Biology subjects

Henshall, D.

Publications and source records attributed to Henshall, D..

8 recordsLinked to original sources

bioRxiv2026

P2X7 receptor antagonism potentiates seizure-suppressive effects of anti-seizure medications in human resected epileptic brain tissue

Resistance to anti-seizure medications (ASMs) remains a major clinical challenge in the management of epilepsy. Neuroinflammation has been implicated as a contributing mechanism and, consistent with this, antagonists of the ATP-gated P2X7 receptor (P2X7R) have been shown to enhance ASM efficacy in animal models. It remains unclear, however, if P2X7R antagonism is effective in human models of drug-resistant epilepsy. Here, we assessed the seizure-suppressive potential of the P2X7R antagonists AFC-5128 and JNJ-47965567 using electrophysiological recordings in acute resected brain slices from patients with epilepsy using artificial cerebrospinal fluid containing low Mg2+ and high K+ to evoked seizure-like events. P2X7R antagonists alone did not suppress seizure-like activity. When co-administered, however, P2X7R antagonists significantly enhanced the anti-seizure efficacy of carbamazepine and lorazepam. Notably, P2X7R antagonist treatment lowered Interleukin-1{beta} levels, and betaine, a drug targeting Interleukin-1{beta} release, mimicked the effects of P2X7R antagonists when combined with carbamazepine. Finally, mice with microglia-specific P2X7R depletion showed improved responsiveness to carbamazepine, suggesting P2X7R-mediated effects are partially mediated via their functions in microglia. These findings suggest that P2X7R-based therapies may represent an effective add-on approach for treating drug-resistant seizures associated with neuroinflammation.

neuroscience↗

bioRxiv2025

Transcriptomic profiles from stereo-EEGs reveal the local cell microenvironment in human epilepsy

Objectives: Our understanding of the pathomechanisms of epilepsy has improved through techniques that access the living human brain. We recently reported that explanted stereo-electroencephalography (SEEG) electrodes from patients with epilepsy carry residual biomolecules and cells which may be utilised for transcriptome and DNA methylation profiling. Methods: Here, we applied bioinformatic and other analyses to explore the transcriptomes (RNA sequencing-based) of those SEEG cases to better understand the types of recovered transcripts in terms of representation of genes expressed by different cell types, brain structures, and the extent to which the signal may reflect local epileptiform activity. Results: Electrodes from all clinical cases retained protein-coding transcripts which reflected the local molecular microenvironment as well as epileptiform activity. Expression of genes involved in housekeeping functions as well as markers of neuronal activity were consistent between patients and between the electrode locations within the brain. We detected transcripts representing various cell types and subtypes including excitatory and inhibitory neurons, all major classes of glia, and endothelial cells, as well as transcripts enriched in specific brain regions. Several genes showed a gradient of expression depending on the electrode position within the brain. We found examples of gene expression that correlated with epileptiform activity as recorded by SEEG. Interpretation: These findings extend the evidence that SEEG electrodes reflect the molecular microenvironments of brain activity in patients with epilepsy, both at sites of seizure onset and within the wider seizure network. The approach has potential applications in intraoperative surgical decision-making as well as to identify molecular biomarkers or therapeutic targets for the drug-resistant epilepsies.

neuroscience↗

bioRxiv2025

Attenuated single neuron and network hyperexcitability following microRNA-134 inhibition in mice with drug-resistant temporal lobe epilepsy

The multi-factorial pathophysiology of acquired epilepsies lends itself to a multi-targeting therapeutic approach. MicroRNAs (miRNA) are short noncoding RNAs that individually can negatively regulate dozens of protein-coding transcripts. Previously, we reported that central injection of antisense oligonucleotides targeting microRNA-134 (Ant-134) shortly after status epilepticus potently suppressed the development of recurrent spontaneous seizures in rodent models of temporal lobe epilepsy. The mechanism(s) of these anti-seizure effects remain, however, incompletely understood. Here we show that intracerebroventricular microinjection of Ant-134 in male mice with pre-existing epilepsy caused by intraamygdala kainic acid-induced status epilepticus potently reduces the occurrence of spontaneous seizures. Recordings from ex vivo brain slices collected 2-4 days after Ant-134 injection in epileptic mice, detected a number of electrophysiological phenotypic changes consistent with reduced excitability. Specifically, Ant-134 reduced action potential bursts after current injection in CA1 neurons and reduced miniature excitatory post-synaptic current frequencies in CA1 neurons. Ant-134 also reduced general network excitability, including attenuating pro-excitatory CA1 responses to Schaffer collateral stimulation in hippocampal slices from epileptic mice. Together, the present study demonstrates inhibiting miR-134 reduces single neuron and network hyperexcitability in mice and extends support for this approach to treat drug-resistant epilepsies. Significance statementTemporal lobe epilepsy is one of the most common forms of drug-resistant epilepsy. Identifying molecular regulators of enduring states of hyperexcitability may lead to new therapeutic approaches. MicroRNAs are short noncoding RNAs that act post-transcriptionally to lower levels of sets of protein-coding genes. Here we show that inhibiting miR-134 reduces spontaneous seizures in mice with active epilepsy. Electrophysiologic recordings from brain slices collected when mice were transitioning to fewer seizures revealed changes to both single neuron and inter-regional communication properties that may explain the reduction in hippocampal network excitability. The findings support the development of this microRNA-targeting approach for epilepsy.

