Scraping Assay as a Novel Strategy to Evaluate Axonal Regeneration Using Human-Induced Pluripotent Stem Cell-Derived Neurons.

Neuronal regrowth after traumatic injury is strongly inhibited in the central nervous system (CNS) of adult mammals. Cell-intrinsic and extrinsic factors limit the regulation of axonal growth and regrowth of fibers is minimal despite nearly all neurons surviving. Developing medical drugs to promote neurological recovery is crucial since neuronal injuries have few palliative cares and no pharmacological interventions. Herein, we developed a novel in vitro axonal regeneration assay system to screen the chemical reagents using human-induced pluripotent stem cell (hiPSC)-derived neurons. These neurons were cultured in a 96-well plate to form a monolayer and were scraped using a floating metal pin tool for axotomy. The cell number and plate coating conditions were optimized to score the regenerating axon. Treatment using the Rho-associated kinase (ROCK) inhibitor Y-27632 enhanced axonal regeneration in this regeneration assay system with hiPSC-derived neurons. Therefore, our novel screening method is suitable for drug screening to identify the chemical compounds that promote axonal regeneration after axotomy under in vitro conditions.

Suppression of Sleeping Beauty-induced Gliomagenicity in Ts1Cje Mice, a Model of Down Syndrome.

BACKGROUND/AIM: Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. MATERIALS AND METHODS: We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. RESULTS: Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. CONCLUSION: Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.

Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons.

Introduction: Cell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression. Methods: We induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA-/- hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA-/- hiPSC-derived mDA neurons using in vitro culture system. Results: We detected human α-synuclein-immunoreactivity in SNCA-/- hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA-/- hiPSC-derived mDA neurons. Conclusion: We have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA-/- hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.

Expression and role of nicotinic acetylcholine receptors during midbrain dopaminergic neuron differentiation from human induced pluripotent stem cells.

AIM: Nicotinic acetylcholine receptors (nAChRs) expressed in midbrain dopaminergic (mDA) neurons modulate mDA neuronal activity. However, their expression patterns and functional roles during mDA neuronal development remain unknown. Here, we profiled the expression and function of nAChR subtypes during mDA neuron differentiation from human induced pluripotent stem cells (hiPSCs). METHODS: Midbrain dopaminergic neurons were differentiated from hiPSCs using a recently developed proprietary method that replicates midbrain development. The expression patterns of developmental marker proteins were monitored during mDA neuronal differentiation using immunohistochemical analysis. Gene expression of nAChR subtypes was analyzed by reverse transcription polymerase chain reaction. Pharmacological nAChR agonists and antagonists were used to reveal the role of the α6 nAChR subunit in the differentiation of mDA neurons from hiPSCs. RESULTS: CHRNA4 expression was detected at the mDA neural progenitor stage, whereas CHRNA6 expression began during the mDA neuronal stage. CHRNA7 was expressed throughout the differentiation process, including in the undifferentiated hiPSCs. We also found that LMO3, a gene expressed in a subset of substantia nigra pars compacta (SNC) DA neurons in the midbrain, showed increased expression following nicotine treatment in a concentration-dependent manner. Additionally, 5-iodo A85380, a selective α6 nAChR agonist, also increased LMO3 expression in hiPSC-derived mDA neurons, and this increase was suppressed by simultaneous treatment with bPiDi, a selective α6 nAChR antagonist. CONCLUSION: Our findings suggest that stimulating the α6 nAChR subunit on hiPSC-derived mDA neurons may induce neuronal maturation that is biased toward SNC DA neurons.

CRISPR-Cas9-Edited SNCA Knockout Human Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons and Their Vulnerability to Neurotoxicity.

