The role of neuroinflammation in canine epilepsy.

The lack of therapeutics that prevent the development of epilepsy, improve disease prognosis or overcome drug resistance represents an unmet clinical need in veterinary as well as in human medicine. Over the past decade, experimental studies and studies in human epilepsy patients have demonstrated that neuroinflammatory processes are involved in epilepsy development and play a key role in neuronal hyperexcitability that underlies seizure generation. Targeting neuroinflammatory signaling pathways may provide a basis for clinically relevant disease-modification strategies in general, and moreover, could open up new therapeutic avenues for human and veterinary patients with drug-resistant epilepsy. A sound understanding of the neuroinflammatory mechanisms underlying seizure pathogenesis in canine patients is therefore essential for mechanism-based discovery of selective epilepsy therapies that may enable the development of new disease-modifying treatments. In particular, subgroups of canine patients in urgent needs, e.g. dogs with drug-resistant epilepsy, might benefit from more intensive research in this area. Moreover, canine epilepsy shares remarkable similarities in etiology, disease manifestation, and disease progression with human epilepsy. Thus, canine epilepsy is discussed as a translational model for the human disease and epileptic dogs could provide a complementary species for the evaluation of antiepileptic and antiseizure drugs. This review reports key preclinical and clinical findings from experimental research and human medicine supporting the role of neuroinflammation in the pathogenesis of epilepsy. Moreover, the article provides an overview of the current state of knowledge regarding neuroinflammatory processes in canine epilepsy emphasizing the urgent need for further research in this specific field. It also highlights possible functional impact, translational potential and future perspectives of targeting specific inflammatory pathways as disease-modifying and multi-target treatment options for canine epilepsy.

An exploratory approach to identify microRNAs as circulatory biomarker candidates for epilepsy-associated psychiatric comorbidities in an electrical post-status epilepticus model.

Patients with epilepsy have a high risk of developing psychiatric comorbidities, and there is a particular need for early detection of these comorbidities. Here, in an exploratory, hypothesis-generating approach, we aimed to identify microRNAs as potential circulatory biomarkers for epilepsy-associated psychiatric comorbidities across different rat models of epilepsy. The identification of distress-associated biomarkers can also contribute to animal welfare assessment. MicroRNA expression profiles were analyzed in blood samples from the electrical post-status epilepticus (SE) model. Preselected microRNAs were correlated with behavioral and biochemical parameters in the electrical post-SE model, followed by quantitative real-time PCR validation in three additional well-described rat models of epilepsy. Six microRNAs (miR-376a, miR-429, miR-494, miR-697, miR-763, miR-1903) were identified showing a positive correlation with weight gain in the early post-insult phase as well as a negative correlation with social interaction, saccharin preference, and plasma BDNF. Real-time PCR validation confirmed miR-203, miR-429, and miR-712 as differentially expressed with miR-429 being upregulated across epilepsy models. While readouts from the electrical post-SE model suggest different microRNA candidates for psychiatric comorbidities, cross-model analysis argues against generalizability across models. Thus, further research is necessary to compare the predictive validity of rodent epilepsy models for detection and management of psychiatric comorbidities.

Hippocampal and Septal 5-HT1A Receptor Expression in Two Rat Models of Temporal Lobe Epilepsy.

Experimental and clinical data suggest an impact of serotonergic signaling on seizure susceptibility and epilepsy-associated psychiatric comorbidities. Previous µPET studies revealed increased binding of the 5-HT1A receptor ligand [18F]MPPF in two rat models with spontaneous recurrent seizures. These findings raised the question whether these alterations are due to altered 5-HT1A receptor expression or a modification of extracellular serotonin concentrations. 5-HT1A receptor expression rates were quantitatively analyzed in rat brain tissue from an electrical and a chemical post-status epilepticus model. Based on the µPET findings, stereological analysis was focused on hippocampal subregions and the septum. Evaluation of 5-HT1A receptor expression in the electrical post-status epilepticus model revealed a decreased optical density in hippocampal CA3 region. In all other brain regions of interest, the analysis demonstrated comparable 5-HT1A receptor expression rates among all experimental groups in the brain regions evaluated. Moreover, 5-HT1A total receptor volume did not differ between groups. A model-specific correlation was demonstrated between 5-HT1A receptor expression and selected seizure and behavioral parameters. In conclusion, analysis in post-status epilepticus models in rats argued against widespread and pronounced alterations in 5-HT1A receptor expression. In view of previous µPET findings, the present data indicate that alterations in in-vivo receptor binding are due to a reduction in extracellular serotonin concentrations rather than changes in receptor density. Correlation analysis points to a possible link between 5-HT1A receptor expression and ictogenesis, seizure termination and behavioral patterns. However, as these findings proved to be model specific, the relevance needs to be further assessed in future studies focusing on other models and species.

Molecular alterations of the TLR4-signaling cascade in canine epilepsy.