neuroscience↗

bioRxiv2024

High-Resolution Multimodal Profiling of Human Epileptic Brain Activity via Explanted Depth Electrodes

Understanding neurological disorders necessitates systems-level approaches that integrate multimodal data, but progress has been hindered by limited sample availability, and the absence of combined electrophysiological and molecular data from live patients. Here, we demonstrate that intracranial stereoelectroencephalography (SEEG) electrodes used for identifying the seizure focus in epilepsy patients enable the integration of RNA sequencing, genomic variants and epigenome maps with in vivo recordings and brain imaging. Specifically, we report a method, MoPEDE (Multimodal Profiling of Epileptic Brain Activity via Explanted Depth Electrodes) that recovers extensive protein-coding transcripts, DNA methylation and mutation profiles from explanted SEEG electrodes matched with electrophysiological and radiological data allowing for high-resolution reconstructions of brain structure and function in human patients. Our study shows that epilepsies of different aetiologies have distinct molecular landscapes and identify transcripts correlating with neurophysiological signals, including immediate early genes, inflammation markers, and axon guidance molecules. Additionally, we identify DNA methylation profiles indicative of transcriptionally permissive or restrictive chromatin states. While gene expression gradients corresponded with the assigned epileptogenicity index, we found outlier molecular fingerprints in some electrodes, potentially indicating seizure generation or propagation zones not detected during electroclinical assessments. These findings validate that RNA profiles, genetic variation and genome-wide epigenetic data from explanted SEEG electrodes offer high-resolution surrogate molecular landscapes of brain activity. Our transformative MoPEDE approach has the potential to enhance diagnostic decisions and deepen our understanding of epileptogenic network processes in the human brain.

genomics↗

bioRxiv2023

Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy.

neuroscience↗

bioRxiv2023

Immunoproteasome deficiency results in accelerated brain aging and epilepsy

The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly inactive in the healthy brain. Here, we show an age-dependent increase in polyubiquitination in the brain of wild-type mice, accompanied with induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout (TKO) mice deficient for LMP2, LMP7 and MECL-1), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged TKO mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, aged TKO mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during aging.

neuroscience↗

bioRxiv2022

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control

There remains an urgent need for new therapies for drug-resistant epilepsy (DRE). Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bi-directional control of sodium channel expression are needed. MicroRNAs (miRNA) are small non-coding RNAs which negatively regulate gene expression. Here, we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the novel anti-seizure medicine cannabidiol (CBD) and plasma from patients with DRE, converge on a single target, miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking of miR-335-5p also increased voltage-gated sodium currents in human iPSC-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazole seizure model, whereas AAV9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect brain excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide new biomarker or therapeutic strategies for different types of drug-resistant epilepsy. Significance StatementDespite the clinical availability of over 30 anti-seizure medications (ASMs), around 30% of people with epilepsy do not achieve seizure freedom. MicroRNAs are small non-coding RNAs which negatively regulate protein expression by binding to target mRNAs. Here, we identified the brain-enriched miR-335-5p to be commonly altered in three heterogenous miRNA profiling datasets. Bi-directional modulation of miR-335-5p identified a potential homeostatic role of miR-335-5p in brain excitability involving voltage-gated sodium channels. Electrophysiological and in vivo approaches revealed pro-epileptic activity of miR-335-5p inhibition whereas overexpression of miR-335-5p resulted in anti-epileptic activity. Overall, targeting miR-335-5p could provide a new approach in the modulation of brain excitability, with possible therapeutic applications in drug-resistant epilepsies and other neurological diseases.

neuroscience↗

bioRxiv2021

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived neurons and astrocytes

The P2X7 receptor (P2X7R) is a cation membrane channel activated by extracellular adenosine 5'-triphosphate. Activation of this receptor results in numerous downstream events including the modulation of neurotransmission, release of pro-inflammatory mediators, cell proliferation or cell death. While the expression of P2X7Rs is well documented on microglia and oligodendrocytes, the presence of functional P2X7Rs on neurons and astrocytes remains debated. Furthermore, to date, functional studies on the P2X7R are mostly limited to studies in cells from rodents and immortalised cell lines expressing human P2X7Rs. To assess the functional expression of P2X7Rs in human neurons and astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into forebrain cortical neurons that co-express FOXG1 and {beta}III-tubulin as well as S100 {beta}-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining on the soma and processes of hiPSC-derived neurons and astrocytes. In addition, our data show that stimulation with the potent nonselective P2X7R agonist BzATP induces robust calcium rises in hiPSC-derived neurons and astrocytes, which were blocked by the selective P2X7R antagonist AFC-5128. Together, our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived forebrain cortical neurons and astrocytes demonstrating that these cells offer the potential for investigating P2X7R-mediated pathophysiology and drug screening in vitro.

neuroscience↗