Transcriptomic and phenotypic convergence of neurodevelopmental disorder risk genes in vitro and in vivo

JOURNAL/nrgr/04.03/01300535-202406000-00036/inline-graphic1/v/2025-03-16T163725Z/r/image-tiff We have previously shown the neuroprotective and pro-neurogenic activity of microneurotrophin BNN-20 in the substantia nigra of the “weaver” mouse, a model of progressive nigrostriatal degeneration. Here, we extended our investigation in two clinically-relevant ways. First, we assessed the effects of BNN-20 on human induced pluripotent stem cell-derived neural progenitor cells and neurons derived from healthy and parkinsonian donors. Second, we assessed if BNN-20 can boost the outcome of mouse neural progenitor cell intranigral transplantations in weaver mice, at late stages of degeneration. We found that BNN-20 has limited direct effects on cultured human induced pluripotent stem cell-derived neural progenitor cells, marginally enhancing their differentiation towards neurons and partially reversing the pathological phenotype of dopaminergic neurons generated from parkinsonian donors. In agreement, we found no effects of BNN-20 on the mouse neural progenitor cells grafted in the substantia nigra of weaver mice. However, the graft strongly induced an endogenous neurogenic response throughout the midbrain, which was significantly enhanced by the administration of microneurotrophin BNN-20. Our results provide straightforward evidence of the existence of an endogenous midbrain neurogenic system that can be specifically strengthened by BNN-20. Interestingly, the lack of major similar activity on cultured human induced pluripotent stem cell-derived neural progenitors and their progeny reveals the in vivo specificity of the aforementioned pro-neurogenic effect.

Differentiation, polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.

An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells

Over three hundred and seventy-three risk genes, broadly enriched for roles in neuronal communication and gene expression regulation, underlie risk for autism spectrum disorder (ASD) and developmental delay (DD). Functional genomic studies of subsets of these genes consistently indicate a convergent role in neurogenesis, but how these diverse risk genes converge on a smaller number of biological pathways in mature neurons is unclear. To uncover shared downstream impacts between neurodevelopmental disorder (NDD) risk genes, here we apply a pooled CRISPR approach to contrast the transcriptomic impacts of targeting 29 NDD loss-of-function genes across human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Points of convergence vary between the cell types of the brain and are greatest in mature glutamatergic neurons, where they broadly target not just synaptic and epigenetic, but unexpectedly, mitochondrial biology. The strongest convergent networks occur between NDD genes with common co-expression patterns in the post-mortem brain, biological annotations, and clinical associations, suggesting that convergence may one-day inform patient stratification and treatment. Towards this, ten out of eleven drugs tested that were predicted to reverse convergent signatures in human cells and/or arousal and sensory processing behaviors in zebrafish ameliorated at least one behavioral phenotype in vivo. Altogether, robust convergence in post-mitotic neurons represents a clinically actionable therapeutic window.

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

Protocol for the generation and xenotransplantation of human induced pluripotent stem cell-derived neural progenitor cells into the mouse forebrain.

Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

Intracerebral hemorrhage (ICH) induces secondary brain injury, driven in part by oxidative stress caused by hemin, a toxic hemoglobin breakdown product. Cortical astrocytes, critical for maintaining redox homeostasis, are vulnerable to hemin-induced oxidative damage, exacerbating neuronal injury. Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) have shown therapeutic potential in brain injury models by promoting neural repair, but their ability to protect astrocytes against hemin toxicity remains unexplored. We hypothesized that iPSC-NPCs coculture mitigates hemin-induced oxidative stress in astrocytes by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway. Astrocytes were exposed to hemin with or without iPSC-NPC coculture. We assessed cell viability, reactive oxygen species (ROS) accumulation, apoptosis, and the role of the Nrf2/HO-1 pathway using small-interfering RNA. iPSC-NPC coculture significantly reduced hemin-induced oxidative damage by promoting Nrf2 nuclear translocation and upregulating HO-1, thereby decreasing ROS and apoptosis. Silencing Nrf2 abolished these protective effects. Our findings demonstrate that iPSC-NPCs protect astrocytes from hemin toxicity via the Nrf2/HO-1 pathway, suggesting a novel therapeutic strategy for ICH-induced oxidative injury.