BACKGROUND: Cumulating evidence from rodent models points to a pathophysiological role of inflammatory signaling in the epileptic brain with Toll-like receptor-4 signaling acting as one key factor. However, there is an apparent lack of information about expression alterations affecting this pathway in canine patients with epilepsy. Therefore, we have analyzed the expression pattern of Toll-like receptor 4 and its ligands in brain tissue of canine patients with structural or idiopathic epilepsy in comparison with tissue from laboratory dogs or from owner-kept dogs without neurological diseases. RESULTS: The analysis revealed an overexpression of Toll-like receptor-4 in the CA3 region of dogs with structural epilepsy. Further analysis provided evidence for an upregulation of Toll-like receptor-4 ligands with high mobility group box-1 exhibiting increased expression levels in the CA1 region of dogs with idiopathic and structural epilepsy, and heat shock protein 70 exhibiting increased expression levels in the piriform lobe of dogs with idiopathic epilepsy. In further brain regions, receptor and ligand expression rates proved to be either in the control range or reduced below control levels. CONCLUSIONS: Our study reveals complex molecular alterations affecting the Toll-like receptor signaling cascade, which differ between epilepsy types and between brain regions. Taken together, the data indicate that multi-targeting approaches modulating Toll-like receptor-4 signaling might be of interest for management of canine epilepsy. Further studies are recommended to explore respective molecular alterations in more detail in dogs with different etiologies and to confirm the role of the pro-inflammatory signaling cascade as a putative target.

Profiling the Expression of Endoplasmic Reticulum Stress Associated Heat Shock Proteins in Animal Epilepsy Models.

Unfolded protein response is a signaling cascade triggered by misfolded proteins in the endoplasmic reticulum. Heat shock protein H4 (HSPH4) and A5 (HSPA5) are two chaperoning proteins present within the organelle, which target misfolded peptides during prolonged stress conditions. Epileptogenic insults and epileptic seizures are a notable source of stress on cells. To investigate whether they influence expression of these chaperones, we performed immunohistochemical stainings in brains from rats that experienced a status epilepticus (SE) as a trigger of epileptogenesis and from canine epilepsy patients. Quantification of HSPA5 and HSPH4 revealed alterations in hippocampus and parahippocampal cortex. In rats, SE induced up-regulation of HSPA5 in the piriform cortex and down-regulation of HSPA5 and HSPH4 in the hippocampus. Regionally restricted increases in expression of the two proteins has been observed in the chronic phase with spontaneous recurrent seizures. Confocal microscopy revealed a predominant expression of both proteins in neurons, no expression in microglia and circumscribed expression in astroglia. In canine patients, only up-regulation of HSPH4 expression was observed in Cornu Ammonis 1 region in animals diagnosed with structural epilepsy. This characterization of HSPA5 and HSPH4 expression provided extensive information regarding spatial and temporal alterations of the two proteins during SE-induced epileptogenesis and following epilepsy manifestations. Up-regulation of both proteins implies stress exerted on ER during these disease phases. Taken together suggest a differential impact of epileptogenesis on HSPA5 and HSPH4 expression and indicate them as a possible target for pharmacological modulation of unfolded protein response.

Regulation of Alzheimer's disease-associated proteins during epileptogenesis.

Clinical evidence and pathological studies suggest a bidirectional link between temporal lobe epilepsy and Alzheimer's disease (AD). Data analysis from omic studies offers an excellent opportunity to identify the overlap in molecular alterations between the two pathologies. We have subjected proteomic data sets from a rat model of epileptogenesis to a bioinformatics analysis focused on proteins functionally linked with AD. The data sets have been obtained for hippocampus (HC) and parahippocampal cortex samples collected during the course of epileptogenesis. Our study confirmed a relevant dysregulation of proteins linked with Alzheimer pathogenesis. When comparing the two brain areas, a more prominent regulation was evident in parahippocampal cortex samples as compared to the HC. Dysregulated protein groups comprised those affecting mitochondrial function and calcium homeostasis. Differentially expressed mitochondrial proteins included proteins of the mitochondrial complexes I, III, IV, and V as well as of the accessory subunit of complex I. The analysis also revealed a regulation of the microtubule associated protein Tau in parahippocampal cortex tissue during the latency phase. This was further confirmed by immunohistochemistry. Moreover, we demonstrated a complex epileptogenesis-associated dysregulation of proteins involved in amyloid ß processing and its regulation. Among others, the amyloid precursor protein and the α-secretase alpha disintegrin metalloproteinase 17 were included. Our analysis revealed a relevant regulation of key proteins known to be associated with AD pathogenesis. The analysis provides a comprehensive overview of shared molecular alterations characterizing epilepsy development and manifestation as well as AD development and progression.

Epileptogenesis-Associated Alterations of Heat Shock Protein 70 in a Rat Post-Status Epilepticus Model.

