The transcriptional response of cortical neurons to concussion reveals divergent fates after injury.

Traumatic brain injury (TBI) is a risk factor for neurodegeneration, however little is known about how different neuron types respond to this kind of injury. In this study, we follow neuronal populations over several months after a single mild TBI (mTBI) to assess long ranging consequences of injury at the level of single, transcriptionally defined neuronal classes. We find that the stress responsive Activating Transcription Factor 3 (ATF3) defines a population of cortical neurons after mTBI. We show that neurons that activate ATF3 upregulate stress-related genes while repressing many genes, including commonly used markers for these cell types. Using an inducible reporter linked to ATF3, we genetically mark damaged cells to track them over time. Notably, we find that a population in layer V undergoes cell death acutely after injury, while another in layer II/III survives long term and retains the ability to fire action potentials. To investigate the mechanism controlling layer V neuron death, we genetically silenced candidate stress response pathways. We found that the axon injury responsive kinase MAP3K12, also known as dual leucine zipper kinase (DLK), is required for the layer V neuron death. This work provides a rationale for targeting the DLK signaling pathway as a therapeutic intervention for traumatic brain injury. Beyond this, our novel approach to track neurons after a mild, subclinical injury can inform our understanding of neuronal susceptibility to repeated impacts.

DLK-dependent mitochondrial fission drives axon degeneration and neuronal cell death.

Currently there are no effective treatments for an array of neurodegenerative disorders to a large part because cell-based models fail to recapitulate disease. Here we developed a robust human iPSCbased model where laser axotomy causes retrograde axon degeneration leading to neuronal cell death. Time-lapse confocal imaging revealed that damage triggers a wave of mitochondrial fission proceeding from the site of injury to the soma. We demonstrated that mitochondrial fission and resultant cell death is entirely dependent on phosphorylation of dynamin related protein 1 (DRP1) by dual leucine zipper kinase (DLK). Importantly, we show that CRISPR mediated Drp1 depletion protected mouse retinal ganglion neurons from mitochondrial fission and degeneration after optic nerve crush. Our results provide a powerful platform for studying degeneration of human neurons, pinpoint key early events in damage related neural death and new focus for therapeutic intervention.

A cellular taxonomy of the adult human spinal cord.

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation. We identified 29 glial clusters and 35 neuronal clusters, organized principally by anatomical location. To demonstrate the relevance of this resource to human disease, we analyzed spinal motoneurons, which degenerate in amyotrophic lateral sclerosis (ALS) and other diseases. We found that compared with other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, suggesting a specialized molecular repertoire underlying their selective vulnerability. We include a web resource to facilitate further investigations into human spinal cord biology.

Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord.

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

A harmonized atlas of mouse spinal cord cell types and their spatial organization.

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

Poor Corticospinal Motor Neuron Health Is Associated with Increased Symptom Severity in the Acute Phase Following Repetitive Mild TBI and Predicts Early ALS Onset in Genetically Predisposed Rodents.

Traumatic brain injury (TBI) is a well-established risk factor for several neurodegenerative disorders including Alzheimer's disease and Parkinson's disease, however, a link between TBI and amyotrophic lateral sclerosis (ALS) has not been clearly elucidated. Using the SOD1G93A rat model known to recapitulate the human ALS condition, we found that exposure to mild, repetitive TBI lead ALS rats to experience earlier disease onset and shortened survival relative to their sham counterparts. Importantly, increased severity of early injury symptoms prior to the onset of ALS disease symptoms was linked to poor health of corticospinal motor neurons and predicted worsened outcome later in life. Whereas ALS rats with only mild behavioral injury deficits exhibited no observable changes in corticospinal motor neuron health and did not present with early onset or shortened survival, those with more severe injury-related deficits exhibited alterations in corticospinal motor neuron health and presented with significantly earlier onset and shortened lifespan. While these studies do not imply that TBI causes ALS, we provide experimental evidence that head injury is a risk factor for earlier disease onset in a genetically predisposed ALS population and is associated with poor health of corticospinal motor neurons.