Parkinson's disease (PD) is an age-related disorder with selective dopaminergic (DA) neuronal degeneration in the substantia nigra pars compacta. The presence of mainly α-synuclein-composed Lewy bodies in DA neurons is among the disease hallmarks in the brain of patients with PD. Human induced pluripotent stem cells (hiPSCs) are powerful tools to investigate PD pathophysiology and understand its molecular and cellular mechanisms better. In this study, we generated an α-synuclein-null hiPSC line introducing a nonsense mutation in the α-synuclein-encoding SNCA alleles using clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9)-mediated gene editing. Our Western blotting analysis revealed the lack of α-synuclein protein expression in SNCA knockout hiPSC-derived cells. In addition, SNCA knockout hiPSCs retained healthy cell morphology, undifferentiated marker gene (e.g., NANOG, POU5F1, and SOX2) expression, and differentiation ability (based on the marker gene expression levels of the three germ layers). Finally, SNCA knockout hiPSC-derived DA neurons exhibited reduced vulnerability to the DA neurotoxin, 1-methyl-4-phenylpyridinium. In conclusion, the SNCA knockout hiPSC line we generated would provide a useful experimental tool for studying the physiological and pathological role of α-synuclein in PD.

Plantainoside B in Bacopa monniera Binds to Aß Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aß.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-ß (Aß) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aß oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aß-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aß aggregates, including its oligomers, using Aß oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aß attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aß injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aß aggregates including its oligomers and brain tissue from a mouse model of Aß pathology. In addition, plantainoside B could delay the Aß aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aß oligomers, thus interrupting the binding of Aß oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.

Involvement of an Aberrant Vascular System in Neurodevelopmental, Neuropsychiatric, and Neuro-Degenerative Diseases.

The vascular system of the prenatal brain is crucial for the development of the central nervous system. Communication between vessels and neural cells is bidirectional, and dysfunctional communication can lead to neurodevelopmental diseases. In the present review, we introduce neurodevelopmental and neuropsychiatric diseases potentially caused by disturbances in the neurovascular system and discuss candidate genes responsible for neurovascular system impairments. In contrast to diseases that can manifest during the developing stage, we have also summarized the disturbances of the neurovascular system in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Furthermore, we discussed the role of abnormal vascularization and dysfunctional vessels in the development of neurovascular-related diseases.

Analysis of Aß-induced neurotoxicity and microglial responses in simple two- and three-dimensional human iPSC-derived cortical culture systems.

The extracellular accumulation of amyloid-ß (Aß) in plaques and associated neurodegeneration are the pathological hallmarks of Alzheimer's disease (AD). These plaques are surrounded by microglia-the resident tissue macrophages of the brain parenchyma that originate from primitive macrophages from the embryonic yolk sac. Microglia, including a unique subpopulation called "disease-associated microglia" (DAM), are strongly implicated in AD pathology; however, their exact function and physiology remain largely unknown. Notably, simple cell and tissue culture systems that adequately recreate the brain microenvironment and can simulate critical aspects of AD pathology could fundamentally contribute to elucidating microglial function in disease development and progression. Thus, we added human-induced pluripotent stem cell (hiPSC)-induced primitive macrophages (hiMacs) to hiPSC-induced cortical neurons (cell model) and cortical organoids (tissue model). The treatment of these culture systems with the O-acyl isopeptide of Aß1-42, which reverts to natural extracellular Aß1-42 at neutral pH and starts self-aggregation, caused the degeneration of hiPSC-induced cortical neurons in 2D culture and within cortical organoid cultures. Notably, the hiMacs phagocytosed extracellular Aß and exhibited a DAM-like phenotype. In both cell and tissue organoid culture systems, neurodegeneration was attenuated by the addition of hiMacs. Moreover, in cortical organoids, Aß plaques formed more circular and fewer hotspot-like morphological structures in the vicinity of hiMacs. These findings demonstrate the utility of simple hiPSC-induced cortical cell and tissue culture systems supplemented with hiMacs for elucidating critical aspects of AD pathology, such as microglial function and physiology. Adopting such systems in routine research practice may lead to the development of novel therapeutic strategies for AD.

Mumefural prevents insulin resistance and amyloid-beta accumulation in the brain by improving lowered interstitial fluid pH in type 2 diabetes mellitus.

