognitive and memory deficits of these mice. Taken together, the present study highlights the anti-aging effects of B. bifidum BGN4 and B. longum BORI in the aged brain and their beneficial effects for age-related brain disorders.

In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for myocardial infarction.

CRISPR/Cas9-mediated gene-editing technology has gained attention as a new therapeutic method for intractable diseases. However, the use of CRISPR/Cas9 for cardiac conditions such as myocardial infarction remains challenging due to technical and biological barriers, particularly difficulties in delivering the system and targeting genes in the heart. In the present study, we demonstrated the in vivo efficacy of the CRISPR/Cas9 magnetoplexes system for therapeutic genome editing in myocardial infarction. First, we developed CRISPR/Cas9 magnetoplexes that magnetically guided CRISPR/Cas9 system to the heart for efficient in vivo therapeutic gene targeting during heart failures. We then demonstrated that the in vivo gene targeting of miR34a via these CRISPR/Cas9 magnetoplexes in a mouse model of myocardial infarction significantly improved cardiac repair and regeneration to facilitate improvements in cardiac function. These results indicated that CRISPR/Cas9 magnetoplexes represent an effective in vivo therapeutic gene-targeting platform in the myocardial infarction of heart, and that this strategy may be applicable for the treatment of a broad range of cardiac failures.

Electromagnetized gold nanoparticles improve neurogenesis and cognition in the aged brain.

Adult neurogenesis is the lifelong process by which new neurons are generated in the dentate gyrus. However, adult neurogenesis capacity decreases with age, and this decrease is closely linked to cognitive and memory decline. Our study demonstrated that electromagnetized gold nanoparticles (AuNPs) promote adult hippocampal neurogenesis, thereby improving cognitive function and memory consolidation in aged mice. According to single-cell RNA sequencing data, the numbers of neural stem cells (NSCs) and neural progenitors were significantly increased by electromagnetized AuNPs. Additionally, electromagnetic stimulation resulted in specific activation of the histone acetyltransferase Kat2a, which led to histone H3K9 acetylation in adult NSCs. Moreover, in vivo electromagnetized AuNP stimulation efficiently increased hippocampal neurogenesis in aged and Hutchinson-Gilford progeria mouse brains, thereby alleviating the symptoms of aging. Therefore, our study provides a proof-of-concept for the in vivo stimulation of hippocampal neurogenesis using electromagnetized AuNPs as a promising therapeutic strategy for the treatment of age-related brain diseases.

Epitranscriptomic N6-Methyladenosine Modification Is Required for Direct Lineage Reprogramming into Neurons.

N6-methyladenosine (m6A), a conserved epitranscriptomic modification of eukaryotic mRNA (mRNA), plays a critical role in a variety of biological processes. Here, we report that m6A modification plays a key role in governing direct lineage reprogramming into induced neuronal cells (iNs). We found that m6A modification is required for the remodeling of specific mRNAs required for the neuronal direct conversion. Inhibition of m6A methylation by Mettl3 knockdown decreased the efficiency of direct lineage reprogramming, whereas increased m6A methylation by Mettl3 overexpression increased the efficiency of iN generation. Moreover, we found that transcription factor Btg2 is a functional target of m6A modification for efficient iN generation. Taken together, our results suggest the importance of establishing epitranscriptomic remodeling for the cell fate conversion into iNs.

Rad50 mediates DNA demethylation to establish pluripotent reprogramming.

DNA demethylation is characterized by the loss of methyl groups from 5-methylcytosine, and this activity is involved in various biological processes in mammalian cell development and differentiation. In particular, dynamic DNA demethylation in the process of somatic cell reprogramming is required for successful iPSC generation. In the present study, we reported the role of Rad50 in the DNA demethylation process during somatic cell reprogramming. We found that Rad50 was highly expressed in pluripotent stem cells and that Rad50 regulated global DNA demethylation levels. Importantly, the overexpression of Rad50 resulted in the enhanced efficiency of iPSC generation via increased DNA demethylation, whereas Rad50 knockdown led to DNA hypermethylation, which suppressed somatic cell reprogramming into iPSCs. Moreover, we found that Rad50 associated with Tet1 to facilitate the DNA demethylation process in pluripotent reprogramming. Therefore, our findings highlight the novel role of Rad50 in the DNA demethylation process during somatic cell reprogramming.

Valproic Acid Significantly Improves CRISPR/Cas9-Mediated Gene Editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has emerged as a powerful technology, with the potential to generate transgenic animals. Particularly, efficient and precise genetic editing with CRISPR/Cas9 offers immense prospects in various biotechnological applications. Here, we report that the histone deacetylase inhibitor valproic acid (VPA) significantly increases the efficiency of CRISPR/Cas9-mediated gene editing in mouse embryonic stem cells and embryos. This effect may be caused through globally enhanced chromatin accessibility, as indicate by histone hyperacetylation. Taken together, our results suggest that VPA can be used to increase the efficacy of CRISPR/Cas9 in generating transgenic systems.

Direct conversion of fibroblasts to osteoblasts as a novel strategy for bone regeneration in elderly individuals.

Mortality caused by age-related bone fractures or osteoporosis is steadily increasing worldwide as the population ages. The pace of the development of bone regeneration engineering to treat bone fractures has consequently increased in recent years. A range of techniques for bone regeneration, such as immunotherapy, allografts, and hydrogel therapy, have been devised. Cell-based therapies using bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells derived from somatic cells are considered to be suitable approaches for bone repair. However, these cell-based therapies suffer from a number of limitations in terms of efficiency and safety. Somatic cells can also be directly differentiated into osteoblasts by several transcription factors. As osteoblasts play a central role in the process of bone formation, the direct reprogramming of fibroblasts into osteoblasts may hence be a new way to treat bone fractures in elderly individuals. Here, we review recent developments regarding the therapeutic potential of the direct reprogramming of cells for bone regeneration.

In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease.

In vivo gene editing in post-mitotic neurons of the adult brain may be a useful strategy for treating neurological diseases. Here, we develop CRISPR-Cas9 nanocomplexes and show they were effective in the adult mouse brain, with minimal off-target effects. Using this system to target Bace1 suppressed amyloid beta (Aß)-associated pathologies and cognitive deficits in two mouse models of Alzheimer's disease. These results broaden the potential application of CRISPR-Cas9 systems to neurodegenerative diseases.