Biology subjects

Sanz-Rodriguez, A.

Publications and source records attributed to Sanz-Rodriguez, A..

5 recordsLinked to original sources

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↗

bioRxiv2025

Shared and distinct microRNA profiles between HT22, N2A and SH-SY5Y cell lines and primary mouse hippocampal neurons

MicroRNAs (miRNA) are small non-coding RNAs that are key negative regulators of gene expression. Their roles include shaping the gene expression landscape during and after brain development by defining and maintaining levels of proteins that generate the distinct morphological and functional properties of neurons and other brain cell types. HT22, N2A, and SH-SY5Y are common immortalized neuronal cell lines that offer simple, less expensive, and time-saving options for in vitro modelling to evaluate miRNA functions. The extent to which these lines reflect primary neurons remains, however, unclear. Here, we benchmarked the miRNA profiles of cultured mouse hippocampal neurons against Argonaute-loaded miRNAs in the adult mouse hippocampus and miRNA data from the hippocampus of patients with drug-resistant temporal lobe epilepsy. We then compared the miRNA expression landscape in HT22, N2A and SH-SY5Y against mouse hippocampal primary cell cultures. We profiled over 700 miRNAs across the lines and detected 310 miRNAs in all four cell types. This included detection of neuron-enriched miRNAs such as miR-124 and miR-128, although the cell lines typically displayed lower levels of these than in primary neurons and reference adult hippocampal tissue. The miRNA profile in the HT22 cell line showed the highest correlation to the mouse primary neuronal cultures. Together, this study provides a dataset on basal miRNA expression across commonly used cell lines for neuroscience research and evidence for both conserved and distinct profiles that should be used to inform decisions on cell lines for modelling brain and miRNA research.

neuroscience↗

bioRxiv2024

EPILEPTOGENESIS INHIBITS THE CIRCADIAN CLOCK AND RESHAPES THE DIURNAL TRANSCRIPTOMIC RHYTHMICITY IN THE MOUSE HIPPOCAMPUS

Epileptogenesis is the process that leads the brain into epileptic activity. Clinical evidence shows that [~]90% of people with epilepsy present rhythmicity in the timing of their seizures presentation. However, whether the circadian clock is a key player during epileptogenesis remains unknown. Here, we triggered epileptogenesis in mice by the intra-amygdala injection of kainic acid and profiled by RNA sequencing their hippocampal diurnal mRNA rhythmicity. We show that epileptogenesis largely reshapes the hippocampal transcriptomic rhythmicity and that the molecular clock machinery is inhibited due to the disruption of the core clock gene Bmal1. We identified relevant dysregulated pathways and their dynamics in epileptogenesis, predicting a key role for microglial-driven neuroinflammation. We predicted the genes that Bmal1 is directly controlling over time. Finally, we sought for translational relevance evidence by performing RNA sequencing in hippocampal samples resected from patients with mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) and cross-analyzing datasets.

neuroscience↗

bioRxiv2022

MicroRNA inhibition using antimiRs in acute human brain tissue sections

IntroductionAn emerging pre-clinical approach for the treatment of pharmacoresistant epilepsy is targeting the microRNA (miRNA) system. MiRNAs are short noncoding RNAs that suppress gene expression at the post-transcriptional level. Targeting miRNAs, which is possible using antisense oligonucleotide antimiRs can produce broad effects on gene expression suited to the complex pathophysiology in temporal lobe epilepsy. Potent anti-seizure and disease- modifying effects have been reported for antimiRs targeting microRNA-134 (antimiR-134). To date, however, pre-clinical testing has been performed using in vitro cell cultures and rodent models. It is uncertain how well this approach will translate to the clinic. Here, we develop an antimiR testing platform in human brain tissue sections. MethodologyHuman brain specimens were obtained with consent from patients undergoing resective surgery to treat focal drug-resistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF), dissected for clinical neuropathological examination, and unused material transferred for sectioning. Individual tissue sections were incubated in oxygenated ACSF, containing either antimiR-134 or a non-targeting control antimiR, for 24 hours at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels measured using RT-qPCR. ResultsACSF transport had no obvious impact on any clinical neurosurgical or neuropathological procedure and specimens were confirmed to be viable following this process. RNA was well- preserved by transportation of specimens in ACSF, with RNA integrity scores significantly higher than tissue transported without ACSF. AntimiR-134 mediated a specific and dose- dependent knockdown of miR-134 in human neocortical sections, with approximately 75% reduction of miR-134 at 1 {micro}M and 90% reduction at 3 {micro}M. These doses did not have off- target effects on expression of a selection of three other miRNAs (miR-10, miR-129 or miR- 132). SignificanceThis is the first demonstration of antimiR-134 effects in live human brain tissues. The findings lend further support to the preclinical development of miR-134 and offer a flexible platform for the pre-clinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain. Key pointsO_LIASO antimiRs are promising treatments for pharmacoresistant epilepsy C_LIO_LIWe developed a pipeline to preserve live human neocortical brain specimens from people undergoing resective surgery C_LIO_LIRNA integrity was sufficient to measure miRNA levels in human brain tissues transported in modified ACSF C_LIO_LIIncubation of acute human neocortical specimens in antimiR-134 resulted in potent and specific reduction in miR-134 levels C_LIO_LIAcute human brain slices are a promising model for testing ASOs C_LI

neuroscience↗

bioRxiv2019

Potent and lasting seizure suppression by systemic delivery of antagomirs targeting miR-134 timed with blood-brain barrier disruption

RNA therapies such as oligonucleotides (OGNs) offer precision treatments for a variety of neurological diseases, including epilepsy but their deployment is hampered by the blood brain barrier (BBB). Here we used brain imaging and assays of serum proteins and tracer extravasation, to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically-injected antisense OGNs targeting microRNA-134 (Ant-134) into the brain parenchyma. A single intraperitoneal injection of Ant-134 two hours after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at three months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces disease-modifying effects after systemic injection in mice when timed with BBB disruption and may be a clinically-viable approach for this and other disease-modifying microRNA therapies.

neuroscience↗