The present study tried to clarify if mumefural would prevent hyperglycemia, one of the typical symptoms of type 2 diabetes mellitus (T2DM), since mumefural is an extract from Japanese apricots preventing hyperglycemia. To clarify if mumefural would prevent T2DM pathogenesis, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats, T2DM model. Mumefural diminished hyperglycemia, HOMA-IR and plasma triglyceride concentration in OLETF rats under fasting conditions. In addition, mumefural elevated protein expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) in the distal colon participating in absorption of weak organic acids, which behave as bases but not acids after absorption into the body. Mumefural also increased the interstitial fluid pH around the brain hippocampus lowered in OLETF rats compared with non-T2DM LETO rats used as control for OLETF rats. Amyloid-beta accumulation in the brain decreased in accordance with the pH elevation. On the one hand, mumefural didn't affect plasma concentrations of glucagon, GLP-1, GIP or PYY under fasting conditions. Taken together, these observations indicate that: 1) mumefural would be a useful functional food improving hyperglycemia, insulin resistance and the lowered interstitial fluid pH in T2DM; 2) the interstitial fluid pH would be one of key factors influencing the accumulation of amyloid-beta.

[Disease modeling using human induced pluripotent stem cell-derived microglia and region-specific neurons].

Neurodegenerative disorders including Alzheimer's disease (AD) and Parkinson's disease (PD) are hard to treat once they have suffered. Therefore, the establishment of new prevention and treatment methods for neurodegenerative disorders is an urgent issue for Japan's aging society, from the perspective of improving the quality of life of patients and medical staff involved in their care. Human induced pluripotent stem cells (hiPSCs) have contributed to the understanding of the pathology of neurodegenerative diseases, and to the development of new preventive and therapeutic strategies based on the understanding of human diseases. Furthermore, new cross-disciplinary scientific trends together with iPSC technology are emerging in the fields of life science, medical science, and information technology. The fusion of various research knowledges and technologies may provide new scientific progress for better understanding of molecular mechanisms of pathology and the onset of neurodegenerative diseases. Here we have developed new brain model with hiPSC technology for the understanding of pathology of AD and PD based on the induction of region-specific brain organoids and microglia using hiPSCs. These brain organoids technology enables us to provide simpler and reproducible analytical methods by combining with not only developmental biology and pharmacology but also transcriptome analysis and direct conversion. These technological advantages contribute to the creation of new research fields with human brain research to understand higher brain functions and pathophysiology of neurodegenerative diseases such as AD and PD.

Nicotinic Acetylcholine Receptors and Microglia as Therapeutic and Imaging Targets in Alzheimer's Disease.

Amyloid-ß (Aß) accumulation and tauopathy are considered the pathological hallmarks of Alzheimer's disease (AD), but attenuation in choline signaling, including decreased nicotinic acetylcholine receptors (nAChRs), is evident in the early phase of AD. Currently, there are no drugs that can suppress the progression of AD due to a limited understanding of AD pathophysiology. For this, diagnostic methods that can assess disease progression non-invasively before the onset of AD symptoms are essential, and it would be valuable to incorporate the concept of neurotheranostics, which simultaneously enables diagnosis and treatment. The neuroprotective pathways activated by nAChRs are attractive targets as these receptors may regulate microglial-mediated neuroinflammation. Microglia exhibit both pro- and anti-inflammatory functions that could be modulated to mitigate AD pathogenesis. Currently, single-cell analysis is identifying microglial subpopulations that may have specific functions in different stages of AD pathologies. Thus, the ability to image nAChRs and microglia in AD according to the stage of the disease in the living brain may lead to the development of new diagnostic and therapeutic methods. In this review, we summarize and discuss the recent findings on the nAChRs and microglia, as well as their methods for live imaging in the context of diagnosis, prophylaxis, and therapy for AD.

