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

A Behavioural, Molecular and Lesion Examination of Hippocampal Contributions to Morris Water Maze Acquisition

The Morris water maze (MWM) is a widely known, simple and effective task in the examination of spatial learning and memory. Successful acquisition of the task is thought to rely on retained representations of allocentric spatial relations, whereby animals learn to associate the location of a hidden platform with surrounding distal cues and subsequently use this information to navigate towards the hidden goal. As the distal cues are critical in this process, features of the cues, such as location, are an important factor to consider in examining how the task is solved. It has also been well documented that the hippocampus is a critical structure in the processing of allocentric representations. However, there has been debate surrounding the exact nature of this involvement, with suggestions that hippocampal damage leads to deficiencies in navigational aspects of the task rather than purely spatial processing impairments. To assess this, we adopted novel methods of analyses which include sub-second monitoring of each individual animal’s behaviour as they navigate during a training trial. From this analysis we initially determine that positioning of the distal cues around the maze can impact on intact animals’ performance. Specifically, we noted that animals with cues positioned close to their goal are more efficient in reaching the target and use more view-dependent strategies, over animals whose cues are in a position further away, who, instead, are more reliant on view-independent behaviours in order to reach their goal. Molecular examinations of both groups of animals reveal higher BDNF expression in the dorsal hippocampus in the group whose cues are positioned further away from their goal, which we suggest reflects the Far cue groups need to infer their position more than the Near cue group. Following this, assessment of animal behaviour following lesions to the dorsal hippocampus indicated that both the Near and Far lesioned groups were significantly impaired in the MWM. Behavioural analysis highlighted lesioned animals’ deficits in accurately monitoring and adapting their motor movements in response to task demands, suggesting that the impairments seen in the maze are due deficits in integrating exploratory behaviours, rather than a purely spatial memory impairment. While there were few differences in performance of the Near and Far lesioned animals, further assessment of the intact hippocampus using immunohistochemical procedures revealed increased c-Fos expression in the Far cue group in area CA1 of the hippocampus. Further to this, subregional assessment using lesion and IEG methodologies led to the distinction that the dentate gyrus, in particular, is critical in performance in the water maze. Together, the behavioural, molecular and lesion data assessing hippocampal contributions to acquisition of the MWM are discussed in terms of models of navigation. From this, we suggest that the water maze task is solved using a vectormodel of navigation, rather than the widely reported, and accepted, cognitive mapping theory of spatial learning. The behavioural lesion data also supports a role for the hippocampus in this model, specifically as lesioned animals’ display clear impairments in the accurate judgement of distance and direction to their goal when in the maze; a critical feature of the vector-mode

The effects of overtraining in the Morris water maze on allocentric and egocentric learning strategies in rats

Animals can use both allocentric and egocentric strategies to learn a spatial task. Our results suggest that allocentric cues are more dominant than idiothetic cues in guiding navigation. Animals do not necessarily learn an egocentric strategy automatically, instead they probably hold just one solution to any particular task at a time until forced to learn an alternative strategy. Further, with overtraining animals do not always switch from allocentric to an egocentric learning strategy perhaps challenging suggestions of a stored hierarchy of strategies

Altered biogenesis and microRNA content of hippocampal exosomes following experimental status epilepticus.

Repetitive or prolonged seizures (status epilepticus) can damage neurons within the hippocampus, trigger gliosis, and generate an enduring state of hyperexcitability. Recent studies have suggested that microvesicles including exosomes are released from brain cells following stimulation and tissue injury, conveying contents between cells including microRNAs (miRNAs). Here, we characterized the effects of experimental status epilepticus on the expression of exosome biosynthesis components and analyzed miRNA content in exosome-enriched fractions. Status epilepticus induced by unilateral intra-amygdala kainic acid in mice resulted in acute subfield-specific, bi-directional changes in hippocampal transcripts associated with exosome biosynthesis including up-regulation of endosomal sorting complexes required for transport (ESCRT)-dependent and -independent pathways. Increased expression of exosome components including Alix were detectable in samples obtained 2 weeks after status epilepticus and changes occurred in both the ipsilateral and contralateral hippocampus. RNA sequencing of exosome-enriched fractions prepared using two different techniques detected a rich diversity of conserved miRNAs and showed that status epilepticus selectively alters miRNA contents. We also characterized editing sites of the exosome-enriched miRNAs and found six exosome-enriched miRNAs that were adenosine-to-inosine (ADAR) edited with the majority of the editing events predicted to occur within miRNA seed regions. However, the prevalence of these editing events was not altered by status epilepticus. These studies demonstrate that status epilepticus alters the exosome pathway and its miRNA content, but not editing patterns. Further functional studies will be needed to determine if these changes have pathophysiological significance for epileptogenesis

RNA sequencing of synaptic and cytoplasmic Upf1-bound transcripts supports contribution of nonsense-mediated decay to epileptogenesis.

The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis.</p

Data_Sheet_1_Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.PDF

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.</p