Advanced Immunolabeling Method for Optical Volumetric Imaging Reveals Dystrophic Neurites of Dopaminergic Neurons in Alzheimer's Disease Mouse Brain.

Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer's disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions. The axonal projection of DA neurons in the septum and the NAc showed dystrophic phenotypes such as growth cone-like enlargement of the axonal terminus and aggregated neurites. Brain-wide imaging of structural defects in the neural circuits was facilitated with brain clearing and antibody penetration assisted with SB3-12 and Quadrol pre-treatment. The whole volumetric imaging process could be completed in a week with the robust iACT method. Created with https://www.biorender.com/ .

Abnormal accumulation of extracellular vesicles in hippocampal dystrophic axons and regulation by the primary cilia in Alzheimer's disease.

Dystrophic neurites (DNs) are abnormal axons and dendrites that are swollen or deformed in various neuropathological conditions. In Alzheimer's disease (AD), DNs play a crucial role in impairing neuronal communication and function, and they may also contribute to the accumulation and spread of amyloid beta (Aß) in the brain of AD patients. However, it is still a challenge to understand the DNs of specific neurons that are vulnerable to Aß in the pathogenesis of AD. To shed light on the development of radiating DNs, we examined enriched dystrophic hippocampal axons in a mouse model of AD using a three-dimensional rendering of projecting neurons. We employed the anterograde spread of adeno-associated virus (AAV)1 and conducted proteomic analysis of synaptic compartments obtained from hippocampo-septal regions. Our findings revealed that DNs were formed due to synaptic loss at the axon terminals caused by the accumulation of extracellular vesicle (EV). Abnormal EV-mediated transport and exocytosis were identified in association with primary cilia, indicating their involvement in the accumulation of EVs at presynaptic terminals. To further address the regulation of DNs by primary cilia, we conducted knockdown of the Ift88 gene in hippocampal neurons, which impaired EV-mediated secretion of Aß and promoted accumulation of axonal spheroids. Using single-cell RNA sequencing, we identified the septal projecting hippocampal somatostatin neurons (SOM) as selectively vulnerable to Aß with primary cilia dysfunction and vesicle accumulation. Our study suggests that DNs in AD are initiated by the ectopic accumulation of EVs at the neuronal axon terminals, which is affected by neuronal primary cilia.

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.

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.

Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells.

It has been reported that hypomagnetic fields (HMFs) have a negative influence on mammalian physiological functions. We previously reported that HMFs were detrimental to cell fate changes during reprogramming into pluripotency. These studies led us to investigate whether HMFs affect cell fate determination during direct differentiation. Here, we found that an HMF environment attenuates differentiation capacity and is detrimental to cell fate changes during the in vitro differentiation of embryonic stem cells (ESCs). Moreover, HMF conditions cause abnormal DNA methylation through the dysregulation of DNA methyltransferase3b (Dnmt3b) expression, eventually resulting in incomplete DNA methylation during differentiation. Taken together, these results suggest that an appropriate electromagnetic field (EMF) environment may be essential for favorable epigenetic remodeling during cell fate determination via differentiation.

Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy.

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

Generation of Integration-Free Induced Neurons Using Graphene Oxide-Polyethylenimine.

Direct conversion of somatic cells into induced neurons (iNs) without inducing pluripotency has great therapeutic potential for treating central nervous system diseases. Reprogramming of somatic cells to iNs requires the introduction of several factors that drive cell-fate conversion, and viruses are commonly used to deliver these factors into somatic cells. However, novel gene-delivery systems that do not integrate transgenes into the genome are required to generate iNs for safe human clinical applications. In this study, it is investigated whether graphene oxide-polyethylenimine (GO-PEI) complexes are an efficient and safe system for messenger RNA delivery for direct reprogramming of iNs. The GO-PEI complexes show low cytotoxicity, high delivery efficiency, and directly converted fibroblasts into iNs without integrating factors into the genome. Moreover, in vivo transduction of reprogramming factors into the brain with GO-PEI complexes facilitates the production of iNs that alleviated Parkinson's disease symptoms in a mouse model. Thus, the GO-PEI delivery system may be used to safely obtain iNs and could be used to develop direct cell reprogramming-based therapies for neurodegenerative diseases.