Rapid Conversion of Human Induced Pluripotent Stem Cells into Dopaminergic Neurons by Inducible Expression of Two Transcription Factors.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs), provide promising sources for regenerative therapy, disease modeling, and drug screening. Relevant efforts have been invested in establishing robust induction protocols for PSC-derived dopaminergic (DA) neuron generation by mimicking brain development-related signaling pathways. However, these protocols require fully trained techniques and a long time to yield mature DA neurons. In this study, to accelerate the entire process, we generated a hiPSC line differentiating into DA neurons by the inducible force expression of two transcription factors ASCL1 and LMX1A. Using this hiPSC line, we established a rapid and simple induction protocol to generate mature DA neurons in 28 days. The induced DA neurons were characterized by gene expression and immunohistochemical analyses of fundamental DA neuronal markers. Moreover, the cell functional properties were analyzed by a multielectrode array system on day 28. This resource offers future applications for high-throughput screening, such as drug development and toxicology that require highly validated DA neurons.

MEK/ERK Signaling Regulates Reconstitution of the Dopaminergic Nerve Circuit in the Planarian Dugesia japonica.

Planarian Dugesia japonica is a flatworm that can autonomously regenerate its own body after an artificial amputation. A recent report showed the role of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway in the head morphogenesis during the planarian regeneration process after amputation; however, neuron-specific regeneration mechanisms have not yet been reported. Here, whether MEK/ERK pathway was involved in the dopaminergic neuronal regeneration in planarians was investigated. Planarians regenerated their body within 14 days after amputation; however, the head region morphogenesis was inhibited by MEK inhibitor U0126 (3 or 10 µM). Furthermore, the number of planarian tyrosine hydroxylase (DjTH)-positive dopaminergic neurons in the regenerated head region was also decreased by U0126. The 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin, can decrease the number of dopaminergic neurons; however, planarians can regenerate dopaminergic neurons after injecting 6-OHDA into the intestinal tract. MEK inhibitor PD98059 (30 µM) or U0126 (10 µM) significantly decreased dopaminergic neurons 5 days after the 6-OHDA injection. During the regeneration process of dopaminergic neurons, phosphorylated histone H3 (H3P)-positive stem cells known as "neoblasts" were increased in the head region; however, MEK inhibitors significantly decreased the number of H3P-positive neoblasts. These results suggested that dopaminergic neuronal regeneration in planarian was regulated by the MEK/ERK pathway.

Orthogonal characterization and pharmacokinetic studies of polylactide-polyethyleneglycol polymeric nanoparticles with different physicochemical properties.

To optimize prolonged and sustained delivery of polylactide-block-polyethyleneglycol polymeric nanoparticles (PLA-PEG NPs), in terms of the PLA isomer and molecular weight, we performed orthogonal physicochemical characterization and evaluated the pharmacokinetics of tamoxifen (TAM)-loaded PLA-PEG NPs. DL-lactide- (DL-PEG NP), L-lactide- (L-PEG NPs), and stereocomplex-based (SC-PEG NPs) PLA-PEGs, with two different PLA to PEG ratios (12k-5k and 5k-5k Da) were synthesized, and NPs were prepared by anti-solvent precipitation. Size exclusion chromatography, multi-angle light scattering, dynamic light scattering, and 1H nuclear magnetic resonance studies revealed that SC-PEG NPs (12k-5k) had a compact structure and the highest PEG density, followed by L-PEG NPs (12k-5k), DL-PEG NPs (12k-5k), and all PLA-PEG NPs (5k-5k). Additionally, solid-phase extraction indicated that SC-PEG NPs (12k-5k) had the highest drug loading content and the lowest surface TAM adsorption, of the PLA-PEGs evaluated. These results were explained by the crystallinity of the PLA core, which was analyzed by X-ray diffraction. In the pharmacokinetic studies, 14C-TAM-loaded 111In-SC-PEG NPs (12k-5k) exhibited the highest area under the plasma concentration-time curve, followed by L-PEG NPs (12k-5k) and DL-PEG NPs (12k-5k), after intravenous injection in mice. These results indicate that SC-PEG NPs (12k-5k) are promising drug carriers for the sustained and prolonged delivery of TAM.

Roles of microglia in Alzheimer's disease and impact of new findings on microglial heterogeneity as a target for therapeutic intervention.

