Early life phenobarbital exposure dysregulates the hippocampal transcriptome.

Introduction: Phenobarbital (PB) and levetiracetam (LEV) are the first-line therapies for neonates with diagnosed seizures, however, a growing body of evidence shows that these drugs given during critical developmental windows trigger lasting molecular changes in the brain. While the targets and mechanism of action of these drugs are well understood-what is not known is how these drugs alter the transcriptomic landscape, and therefore molecular profile/gene expression during these critical windows of neurodevelopment. PB is associated with a range of neurotoxic effects in developing animals, from cell death to altered synaptic development to lasting behavioral impairment. LEV does not produce these effects. Methods: Here we evaluated the effects of PB and Lev on the hippocampal transcriptome by RNA sequencing. Neonatal rat pups were given a single dose of PB, Lev or vehicle and sacrificed 72 h later-at time at which drug is expected to be cleared. Results: We found PB induces broad changes in the transcriptomic profile (124 differentially expressed transcripts), as compared to relatively small changes in LEV-treated animals (15 transcripts). PB exposure decreased GABAergic and oligodendrocyte markers pvalb and opalin, and increased the marker of activated microglia, cd68 and the astrocyte- associated gene vegfa. These data are consistent with the existing literature showing developmental neurotoxicity associated with PB, but not LEV. Discussion: The widespread change in gene expression after PB, which affected transcripts reflective of multiple cell types, may provide a link between acute drug administration and lasting drug toxicity.

Slow motor neurons resist pathological TDP-43 and mediate motor recovery in the rNLS8 model of amyotrophic lateral sclerosis.

In the intermediate stages of amyotrophic lateral sclerosis (ALS), surviving motor neurons (MNs) that show intrinsic resistance to TDP-43 proteinopathy can partially compensate for the loss of their more disease-susceptible counterparts. Elucidating the mechanisms of this compensation may reveal approaches for attenuating motor impairment in ALS patients. In the rNLS8 mouse model of ALS-like pathology driven by doxycycline-regulated neuronal expression of human TDP-43 lacking a nuclear localization signal (hTDP-43ΔNLS), slow MNs are more resistant to disease than fast-fatigable (FF) MNs and can mediate recovery following transgene suppression. In the present study, we used a viral tracing strategy to show that these disease-resistant slow MNs sprout to reinnervate motor endplates of adjacent muscle fibers vacated by degenerated FF MNs. Moreover, we found that neuromuscular junctions within fast-twitch skeletal muscle (tibialis anterior, TA) reinnervated by SK3-positive slow MNs acquire resistance to axonal dieback when challenged with a second course of hTDP-43ΔNLS pathology. The selective resistance of reinnervated neuromuscular junctions was specifically induced by the unique pattern of reinnervation following TDP-43-induced neurodegeneration, as recovery from unilateral sciatic nerve crush did not produce motor units resistant to subsequent hTDP-43ΔNLS. Using cross-reinnervation and self-reinnervation surgery in which motor axons are disconnected from their target muscle and reconnected to a new muscle, we show that FF MNs remain hTDP-43ΔNLS-susceptible and slow MNs remain resistant, regardless of which muscle fibers they control. Collectively, these findings demonstrate that MN identity dictates the susceptibility of neuromuscular junctions to TDP-43 pathology and slow MNs can drive recovery of motor systems due to their remarkable resilience to TDP-43-driven degeneration. This study highlights a potential pathway for regaining motor function with ALS pathology in the advent of therapies that halt the underlying neurodegenerative process.

Descending projections from the substantia nigra pars reticulata differentially control seizures.

Three decades of studies have shown that inhibition of the substantia nigra pars reticulata (SNpr) attenuates seizures, yet the circuits mediating this effect remain obscure. SNpr projects to the deep and intermediate layers of the superior colliculus (DLSC) and the pedunculopontine nucleus (PPN), but the contributions of these projections are unknown. To address this gap, we optogenetically silenced cell bodies within SNpr, nigrotectal terminals within DLSC, and nigrotegmental terminals within PPN. Inhibition of cell bodies in SNpr suppressed generalized seizures evoked by pentylenetetrazole (PTZ), partial seizures evoked from the forebrain, absence seizures evoked by gamma-butyrolactone (GBL), and audiogenic seizures in genetically epilepsy-prone rats. Strikingly, these effects were fully recapitulated by silencing nigrotectal projections. By contrast, silencing nigrotegmental terminals reduced only absence seizures and exacerbated seizures evoked by PTZ. These data underscore the broad-spectrum anticonvulsant efficacy of this circuit, and demonstrate that specific efferent projection pathways differentially control different seizure types.

Electroconvulsive Shock Enhances Responsive Motility and Purinergic Currents in Microglia in the Mouse Hippocampus.

Microglia are in a privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. Hippocampal microglia become activated after prolonged, damaging seizures known as status epilepticus (SE). However, since SE causes both hyperactivity and injury of neurons, the mechanisms triggering this activation remain unclear, as does the relevance of the microglial activation to the ensuing epileptogenic processes. In this study, we use electroconvulsive shock (ECS) to study the effect of neuronal hyperactivity without neuronal degeneration on mouse hippocampal microglia. Unlike SE, ECS did not alter hippocampal CA1 microglial density, morphology, or baseline motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices, and similarly upregulated expression of the chemokine C-C motif chemokine ligand 2 (CCL2). Whole-cell patch-clamp recordings of hippocampal CA1sr microglia showed that ECS enhanced purinergic currents mediated by P2X7 receptors in the absence of changes in passive properties or voltage-gated currents, or changes in receptor expression. This differs from previously described alterations in intrinsic characteristics which coincided with enhanced purinergic currents following SE. These ECS-induced effects point to a "seizure signature" in hippocampal microglia characterized by altered purinergic signaling. These data demonstrate that ictal activity per se can drive alterations in microglial physiology without neuronal injury. These physiological changes, which up until now have been associated with prolonged and damaging seizures, are of added interest as they may be relevant to electroconvulsive therapy (ECT), which remains a gold-standard treatment for depression.

