Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice.
Nature neuroscience
Whole-brain genome editing to correct single-base mutations and reduce or reverse behavioral changes in animal models of autism spectrum disorder (ASD) has not yet been achieved. We developed an apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptide-embedded cytosine base editor (AeCBE) system for converting C·G to T·A base pairs. We demonstrate its effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo in mice. We first constructed Mef2cL35P heterozygous mice. Male heterozygous mice exhibited hyperactivity, repetitive behavior and social abnormalities. We then programmed AeCBE to edit the mutated C·G base pairs of Mef2c in the mouse brain through the intravenous injection of blood-brain barrier-crossing adeno-associated virus. This treatment successfully restored Mef2c protein levels in several brain regions and reversed the behavioral abnormalities in Mef2c-mutant mice. Our work presents an in vivo base-editing paradigm that could potentially correct single-base genetic mutations in the brain.
10.1038/s41593-023-01499-x
NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming.
Neuron
The regenerative capacity of neurons is limited in the central nervous system (CNS), with irreversible neuronal loss upon insult. In contrast, microglia exhibit extraordinary capacity for repopulation. Matsuda et al. (2019) recently reported NeuroD1-induced microglia-to-neuron conversion, aiming to provide an "unlimited" source to regenerate neurons. However, the extent to which NeuroD1 can exert cross-lineage reprogramming of microglia (myeloid lineage) to neurons (neuroectodermal lineage) is unclear. In this study, we unexpectedly found that NeuroD1 cannot convert microglia to neurons in mice. Instead, NeuroD1 expression induces microglial cell death. Moreover, lineage tracing reveals non-specific leakage of similar lentiviruses as previously used for microglia-to-neuron conversion, which confounds the microglia-to-neuron observation. In summary, we demonstrated that NeuroD1 cannot induce microglia-to-neuron cross-lineage reprogramming. We here propose rigid principles for verifying glia-to-neuron conversion. This Matters Arising paper is in response to Matsuda et al. (2019), published in Neuron.
10.1016/j.neuron.2021.11.008
Revisiting astrocyte to neuron conversion with lineage tracing in vivo.
Cell
In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.
10.1016/j.cell.2021.09.005
Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice.
Zhou Haibo,Su Jinlin,Hu Xinde,Zhou Changyang,Li He,Chen Zhaorong,Xiao Qingquan,Wang Bo,Wu Wenyan,Sun Yidi,Zhou Yingsi,Tang Cheng,Liu Fei,Wang Linhan,Feng Canbin,Liu Mingzhe,Li Sanlan,Zhang Yifeng,Xu Huatai,Yao Haishan,Shi Linyu,Yang Hui
Cell
Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.
10.1016/j.cell.2020.03.024
In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model.
Guo Ziyuan,Zhang Lei,Wu Zheng,Chen Yuchen,Wang Fan,Chen Gong
Cell stem cell
Loss of neurons after brain injury and in neurodegenerative disease is often accompanied by reactive gliosis and scarring, which are difficult to reverse with existing treatment approaches. Here, we show that reactive glial cells in the cortex of stab-injured or Alzheimer's disease (AD) model mice can be directly reprogrammed into functional neurons in vivo using retroviral expression of a single neural transcription factor, NeuroD1. Following expression of NeuroD1, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and GABAergic neurons. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting that they integrated into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. Our studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide an alternative approach for repair of injured or diseased brain.
10.1016/j.stem.2013.12.001
Generation of Functional Human 3D Cortico-Motor Assembloids.
Andersen Jimena,Revah Omer,Miura Yuki,Thom Nicholas,Amin Neal D,Kelley Kevin W,Singh Mandeep,Chen Xiaoyu,Thete Mayuri Vijay,Walczak Elisabeth M,Vogel Hannes,Fan H Christina,Paşca Sergiu P
Cell
Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.
10.1016/j.cell.2020.11.017
Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes.
Nature biomedical engineering
Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.
10.1038/s41551-022-00963-6
Microglia-organized scar-free spinal cord repair in neonatal mice.
Li Yi,He Xuelian,Kawaguchi Riki,Zhang Yu,Wang Qing,Monavarfeshani Aboozar,Yang Zhiyun,Chen Bo,Shi Zhongju,Meng Huyan,Zhou Songlin,Zhu Junjie,Jacobi Anne,Swarup Vivek,Popovich Phillip G,Geschwind Daniel H,He Zhigang
Nature
Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal-but not adult-microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.
10.1038/s41586-020-2795-6
Reprogramming to recover youthful epigenetic information and restore vision.
Nature
Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity. Changes to DNA methylation patterns over time form the basis of ageing clocks, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.
10.1038/s41586-020-2975-4
Walking naturally after spinal cord injury using a brain-spine interface.
Nature
A spinal cord injury interrupts the communication between the brain and the region of the spinal cord that produces walking, leading to paralysis. Here, we restored this communication with a digital bridge between the brain and spinal cord that enabled an individual with chronic tetraplegia to stand and walk naturally in community settings. This brain-spine interface (BSI) consists of fully implanted recording and stimulation systems that establish a direct link between cortical signals and the analogue modulation of epidural electrical stimulation targeting the spinal cord regions involved in the production of walking. A highly reliable BSI is calibrated within a few minutes. This reliability has remained stable over one year, including during independent use at home. The participant reports that the BSI enables natural control over the movements of his legs to stand, walk, climb stairs and even traverse complex terrains. Moreover, neurorehabilitation supported by the BSI improved neurological recovery. The participant regained the ability to walk with crutches overground even when the BSI was switched off. This digital bridge establishes a framework to restore natural control of movement after paralysis.
10.1038/s41586-023-06094-5
The neurons that restore walking after paralysis.
Nature
A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord applied during neurorehabilitation (EES) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential for a patient to walk after spinal cord injury. To identify these putative neurons, we modelled the technological and therapeutic features underlying EES in mice. We applied single-nucleus RNA sequencing and spatial transcriptomics to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type and spatial prioritization to identify the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons are not required for walking before spinal cord injury, we demonstrate that they are essential for the recovery of walking with EES following spinal cord injury. Augmenting the activity of these neurons phenocopied the recovery of walking enabled by EES, whereas ablating them prevented the recovery of walking that occurs spontaneously after moderate spinal cord injury. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after paralysis. Moreover, our methodology establishes a framework for using molecular cartography to identify the neurons that produce complex behaviours.
10.1038/s41586-022-05385-7