Targeted disruption of dual leucine zipper kinase and leucine zipper kinase promotes neuronal survival in a model of diffuse traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown. METHODS: Here we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI. RESULTS: Our findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival. CONCLUSION: The mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI.

Optogenetically transduced human ES cell-derived neural progenitors and their neuronal progenies: Phenotypic characterization and responses to optical stimulation.

Optogenetically engineered human neural progenitors (hNPs) are viewed as promising tools in regenerative neuroscience because they allow the testing of the ability of hNPs to integrate within nervous system of an appropriate host not only structurally, but also functionally based on the responses of their differentiated progenies to light. Here, we transduced H9 embryonic stem cell-derived hNPs with a lentivirus harboring human channelrhodopsin (hChR2) and differentiated them into a forebrain lineage. We extensively characterized the fate and optogenetic functionality of hChR2-hNPs in vitro with electrophysiology and immunocytochemistry. We also explored whether the in vivo phenotype of ChR2-hNPs conforms to in vitro observations by grafting them into the frontal neocortex of rodents and analyzing their survival and neuronal differentiation. Human ChR2-hNPs acquired neuronal phenotypes (TUJ1, MAP2, SMI-312, and synapsin 1 immunoreactivity) in vitro after an average of 70 days of coculturing with CD1 astrocytes and progressively displayed both inhibitory and excitatory neurotransmitter signatures by immunocytochemistry and whole-cell patch clamp recording. Three months after transplantation into motor cortex of naïve or injured mice, 60-70% of hChR2-hNPs at the transplantation site expressed TUJ1 and had neuronal cytologies, whereas 60% of cells also expressed ChR2. Transplant-derived neurons extended axons through major commissural and descending tracts and issued synaptophysin+ terminals in the claustrum, endopiriform area, and corresponding insular and piriform cortices. There was no apparent difference in engraftment, differentiation, or connectivity patterns between injured and sham subjects. Same trends were observed in a second rodent host, i.e. rat, where we employed longer survival times and found that the majority of grafted hChR2-hNPs differentiated into GABAergic neurons that established dense terminal fields and innervated mostly dendritic profiles in host cortical neurons. In physiological experiments, human ChR2+ neurons in culture generated spontaneous action potentials (APs) 100-170 days into differentiation and their firing activity was consistently driven by optical stimulation. Stimulation generated glutamatergic and GABAergic postsynaptic activity in neighboring ChR2- cells, evidence that hChR2-hNP-derived neurons had established functional synaptic connections with other neurons in culture. Light stimulation of hChR2-hNP transplants in vivo generated complicated results, in part because of the variable response of the transplants themselves. Our findings show that we can successfully derive hNPs with optogenetic properties that are fully transferrable to their differentiated neuronal progenies. We also show that these progenies have substantial neurotransmitter plasticity in vitro, whereas in vivo they mostly differentiate into inhibitory GABAergic neurons. Furthermore, neurons derived from hNPs have the capacity of establishing functional synapses with postsynaptic neurons in vitro, but this outcome is technically challenging to explore in vivo. We propose that optogenetically endowed hNPs hold great promise as tools to explore de novo circuit formation in the brain and, in the future, perhaps launch a new generation of neuromodulatory therapies.

Primary Traumatic Axonopathy in Mice Subjected to Impact Acceleration: A Reappraisal of Pathology and Mechanisms with High-Resolution Anatomical Methods.

Traumatic axonal injury (TAI) is a common neuropathology in traumatic brain injury and is featured by primary injury to axons. Here, we generated TAI with impact acceleration of the head in male Thy1-eYFP-H transgenic mice in which specific populations of neurons and their axons are labeled with yellow fluorescent protein. This model results in axonal lesions in multiple axonal tracts along with blood-brain barrier disruption and neuroinflammation. The corticospinal tract, a prototypical long tract, is severely affected and is the focus of this study. Using optimized CLARITY at single-axon resolution, we visualized the entire corticospinal tract volume from the pons to the cervical spinal cord in 3D and counted the total number of axonal lesions and their progression over time. Our results divulged the presence of progressive traumatic axonopathy that was maximal at the pyramidal decussation. The perikarya of injured corticospinal neurons atrophied, but there was no evidence of neuronal cell death. We also used CLARITY at single-axon resolution to explore the role of the NMNAT2-SARM1 axonal self-destruction pathway in traumatic axonopathy. When we interfered with this pathway by genetically ablating SARM1 or by pharmacological strategies designed to increase levels of Nicotinamide (Nam), a feedback inhibitor of SARM1, we found a significant reduction in the number of axonal lesions early after injury. Our findings show that high-resolution neuroanatomical strategies reveal important features of TAI with biological implications, especially the progressive axonopathic nature of TAI and the role of the NMNAT2-SARM1 pathway in the early stages of axonopathy.SIGNIFICANCE STATEMENT In the first systematic application of novel high-resolution neuroanatomical tools in neuropathology, we combined CLARITY with 2-photon microscopy, optimized for detection of single axonal lesions, to reconstruct the injured mouse brainstem in a model of traumatic axonal injury (TAI) that is a common pathology associated with traumatic brain injury. The 3D reconstruction of the corticospinal tract at single-axon resolution allowed for a more advanced level of qualitative and quantitative understanding of TAI. Using this model, we showed that TAI is an axonopathy with a prominent role of the NMNAT2-SARM1 molecular pathway, that is also implicated in peripheral neuropathy. Our results indicate that high-resolution anatomical models of TAI afford a level of detail and sensitivity that is ideal for testing novel molecular and biomechanical hypotheses.