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Traumatic axonal injury (TAI) and the associated axonopathy are common consequences of traumatic brain injury (TBI) and contribute to significant neurological morbidity. It has been previously suggested that TAI activates a highly conserved program of axonal self-destruction known as Wallerian degeneration (WD). In the present study, we utilize our well-established impact acceleration model of TBI (IA-TBI) to characterize the pathology of injured myelinated axons in the white matter tracks traversing the ventral, lateral, and dorsal spinal columns in the mouse and assess the effect of Sterile Alpha and TIR Motif Containing 1 (Sarm1) gene knockout on acute and subacute axonal degeneration and myelin pathology. In silver-stained preparations, we found that IA-TBI results in white matter pathology as well as terminal field degeneration across the rostrocaudal axis of the spinal cord. At the ultrastructural level, we found that traumatic axonopathy is associated with diverse types of axonal and myelin pathology, ranging from focal axoskeletal perturbations and focal disruption of the myelin sheath to axonal fragmentation. Several morphological features such as neurofilament compaction, accumulation of organelles and inclusions, axoskeletal flocculation, myelin degeneration and formation of ovoids are similar to profiles encountered in classical examples of WD. Other profiles such as excess myelin figures and inner tongue evaginations are more typical of chronic neuropathies. Stereological analysis of pathological axonal and myelin profiles in the ventral, lateral, and dorsal columns of the lower cervical cord (C6) segments from wild type and Sarm1 KO mice at 3 and 7 days post IA-TBI (n = 32) revealed an up to 90% reduction in the density of pathological profiles in Sarm1 KO mice after IA-TBI. Protection was evident across all white matter tracts assessed, but showed some variability. Finally, Sarm1 deletion ameliorated the activation of microglia associated with TAI. Our findings demonstrate the presence of severe traumatic axonopathy in multiple ascending and descending long tracts after IA-TBI with features consistent with some chronic axonopathies and models of WD and the across-tract protective effect of Sarm1 deletion.

Diffuse or traumatic axonal injury is one of the principal pathologies encountered in traumatic brain injury (TBI) and the resulting axonal loss, disconnection, and brain atrophy contribute significantly to clinical morbidity and disability. The seminal discovery of the slow Wallerian degeneration mice (Wld) in which transected axons do not degenerate but survive and function independently for weeks has transformed concepts on axonal biology and raised hopes that axonopathies may be amenable to specific therapeutic interventions. Here we review mechanisms of axonal degeneration and also describe how these mechanisms may inform biological therapies of traumatic axonopathy in the context of TBI.In the last decade, SARM1 [sterile a and Toll/interleukin-1 receptor (TIR) motif containing 1] and the DLK (dual leucine zipper bearing kinase) and LZK (leucine zipper kinase) MAPK (mitogen-activated protein kinases) cascade have been established as the key drivers of Wallerian degeneration, a complex program of axonal self-destruction which is activated by a wide range of injurious insults, including insults that may otherwise leave axons structurally robust and potentially salvageable. Detailed studies on animal models and postmortem human brains indicate that this type of partial disruption is the main initial pathology in traumatic axonopathy. At the same time, the molecular dissection of Wallerian degeneration has revealed that the decision that commits axons to degeneration is temporally separated from the time of injury, a window that allows potentially effective pharmacological interventions.Molecular signals initiating and triggering Wallerian degeneration appear to be playing an important role in traumatic axonopathy and recent advances in understanding their nature and significance is opening up new therapeutic opportunities for TBI.

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 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.

Aims Galanin is a highly inducible neuroprotective neuropeptide and in Alzheimer's disease (AD), a network of galaninergic fibres has been reported to hypertrophy and hyperinnervate the surviving cholinergic neurons in the basal forebrain. We aimed to determine (i) the extent of galanin hyperinnervation in patients with AD and Lewy body disease and (ii) whether galanin expression relates to the neuropathological burden and cholinergic losses. Methods Galanin immunohistochemistry was carried out in the anterior nucleus basalis of Meynert of 27 Parkinson's disease (PD) cases without cognitive impairment (mild cognitive impairment [MCI]), 15 with PD with MCI, 42 with Parkinson's disease dementia (PDD), 12 with Dementia with Lewy bodies (DLB), 19 with AD, 12 mixed AD/DLB and 16 controls. Galaninergic innervation of cholinergic neurons was scored semiquantitatively. For a subgroup of cases (n = 60), cholinergic losses were determined from maximum densities of choline acetyltransferase positive (ChAT+ve) neurons and their projection fibres. Quantitative data for α-synuclein, amyloid beta and tau pathology were obtained from tissue microarrays covering cortical/subcortical regions. Results Significant losses of cholinergic neurons and their projection fibres were observed across all diseases. Galaninergic hyperinnervation was infrequent and particularly uncommon in established AD and DLB. We found that hyperinnervation frequencies are significantly higher in the transition between PD without MCI to PDD and that higher burdens of co-existent AD pathology impair this galaninergic response. Conclusions Our results suggest that galanin upregulation represents an intrinsic response early in Lewy body diseases but which fails with increasing burdens of AD related pathology.

Individuals who have sustained a traumatic brain injury (TBI) often report auditory dysfunction including changes to their hearing sensitivity, tinnitus, and hyperacusis. One factor that may have a significant contribution to a person’s post-injury outcomes is whether the TBI occurred in combination with a high intensity noise exposure (e.g., as in the case of a car accident). To date, no study has examined the combined effects of a brain injury and high intensity noise exposure to evaluate whether this would lead to worse overall auditory outcomes than either trauma in isolation. In this study we tested the auditory sensitivity of male and female CBA/CaJ laboratory mice before and 3, 7, 14, 30, 60, and 90 days after acoustic (noise exposure), physical (controlled cortical impact), and combined acoustic and physical injuries. Following completion of the experiment, cochlear and brain tissue was fixed with paraformaldehyde and central and peripheral auditory structures were examined for damage. Our preliminary results show that physical injuries alone cause damage to the auditory sensitivity of mice measured using the auditory brainstem response, reduced acoustic startle responses in quiet and noise, yet hair cells remain intact. Long-term consequences on auditory perception are being tracked in these mice.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington’s disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.