Microglia are specialized macrophages that reside within the central nervous system and play key roles in brain immunity, development and homeostasis. Recent studies also revealed functions of microglia in neuroprotection and neuroinflammation, leading to the discovery that microglia are involved in several brain pathologies including Alzheimer's disease (AD). However, the beneficial and detrimental actions of this intriguing cell population can be challenging to dissect: the advent of single-cell and single-nucleus transcriptomic technologies has revolutionized our understanding of the heterogeneity of multiple cell types and is now being applied to the study of microglia in health and disease. Here, we review recent findings on microglial biology, focusing on insights from single cell transcriptomic studies and the heterogeneity that they reveal, and consider the impact of these findings on our understanding of AD. We also discuss how microglia might represent a next-generation therapeutic target for treatment of AD and other neuroinflammatory conditions.

Generation of striatal neurons from human induced pluripotent stem cells by controlling extrinsic signals with small molecules.

Human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling human brain development and treating neurodegenerative diseases. Here we established a robust protocol with high scalability for generating striatal medium spiny neurons (MSNs) from hiPSCs using small molecules under two- and three-dimensional culture conditions. Using this protocol, GSH2+ lateral ganglionic eminence (LGE) progenitors were generated in two-dimensional culture by Sonic hedgehog signaling activation using purmorphamine, WNT signaling inhibition using XAV939, and dual-SMAD inhibition using LDN193189 and A83-01. Quantitative PCR analysis revealed sequential expression of LGE and striatal genes during differentiation. These LGE progenitors subsequently gave rise to DARPP32+ MSNs exhibiting spontaneous and evoked monophasic spiking activity. Applying this protocol in three-dimensional culture, we generated striatal neurospheres with gene expression profiles and cell layer organization resembling that of the developing striatum, including distinct ventricular and subventricular zones and DARPP32+ neurons at the surface. This protocol provides a useful experimental model for studying striatal development and yields cells potentially applicable for regenerative medicine to treat striatum-related disorders such as Huntington's disease.

Combination of Drugs and Cell Transplantation: More Beneficial Stem Cell-Based Regenerative Therapies Targeting Neurological Disorders.

Cell transplantation therapy using pluripotent/multipotent stem cells has gained attention as a novel therapeutic strategy for treating neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, ischemic stroke, and spinal cord injury. To fully realize the potential of cell transplantation therapy, new therapeutic options that increase cell engraftments must be developed, either through modifications to the grafted cells themselves or through changes in the microenvironment surrounding the grafted region. Together these developments could potentially restore lost neuronal function by better supporting grafted cells. In addition, drug administration can improve the outcome of cell transplantation therapy through better accessibility and delivery to the target region following cell transplantation. Here we introduce examples of drug repurposing approaches for more successful transplantation therapies based on preclinical experiments with clinically approved drugs. Drug repurposing is an advantageous drug development strategy because drugs that have already been clinically approved can be repurposed to treat other diseases faster and at lower cost. Therefore, drug repurposing is a reasonable approach to enhance the outcomes of cell transplantation therapies for neurological diseases. Ideal repurposing candidates would result in more efficient cell transplantation therapies and provide a new and beneficial therapeutic combination.

A novel dipeptide type inhibitor of the Wnt/ß-catenin pathway suppresses proliferation of acute myelogenous leukemia cells.

The Wnt/ß-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/ß-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/ß-catenin signaling pathway.

Neuroprotective effects of 5-aminolevulinic acid against neurodegeneration in rat models of Parkinson's disease and stroke.

Oxidative stress is associated with the progression of the neurodegenerative diseases Parkinson's disease (PD) and cerebral ischemia. Recently, 5-aminolevulinic acid (5-ALA), an intermediate in the porphyrin synthesis pathway, was reported to exert antioxidative effects on macrophages and cardiomyocytes. Here, we demonstrated the neuroprotective effects of 5-ALA using rat models of PD and ischemia as well as in vitro in SH-SY5Y cells. 5-ALA partially prevented neurodegeneration in each condition. These results suggest that 5-ALA has a potential for promising therapeutic agent to protect against neurodegeneration exacerbated by oxidative stress.