Remyelination Pharmacotherapy Investigations Highlight Diverse Mechanisms Underlying Multiple Sclerosis Progression.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by a complex lesion microenvironment. Although much progress has been made in developing immunomodulatory treatments to reduce myelin damage and delay the progression of MS, there is a paucity in treatment options that address the multiple pathophysiological aspects of the disease. Currently available immune-centered therapies are able to reduce the immune-mediated damage exhibited in MS patients, however, they cannot rescue the eventual failure of remyelination or permanent neuronal damage that occurs as MS progresses. Recent advances have provided a better understanding of remyelination processes, specifically oligodendrocyte lineage cell progression following demyelination. Further there have been new findings highlighting various components of the lesion microenvironment that contribute to myelin repair and restored axonal health. In this review we discuss the complexities of myelin repair following immune-mediated damage in the CNS, the contribution of animal models of MS in providing insight on OL progression and myelin repair, and current and potential remyelination-centered therapeutic targets. As remyelination therapies continue to progress into clinical trials, we consider a dual approach targeting the inflammatory microenvironment and intrinsic remyelination mechanisms to be optimal in aiding MS patients.

Reduction of matrix metalloproteinase 9 (MMP-9) protects motor neurons from TDP-43-triggered death in rNLS8 mice.

Therapeutic strategies are needed for the treatment of amyotrophic lateral sclerosis (ALS). One potential target is matrix metalloproteinase-9 (MMP-9), which is expressed only by fast motor neurons (MNs) that are selectively vulnerable to various ALS-relevant triggers. Previous studies have shown that reduction of MMP-9 function delayed motor dysfunction in a mouse model of familial ALS. However, given that the majority of ALS cases are sporadic, we propose preclinical testing in a mouse model which may be more clinically translatable: rNLS8 mice. In rNLS8 mice, neurodegeneration is triggered by the major pathological hallmark of ALS, TDP-43 mislocalization and aggregation. MMP-9 was targeted in 3 different ways in rNLS8 mice: by AAV9-mediated knockdown, using antisense oligonucleotide (ASO) technology, and by genetic modification. All 3 strategies preserved the motor unit during disease, as measured by MN counts, tibialis anterior (TA) muscle innervation, and physiological recordings from muscle. However, the strategies that reduced MMP-9 beyond the motor unit lead to premature deaths in a subset of rNLS8 mice. Therefore, selective targeting of MMP-9 in MNs could be beneficial in ALS, but side effects outside of the motor circuit may limit the most commonly used clinical targeting strategies.

Microglia-mediated recovery from ALS-relevant motor neuron degeneration in a mouse model of TDP-43 proteinopathy.

Though motor neurons selectively degenerate in amyotrophic lateral sclerosis, other cell types are likely involved in this disease. We recently generated rNLS8 mice in which human TDP-43 (hTDP-43) pathology could be reversibly induced in neurons and expected that microglia would contribute to neurodegeneration. However, only subtle microglial changes were detected during disease in the spinal cord, despite progressive motor neuron loss; microglia still reacted to inflammatory triggers in these mice. Notably, after hTDP-43 expression was suppressed, microglia dramatically proliferated and changed their morphology and gene expression profiles. These abundant, reactive microglia selectively cleared neuronal hTDP-43. Finally, when microgliosis was blocked during the early recovery phase using PLX3397, a CSF1R and c-kit inhibitor, rNLS8 mice failed to regain full motor function, revealing an important neuroprotective role for microglia. Therefore, reactive microglia exert neuroprotective functions in this amyotrophic lateral sclerosis model, and definition of the underlying mechanism could point toward novel therapeutic strategies.

Progression of motor neuron disease is accelerated and the ability to recover is compromised with advanced age in rNLS8 mice.

In order to treat progressive paralysis in ALS patients, it is critical to develop a mouse that closely models human ALS in both pathology and also in the timing of these events. We have recently generated new TDP-43 bigenic mice (called rNLS8) with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (hTDP-43∆NLS) under the control of the NEFH promoter. Our previous studies characterized the pathology and disease course in young rNLS8 mice following induction of neuronal hTDP-43ΔNLS. We now seek to examine if the order and timing of pathologic events are changed in aged mice. We found that the expression of hTDP-43∆NLS in 12+ month old mice did not accelerate the appearance of neuromuscular abnormalities or motor neuron (MN) death in the lumbar spinal cord (SC), though disease progression was accelerated. However, following suppression of the transgene, important differences between young and aged rNLS8 mice emerged in functional motor recovery. We found that recovery was incomplete in aged mice relative to their younger treatment matched counterparts based on gross behavioral measures and physiological recordings from the animals' gastrocnemius (GC) muscles, despite muscle reinnervation by surviving MNs. This is likely because the reinnervation most often only resulted in partial nerve and endplate connections and the muscle's junctional folds were much more disorganized in aged rNLS8 mice. We believe that these studies will be an important basis for the future design and evaluation of therapies designed to slow denervation and promote re-innervation in adult ALS patients.