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.

Homogeneous generation of iDA neurons with high similarity to bona fide DA neurons using a drug inducible system.

Recent work generating induced dopaminergic (iDA) neurons using direct lineage reprogramming potentially provides a novel platform for the study and treatment Parkinson's disease (PD). However, one of the most important issues for iDA-based applications is the degree to which iDA neurons resemble the molecular and functional properties of their endogenous DA neuron counterparts. Here we report that the homogeneity of the reprogramming gene expression system is critical for the generation of iDA neuron cultures that are highly similar to endogenous DA neurons. We employed an inducible system that carries iDA-inducing factors as defined transgenes for direct lineage reprogramming to iDA neurons. This system circumvents the need for viral transduction, enabling a more efficient and reproducible reprogramming process for the generation of genetically homogenous iDA neurons. We showed that this inducible system generates iDA neurons with high similarity to their bona fide in vivo counterparts in comparison to direct infection methods. Thus, our results suggest that homogenous expression of exogenous genes in direct lineage reprogramming is critical for the generation of high quality iDA neuron cultures, making such culture systems a valuable resource for iDA-based drug screening and, ultimately, potential therapeutic intervention in PD.

p53 signalling mediates acupuncture-induced neuroprotection in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with a selective loss of dopamine (DA) neurons in the substantia nigra of the midbrain. Recently, it has been demonstrated that acupuncture treatment has protective effects in PD. However, to date, the molecular mechanisms underlying acupuncture's effect on DA neuronal protection are largely unknown. In this study, we report that p53 signalling mediates the protective effects of acupuncture treatment in a mouse model of PD. We found that the acupuncture treatment in the mouse PD model results in significant recovery to the normal in the context of behaviour and molecular signatures. We found that the gene network associated with p53 signalling is closely involved in the protective effects of acupuncture treatment in PD. Consistent with this idea, we demonstrated that specific knockout of the p53 gene in the midbrain DA neurons abrogates the acupuncture induced protective effects in the mouse model of PD. Thus, these data suggest that p53 signalling mediates the protective effects of acupuncture treatment in PD.

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3ß and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Electromagnetic fields mediate efficient cell reprogramming into a pluripotent state.

Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency.

Ebf3-miR218 regulation is involved in the development of dopaminergic neurons.

The development of midbrain dopaminergic (DA) neurons is a complex process that requires the precise spatial and temporal expression of numerous genes. Here, we report that Ebf3, a transcription factor, plays a critical role in the terminal development of DA neurons. We found a specific upregulation of Ebf3 in Dicer knockout midbrain DA neurons and dynamic patterns of Ebf3 expression during the development of DA neurons. We further demonstrated that the overexpression of Ebf3 at the neural precursor stage of embryonic stem (ES) differentiation induces a significant increase in the number of TH+ DA neurons, whereas the suppression of Ebf3 leads to significant reduction in the development of TH+ DA neurons. Additionally, we found that Ebf3 is a candidate target for miR218 during DA neuronal development, such that the regulation of Ebf3 expression by miR218 controls the terminal differentiation of DA neurons. Thus, our data suggest that complex transcription factor-miRNA regulation is critical for the development of midbrain DA neurons.

Cell reprogramming into the pluripotent state using graphene based substrates.

Graphene has been attracting considerable interest in the field of biomedical engineering because graphene and its derivatives are considered to be ideal platforms for supporting cell growth and differentiation. Here we report that graphene promotes the reprogramming of mouse somatic fibroblasts into induced pluripotent stem cells (iPSCs). We constructed a layer of graphene film on a glass substrate and characterized it as a monolayer using Raman spectroscopy. We found that the graphene substrate significantly improved cellular reprogramming efficiency by inducing mesenchymal-to-epithelial-transition (MET) which is known to affect H3K4me3 levels. Thus, our results reveal that a graphene substrate directly regulates dynamic epigenetic changes associated with reprogramming, providing an efficient tool for epigenetic pluripotent reprogramming.