Background

Brain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously found that neurons respond to prolonged BDNF exposure (both in vivo (in models of epilepsy and TBI) and in vitro (in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory cytokines in non-neuronal cells.

Results

Here, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is specifically regulated by agents that inhibit JAK/STAT signaling. Surprisingly, the BDNF-induced JAK/STAT transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this dataset has revealed a unique non-canonical mechanism of JAK/STATs in neurons as differential gene expression mediated by STAT3 is not solely dependent upon phosphorylation at residue 705 and may involve a BDNF-induced interaction of STAT3 with Heterochromatin Protein 1 alpha (HP1α).

Conclusions

These findings suggest that the neuronal BDNF-induced JAK/STAT pathway involves more than STAT3 phosphorylation at 705, providing the first evidence for a non-canonical mechanism that may involve HP1α. Our analysis reveals that JAK/STAT signaling regulates many of the genes associated with epilepsy syndromes where BDNF levels are markedly elevated. Uncovering the mechanism of this novel form of BDNF signaling in the brain may provide a new direction for epilepsy therapeutics and open a window into the complex mechanisms of STAT3 transcriptional regulation in neurological disease.

While the exact role of β1 subunit-containing GABA-A receptors (GABARs) in brain function is not well understood, altered expression of the β1 subunit gene (GABRB1) is associated with neurological and neuropsychiatric disorders. In particular, down-regulation of β1 subunit levels is observed in brains of patients with epilepsy, autism, bipolar disorder and schizophrenia. A pathophysiological feature of these disease states is imbalance in energy metabolism and mitochondrial dysfunction. The transcription factor, nuclear respiratory factor 1 (NRF-1), has been shown to be a key mediator of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Using a variety of molecular approaches (including mobility shift, promoter/reporter assays, and overexpression of dominant negative NRF-1), we now report that NRF-1 regulates transcription of GABRB1 and that its core promoter contains a conserved canonical NRF-1 element responsible for sequence specific binding and transcriptional activation. Our identification of GABRB1 as a new target for NRF-1 in neurons suggests that genes coding for inhibitory neurotransmission may be coupled to cellular metabolism. This is especially meaningful as binding of NRF-1 to its element is sensitive to the kind of epigenetic changes that occur in multiple disorders associated with altered brain inhibition.

Synaptic inhibition in the adult brain is primarily mediated by the γ-aminobutyric acid (GABA) type A receptor (GABA(A)R). The distribution, properties, and dynamics of these receptors are largely determined by their subunit composition. Alteration of subunit composition after a traumatic brain injury (TBI) may result in abnormal increased synaptic firing and possibly contribute to injury-related pathology. Several studies have shown that the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway can alter GABA(A)R subunit expression. The present study investigated changes in JAK/STAT pathway activation after two different severities of experimental TBI in the mouse using the controlled cortical impact (CCI) model. It also investigated whether modulating the activation of the JAK/STAT pathway after severe controlled cortical impact (CCI-S) with a JAK/STAT inhibitor (WP1066) alters post-traumatic epilepsy development and/or neurological recovery after injury. Our results demonstrated differential changes in both the activation of STAT3 and the expression of the GABA(A)R α1 and γ2 subunit levels that were dependent on the severity of the injury. The change in the GABA(A)R α1 subunit levels appeared to be at least partly transcriptionally mediated. We were able to selectively reverse the decrease in GABA(A)R α1 protein levels with WP1066 treatment after CCI injury. WP1066 treatment also improved the degree of recovery of vestibular motor function after injury. These findings suggest that the magnitude of JAK/STAT pathway activation and GABA(A)R α1 subunit level decrease is dependent on injury severity in this mouse model of TBI. In addition, reducing JAK/STAT pathway activation after severe experimental TBI reverses the decrease in the GABA(A)R α1 protein levels and improves vestibular motor recovery.

Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABAA receptor α1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

Objective

Temporal lobe epilepsy (TLE) is frequently medically intractable and often progressive. Compromised inhibitory neurotransmission due to altered γ-aminobutyric acid (GABA)A receptor α4 subunit (GABAA Rα4) expression has been emphasized as a potential contributor to the initial development of epilepsy following a brain insult (primary epileptogenesis), but the regulation of GABAA Rα4 during chronic epilepsy, specifically, how expression is altered following spontaneous seizures, is less well understood.

Methods

Continuous video-electroencephalography (EEG) recordings from rats with pilocarpine-induced TLE were used to capture epileptic animals within 3 h of a spontaneous seizure (SS), or >24 h after the last SS, to determine whether recent occurrence of a seizure was associated with altered levels of GABAA Rα4 expression. We further evaluated whether this GABAA Rα4 plasticity is regulated by signaling mechanisms active in primary epileptogenesis, specifically, increases in brain-derived neurotrophic factor (BDNF) and early growth response factor 3 (Egr3).

Results

Elevated levels of GABAA Rα4 messenger RNA (mRNA) and protein were observed following spontaneous seizures, and were associated with higher levels of BDNF and Egr3 mRNA.

Significance

These data suggest that spontaneous, recurrent seizures that define chronic epilepsy may influence changes in GABAA Rα4 expression, and that signaling pathways known to regulate GABAA Rα4 expression after status epilepticus may also be activated after spontaneous seizures in chronically epileptic animals.

RNA binding proteins are a diverse class of proteins that regulate all aspects of RNA metabolism. Accumulating studies indicate that heterogeneous nuclear ribonucleoproteins are associated with cellular adaptations in response to drugs of abuse. We recently mapped and validated heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) as a quantitative trait gene underlying differential behavioral sensitivity to methamphetamine. The molecular mechanisms by which hnRNP H1 alters methamphetamine behaviors are unknown but could involve pre- and/or post-synaptic changes in protein localization and function. Methamphetamine initiates post-synaptic D1 dopamine receptor signaling indirectly by binding to pre-synaptic dopamine transporters and vesicular monoamine transporters of midbrain dopaminergic neurons which triggers reverse transport and accumulation of dopamine at the synapse. Here, we examined changes in neuronal localization of hnRNP H in primary rat cortical neurons that express dopamine receptors that can be modulated by the D1 or D2 dopamine receptor agonists SKF38393 and (-)-Quinpirole HCl, respectively. Basal immunostaining of hnRNP H was localized primarily to the nucleus. D1 dopamine receptor activation induced an increase in hnRNP H nuclear immunostaining as detected by immunocytochemistry with a C-domain directed antibody containing epitope near the glycine-rich domain but not with an N-domain specific antibody. Although there was no change in hnRNP H protein in the nucleus or cytoplasm, there was a decrease in Hnrnph1 transcript following D1 receptor stimulation. Taken together, these results suggest that D1 receptor activation increases availability of the hnRNP H C-terminal epitope, which could potentially reflect changes in protein-protein interactions. Thus, D1 receptor signaling could represent a key molecular post-synaptic event linking Hnrnph1 polymorphisms to drug-induced behavior.

Objective

Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; a more detailed understanding of JAK/STAT participation in epileptogenic responses is required, however, to increase the therapeutic efficacy and reduce adverse effects associated with global inhibition.

Methods

We developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wild-type and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Hippocampal RNA was obtained 24 h after IHKA and subjected to deep sequencing.

Results

Selective STAT3 knock-out in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. Gene expression was rescued in major networks associated with response to brain injury, neuronal plasticity, and learning and memory. We also provide the first evidence that neuronal STAT3 may directly influence brain inflammation.

Interpretation

Inhibiting neuronal STAT3 signaling improved outcomes in an animal model of TLE, prevented progression of seizures and cognitive co-morbidities while rescuing pathogenic changes in gene expression of major networks associated with epileptogenesis. Specifically targeting neuronal STAT3 may be an effective disease-modifying strategy for TLE. ANN NEUROL 2023;94:106-122.