Kazuyuki Takata

Parkinson′s disease is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra that project to the striatum. Postmortem studies have found that α-synuclein pathology spreads across brain regions in a prion-like manner. Human three-dimensional neural tissues provide experimentally tractable platforms for clarifying the molecular pathology of synucleinopathy that overcome the limitations of postmortem brain studies. In this study, we established a human striatal-midbrain assembloid model enabling spatially and temporally controlled α-synuclein induction after formation of the nigrostriatal pathway within hStrMAs. Human induced pluripotent stem cells (hiPSCs) were differentiated into striatal spheroids and midbrain spheroids, which were fused to reconstruct the nigrostriatal pathway in vitro. Histological, electrophysiological, and single-cell RNA sequencing analyses confirmed the generation of region-specific neuronal populations, including striatal GABAergic neurons and midbrain DA neurons, and demonstrated that distinct forebrain and ventral midbrain identities were preserved within assembloids. To model synucleinopathy, we generated a doxycycline-inducible α-synuclein hiPSC line, permitting selective induction of α-synuclein expression in the striatal region after assembloid formation. Spatially restricted α-synuclein induction resulted in the progressive accumulation of phosphorylated and fibrillar α-synuclein in midbrain DA neurons, indicating interregional propagation through established neural circuits. Transcriptomic analysis of the midbrain region revealed significant downregulation of DA identity and synaptic function genes, suggesting functional impairment of DA neurons following α-synuclein propagation. These findings demonstrate that the human neural circuit architecture contributes to the interregional propagation of α-synuclein pathology. Our controllable human assembloid platform provides a powerful experimental framework for dissecting the mechanisms of pathological protein spread and developing therapeutic strategies targeting molecular pathology in neurodegenerative diseases.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ) peptide accumulation, leading to neuroinflammation and neurodegeneration. In early AD stages, neurodegeneration of basal forebrain cholinergic neurons occurs. Microglia, which are brain immune cells, contribute to Aβ clearance and neuroinflammation. This study investigated the therapeutic effects of PNU282987, a selective full agonist of α7 nicotinic acetylcholine receptor (nAChR), using human models of microglia (hiMacs) and basal forebrain cholinergic neurons (hiBFChNs), both differentiated from human induced pluripotent stem cells (hiPSCs). Our findings indicated that PNU282987 markedly enhanced Aβ phagocytosis by microglia and extracellular Aβ clearance. Furthermore, PNU282987 injection reduced Aβ accumulation in the brain of a mouse model. Treatment of hiMacs with PNU282987 upregulated the expressions of efferocytosis-related genes, such as ASAP2, OSM, and THBD. Efferocytosis-like activation by PNU282987 in hiMacs was further suggested by an increased release of the anti-inflammatory cytokine interleukin-10 (IL-10), along with suppression of the pro-inflammatory cytokine IL-1β produced from microglia with Aβ treatment. This indicates a transformation from Aβ-induced inflammatory phagocytosis to an efferocytosis-like anti-inflammatory phagocytosis. PNU282987 also exerted direct neuroprotective effects on hiBFChNs against Aβ and tumor necrosis factor-α. Furthermore, PNU282987 changed the extracellular contents released from Aβ-treated hiMacs and attenuated the neurotoxicity. These results suggest that α7 nAChR stimulation by PNU282987 enhances the therapeutic effects against AD by promoting Aβ clearance with anti-neuroinflammatory regulation in the microglia and providing direct protection to neurons, thereby addressing the inflammatory and neurodegenerative aspects of AD.

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.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-β (Aβ) 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 Aβ oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aβ-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aβ aggregates, including its oligomers, using Aβ oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aβ 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 Aβ injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aβ aggregates including its oligomers and brain tissue from a mouse model of Aβ pathology. In addition, plantainoside B could delay the Aβ aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aβ oligomers, thus interrupting the binding of Aβ oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.