Cell transplantation therapy provides a regenerative strategy for neural repair. We tested the hypothesis that selective excitation of transplanted induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) could recapitulate an activity-enriched microenvironment that confers regenerative benefits for the treatment of stroke. Mouse iPS-NPCs were transduced with a novel optochemogenetics fusion protein, luminopsin 3 (LMO3), which consisted of a bioluminescent luciferase, Gaussia luciferase, and an opsin, Volvox Channelrhodopsin 1. These LMO3-iPS-NPCs can be activated by either photostimulation using light or by the luciferase substrate coelenterazine (CTZ). In vitro stimulations of LMO3-iPS-NPCs increased expression of synapsin-1, postsynaptic density 95, brain derived neurotrophic factor (BDNF), and stromal cell-derived factor 1 and promoted neurite outgrowth. After transplantation into the ischemic cortex of mice, LMO3-iPS-NPCs differentiated into mature neurons. Synapse formation between implanted and host neurons was identified using immunogold electron microscopy and patch-clamp recordings. Stimulation of transplanted cells with daily intranasal administration of CTZ enhanced axonal myelination, synaptic transmission, improved thalamocortical connectivity, and functional recovery. Patch-clamp and multielectrode array recordings in brain slices showed that CTZ or light stimulation facilitated synaptic transmission and induced neuroplasticity mimicking the LTP of EPSPs. Stroke mice received the combined LMO3-iPS-NPC/CTZ treatment, but not cell or CTZ alone, showed enhanced neural network connections in the peri-infarct region, promoted optimal functional recoveries after stroke in male and female, young and aged mice. Thus, excitation of transplanted cells via the noninvasive optochemogenetics treatment provides a novel integrative cell therapy with comprehensive regenerative benefits after stroke.SIGNIFICANCE STATEMENT Neural network reconnection is critical for repairing damaged brain. Strategies that promote this repair are expected to improve functional outcomes. This study pioneers the generation and application of an optochemogenetics approach in stem cell transplantation therapy after stroke for optimal neural repair and functional recovery. Using induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) expressing the novel optochemogenetic probe luminopsin (LMO3), and intranasally delivered luciferase substrate coelenterazine, we show enhanced regenerative properties of LMO3-iPS-NPCs in vitro and after transplantation into the ischemic brain of different genders and ages. The noninvasive repeated coelenterazine stimulation of transplanted cells is feasible for clinical applications. The synergetic effects of the combinatorial cell therapy may have significant impacts on regenerative approach for treatments of CNS injuries.

Induced pluripotent stem cell-derived neural progenitor cell transplantation promotes regeneration and functional recovery after post-traumatic stress disorder in rats

Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes

Nanowire arrays used as cell culture substrates build a potent tool for advanced biological applications such as cargo delivery and biosensing. The unique topography of nanowire arrays, however, renders them a challenging growth environment for cells and explains why only basic cell lines have been employed in existing studies. Here, we present the culturing of human induced pluripotent stem cell-derived neural progenitor cells on rectangularly arranged nanowire arrays: In detail, we mapped the impact on proliferation, viability, and topography-induced membrane deformation across a multitude of array pitches (1, 3, 5, 10 μm) and nanowire lengths (1.5, 3, 5 μm). Against the intuitive expectation, a reduced proliferation was found on the arrays with the smallest array pitch of 1 μm and long NWs. Typically, cells settle in a fakir-like state on such densely-spaced nanowires and thus experience no substantial stress caused by nanowires indenting the cell membrane. However, imaging of F-actin showed a distinct reorganization of the cytoskeleton along the nanowire tips in the case of small array pitches interfering with regular proliferation. For larger pitches, the cell numbers depend on the NW lengths but proliferation generally continued although heavy deformations of the cell membrane were observed caused by the encapsulation of the nanowires. Moreover, we noticed a strong interaction of the nanowires with the nucleus in terms of squeezing and indenting. Remarkably, the cell viability is maintained at about 85% despite the massive deformation of the cells. Considering the enormous potential of human induced stem cells to study neurodegenerative diseases and the high cellular viability combined with a strong interaction with nanowire arrays, we believe that our results pave the way to apply nanowire arrays to human stem cells for future applications in stem cell research and regenerative medicine.

The in vivo timeline of differentiation of engrafted human neural progenitor cells

Neural rosettes (NPC rosettes) are radially arranged groups of cells surrounding a central lumen that arise stochastically in monolayer cultures of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPC). Since NPC rosette formation is thought to mimic cell behavior in the early neural tube, these rosettes represent important in vitro models for the study of neural tube morphogenesis. However, using current protocols, NPC rosette formation is not synchronized and results are inconsistent among different hPSC lines, hindering quantitative mechanistic analyses and challenging live cell imaging. Here, we report a rapid and robust protocol to induce rosette formation within 6 h after evenly-sized “colonies” of NPC are generated through physical cutting of uniformly polarized NESTIN+/PAX6+/PAX3+/DACH1+ NPC monolayers. These NPC rosettes show apically polarized lumens studded with primary cilia. Using this assay, we demonstrate reduced lumenal size in the absence of PODXL, an important apical determinant recently identified as a candidate gene for juvenile Parkinsonism. Interestingly, time lapse imaging reveals that, in addition to radial organization and apical lumen formation, cells within cut NPC colonies initiate rapid basally-driven spreading. Further, using chemical, genetic and biomechanical tools, we show that NPC rosette morphogenesis requires this basal spreading activity and that spreading is tightly regulated by Rho/ROCK signaling. This robust and quantitative NPC rosette platform provides a sensitive system for the further investigation of cellular and molecular mechanisms underlying NPC rosette morphogenesis.