Martin L. Doughty

Traumatic brain injury (TBI) results from a primary injury that impacts the brain in a spatially-dependent manner. Here we investigated the topographical relationship of early transcriptional responses to a single, focal TBI in mice by controlled cortical impact (CCI). Guided by the presence of the anterior commissure (AC) in coronal sections at the rostro-caudal point of impact, we compared gene expression changes in the neocortex (CTX) and corpus callosum-external capsule (CC-EC), striatum (STR) and AC. Injury-induced gene expression changes were detected in the CTX, CC-EC and STR but not AC and were principally segregated based on cytoarchitecture, and secondarily by proximity to the site of impact. Additionally, unbiased spatial clustering revealed a positive relationship between proximity to the impact and the number of acutely differentially expressed genes within the laminar CTX. Gene pathways for interferon gamma response and for leukocyte-mediated migration and immunity were acutely enhanced across the injured CTX, CC-EC and STR. Within 1-week post-injury, transcriptional responses to injury in the CTX and CC-EC included gene pathways for adaptive T- and B-cell mediated immunity, whereas gene expression changes in the STR were largely resolved. Next, we examined the effects of systemic depletion of neutrophils and monocytes on spatial gene expression changes in the injured brain. This led to the upregulation of gene pathways functioning in synaptic transmission and an alternating down- and then upregulation of genes functioning in ribosomal messenger RNA translation and aerobic metabolism in mitochondria. These data suggest infiltrating neutrophils and monocytes play an evolving, multifaceted role in modulating the metabolic, transcriptional, and synaptic activity of brain tissue post-injury.

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by cognitive and language impairments, seizures, reduced or fragmented sleep, motor ataxia, and a characteristic happy affect. AS arises due to the neuronal loss of UBE3A, an E3 ligase that regulates protein abundance through the addition of lysine 48 (K48)-linked polyubiquitin chains to proteins targeted for degradation by the ubiquitin proteasome system (UPS). Using a dual SMAD inhibition protocol to derive cortical neurons from human induced pluripotent stem cells, we examined UBE3A deletion effects on the neuronal proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS identified 645 proteins differentially abundant between UBE3A knockout (KO) and isogenic UBE3A wild-type control cortical neurons. Proteins with increased abundance with UBE3A loss of function include GRIPAP1 and PACSIN1, synaptic proteins implicated in AMPA receptor recycling. We provide evidence UBE3A polyubiquitinates PACSIN1 and GRIPAP1 to regulate protein turnover, with potential implications for impaired activity-dependent synaptic plasticity observed in AS.

Traumatic brain injury is a leading cause of mortality and morbidity in the United States. Acute trauma to the brain triggers chronic secondary injury mechanisms that contribute to long‐term neurological impairment. We have developed a single, unilateral contusion injury model of sensorimotor dysfunction in adult mice. By targeting a topographically defined neurological circuit with a mild impact, we are able to track sustained behavioral deficits in sensorimotor function in the absence of tissue cavitation or neuronal loss in the contused cortex of these mice. Stereological histopathology and multiplex enzyme‐linked immunosorbent assay proteomic screening confirm contusion resulted in chronic gliosis and the robust expression of innate immune cytokines and monocyte attractant chemokines IL‐1β, IL‐5, IL‐6, TNFα, CXCL1, CXCL2, CXCL10, CCL2, and CCL3 in the contused cortex. In contrast, the expression of neuroinflammatory proteins with adaptive immune functions was not significantly modulated by injury. Our data support widespread activation of innate but not adaptive immune responses, confirming an association between sensorimotor dysfunction with innate immune activation in the absence of tissue or neuronal loss in our mice.

Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs). Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1β (interleukin-1 beta). We show TNFα and IL-1β induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1β effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity. Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

The transcriptional programs that drive the generation of diverse GABAergic neuron populations from their common progenitor pools in the developing cerebellum remain unclear. Neurog1 is a pro‐neural basic helix‐loop‐helix transcription factor expressed in GABAergic progenitor cells in the ventricular zone (VZ) of embryos and subsequently in the presumptive white matter (pWM) tracts of developing postnatal mice. Genetic inducible fate‐mapping labels Purkinje cells and all inhibitory interneuron cell types of the cerebellar cortex. As conventional Neurog1^Neo knockout (KO) mice are neonatal lethal, we generated Neurog1^loxP mutant mice to examine the effects of conditional Neurog1 deletion on the postnatal development of the cerebellum. Targeted Neurog1 loss‐of‐function in the developing cerebellum does not result in significant differences in cerebellar morphology or in the number of GABAergic neurons in the cerebellar cortex of mice at postnatal day 21 (P21). To determine the effects of Neurog1 deletion on GABAergic progenitors, we quantified rates of cell proliferation and cell cycle progression or re‐entry in embryonic Neurog1^Neo and postnatal Neurog1^loxP mutants. The data revealed no significant effect of Neurog1 loss‐of‐function on embryonic day 12.5 (E12.5) VZ progenitors or on P5 and P6 progenitors in the pWM at P7. However, 4–5 day pulse‐labeling of P5 and P6 progenitors revealed reductions in inhibitory interneuron dispersal from the pWM to the cerebellar cortex in P10 conditional Neurog1^loxP/loxP KO mice. Thus, our conditional Neurog1 KO approach reveals a requirement for Neurog1 activity in inhibitory interneuron cell dispersal from pWM tracts in the developing cerebellum of postnatal mice.

Human induced pluripotent stem (iPS) cell‐derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell‐derived neural stem cells (NSCs). We first demonstrate human iPS cell‐derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co‐culture on feeder layers of isogenic human iPS cell‐derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor‐channel function and occurred without changes in I_Na, I_K, V_m, and AP properties of iPS cell‐derived neurons. These data demonstrate co‐culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell‐derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell‐derived neurons in culture. We present RNA‐sequencing data for an immature, fetal‐like status of our human iPS cell‐derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co‐culture system.

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions, Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion, notably the up-regulation of reelin (Reln), the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln, and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ.