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Neural stem cells (NSCs) differentiate into both neurons and glia, and strategies using human NSCs have the potential to restore function following spinal cord injury (SCI). However, the time period of maturation for human NSCs in adult injured CNS is not well defined, posing fundamental questions about the design and implementation of NSC-based therapies. This work assessed human H9 NSCs that were implanted into sites of SCI in immunodeficient rats over a period of 1.5 years. Notably, grafts showed evidence of continued maturation over the entire assessment period. Markers of neuronal maturity were first expressed 3 months after grafting. However, neurogenesis, neuronal pruning, and neuronal enlargement continued over the next year, while total graft size remained stable over time. Axons emerged early from grafts in very high numbers, and half of these projections persisted by 1.5 years. Mature astrocyte markers first appeared after 6 months, while more mature oligodendrocyte markers were not present until 1 year after grafting. Astrocytes slowly migrated from grafts. Notably, functional recovery began more than 1 year after grafting. Thus, human NSCs retain an intrinsic human rate of maturation, despite implantation into the injured rodent spinal cord, yet they support delayed functional recovery, a finding of great importance in planning human clinical trials.

Biological and engineering strategies for neural repair and recovery from neurotrauma continue to emerge at a rapid pace. Until recently, studies of the impact of neurotrauma and repair strategies on the reorganization of the central nervous system have focused on broadly defined circuits and pathways. Optogenetic modulation and recording methods now enable the interrogation of precisely defined neuronal populations in the brain and spinal cord, allowing unprecedented precision in electrophysiological and behavioral experiments. This mini-review summarizes the spectrum of light-based tools that are currently available to probe the properties and functions of well-defined neuronal subpopulations in the context of neurotrauma. In particular, we highlight the challenges to implement these tools in damaged and reorganizing tissues, and we discuss best practices to overcome these obstacles.

Recent advances in the development of in-body bioelectronic systems are providing new opportunities for the clinical management of various diseases and disorders. These emerging technologies are tailored to specific organs and are beginning to blend both diagnostic sensing and therapeutic actuation. The aim of these systems is to seamlessly integrate with the physiological environment, as illustrated by the diverse device strategies discussed throughout this article. Next generation modalities, such as optogenetics combining gene therapy with devices for photostimulation, are gaining popularity and offer advantages over existing therapeutic strategies. In this perspective, we explore the current state of technological developments, key challenges in the field and potential pathways for translating these innovations into clinical practice.

Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid comigration in diverse organisms, but direct evidence for such fusion has been lacking. We used laser trapping and quantitative fluorescence microscopy to study native kinetochore particles isolated from yeast. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I–specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid comigration, a conserved feature of meiosis that is fundamental to Mendelian inheritance.

The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C(Mif2) protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

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 1 – 3 applied during neurorehabilitation 4 , 5 (EES REHAB ) 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 REHAB in mice. We applied single-nucleus RNA sequencing 6 – 9 and spatial transcriptomics 10 , 11 to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type 12 , 13 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 REHAB , 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. Transcriptomic analysis following epidural electrical stimulation of the lumbar spinal cord during neurorehabilitation in mice identifies a population of neurons that orchestrates the restoration of walking following paralysis.

Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

Neural progenitor cell/neural stem cell (NPC/NSC) grafts integrate into sites of spinal cord injury (SCI) and show anatomical and electrophysiological evidence of forming neuronal relays across lesions. To determine how signals may be propagated through grafts, we performed ex vivo and in vivo calcium imaging of NPC grafts and host spinal cord neurons in mice. Following optogenetic stimulation of corticospinal tract axon terminals ex vivo, we detected consistent calcium responses throughout grafts. Grafts exhibited spontaneous activity in both independently-firing neurons and emergent neuronal networks, which were also activated by corticospinal stimulation. These patterns of activation resembled those present in intact spinal cord. In turn, optogenetic stimulation of graft axons extending out of lesion sites triggered excitatory post-synaptic responses in caudal host spinal cord neurons. Finally, distinct sensory stimuli elicited responses in the dorsal aspect of grafts in vivo. Thus, NPC/NSC grafts form local networks that are activated by host inputs and can activate host neurons on the distal side of spinal cord lesions.In order to properly assess the potential benefit of neural stem cell grafts in humans, we sought to understand the rate at which human grafts mature in the injury site. We grafted human NSCs into sites of SCI in immunodeficient rats and assessed anatomical and functional outcomes over a 1.5-year period. Although mature neuron markers appeared 3 months after grafting; neurogenesis, neuronal pruning, and neuronal soma enlargement progressed over the next year. Graft size remained stable as axons emerged in large numbers early on, with half of these projections persisting after 1.5 years. Astrocytes expressed mature markers after 6 months, while oligodendrocytes did not display mature markers until 1 year after grafting. A slow migration of astrocytes from graft sites into host tissue was observed. Importantly, functional improvement did not occur until over 1 year after grafting. Thus, human NSCs mature at their intrinsic rate even when grafted into a rodent environment, and they support functional recovery only at this delayed timepoint, a key finding in human clinical trial planning.

The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-CMif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

Autonomic dysreflexia is a life-threatening medical condition characterized by episodes of uncontrolled hypertension that occur in response to sensory stimuli after spinal cord injury (SCI)1–7. The fragmented understanding of the mechanisms underlying autonomic dysreflexia hampers the development of therapeutic strategies to manage this condition, leaving people with SCI at daily risk of heart attack and stroke8–18. Here, we expose the complete de novo neuronal architecture that develops after SCI and causes autonomic dysreflexia. In parallel, we uncover a competing, yet overlapping neuronal architecture activated by epidural electrical stimulation of the spinal cord that safely regulates blood pressure after SCI. The discovery that these adversarial neuronal architectures converge onto a single neuronal subpopulation provided a blueprint for the design of a mechanism-based intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI. These results establish a path for the effective treatment of autonomic dysreflexia in people with SCI.