Temporal lobe epilepsy is triggered by an initial insult, such as status epilepticus, that initiates the process of epilepsy development. Heat shock protein 70 (Hsp70) is a ubiquitously expressed molecular chaperone, involved in the inflammatory response that is upregulated after status epilepticus. Hsp70 has been described as an endogenous intracellular ligand of Toll-like receptor 4. It is released from damaged or necrotic tissue and by activated immune cells after an inflammatory event. So far, the time course and the pattern of epileptogenesis-associated alterations in Hsp70 expression have not been described in detail. Thus, we investigated immunohistochemical expression of Hsp70 in hippocampus, parahippocampal cortex, parietal cortex, amygdala, and thalamus following status epilepticus in a rat model of temporal lobe epilepsy. The impact of status epilepticus on Hsp70 expression varied during different phases of epileptogenesis, displaying a stronger effect in the early post-insult phase, a milder and more localized effect in the latency phase and no relevant effect in the chronic phase. Cellular-level characterization revealed that Hsp70 colocalized with the neuronal marker NeuN and with Toll-like receptor 4. No colocalization with the astrocytic marker GFAP or the microglia marker Iba1 was found. The intense neuronal Hsp70 upregulation during the early post-insult phase might contribute to the onset of excessive inflammation triggering molecular and cellular reorganization and generation of a hyperexcitable epileptic network. Therefore, development of multi-targeting strategies aiming at prevention of epileptogenesis should consider Hsp70 modulation in the early days following an epileptogenic insult.

Genetic and Pharmacological Targeting of Heat Shock Protein 70 in the Mouse Amygdala-Kindling Model.

Inflammatory responses involving Toll-like receptor signaling represent a key factor contributing to epileptogenesis. Thus, it is of particular interest to explore the relevance of toll-like receptor ligands and modulators, such as heat shock protein 70 (HSP70). Motivated by recent findings demonstrating an upregulation of HSP70 in a model of epileptogenesis, we analyzed the consequences of genetic and pharmacological targeting of HSP70 expression in a mouse kindling paradigm. Lack of inducible HSP70 resulted in increased prekindling seizure thresholds. However, at threshold stimulation the deficiency-promoted seizure spread, as indicated by an increased seizure severity. Subsequent kindling stimulations elicited more severe seizures in knockout mice, whereas endogenous termination of seizure activity remained unaffected with duration of behavioral and electrographic seizure activity comparable to that of wild-type mice. Interestingly, HSP70 deficiency resulted in enhanced microglia activation in the CA1 region. Next, we assessed a pharmacological targeting approach aiming to promote HSP70 expression. Celastrol treatment had no impact on kindling progression but reduced postkindling seizure thresholds and enhanced microglia activation in CA1 and CA3. In conclusion, the findings from HSP70-knockout mice support a protective role of HSP70 with an effect on microglia activation and spread of seizure activity. Unexpectedly, celastrol administration resulted in detrimental consequences. These findings should be considered as a warning about the general safety of celastrol as a drug candidate. The impact of pathophysiological mechanisms on the quality of celastrol effects requires comprehensive future studies exploring influencing factors. Moreover, alternate strategies to increase HSP70 expression should be further developed and validated.

Genetic Modulation of HSPA1A Accelerates Kindling Progression and Exerts Pro-convulsant Effects.

Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional states can promote ictogenesis and epileptogenesis. So far, research has focused on the role of high-mobility group box protein 1 as an activator of TLRs. However, the development of targeting strategies might need to consider a role of additional receptor ligands. Considering the fact that heat shock protein A1 (hsp70) has been confirmed as a TLR 2 and 4 ligand, we have explored the consequences of its overexpression in a mouse kindling paradigm. The genetic modulation enhanced seizure susceptibility with lowered seizure thresholds prior to kindling. In contrast to wildtype (WT) mice, HSPA1A transgenic (TG) mice exhibited generalized seizures very early during the kindling paradigm. Along with an increased seizure severity, seizure duration proved to be prolonged in TG mice during this phase. Toward the end of the stimulation phase seizure parameters of WT mice reached comparable levels. However, a difference between genotypes was still evident when comparing seizure parameters during the post-kindling threshold determination. Surprisingly, HSPA1A overexpression did not affect microglia activation in the hippocampus. In conclusion, the findings demonstrate that hsp70 can exert pro-convulsant effects promoting ictogenesis in naïve animals. The pronounced impact on the response to subsequent stimulations gives first evidence that genetic HSPA1A upregulation may also contribute to epileptogenesis. Thus, strategies inhibiting hsp70 or its expression might be of interest for prevention of seizures and epilepsy. However, conclusions about a putative pro-epileptogenic effect of hsp70 require further investigations in models with development of spontaneous recurrent seizures.

Proteomic profiling of epileptogenesis in a rat model: Focus on cell stress, extracellular matrix and angiogenesis.

Information about epileptogenesis-associated changes in protein expression patterns is of particular interest for future selection of target and biomarker candidates. Bioinformatic analysis of proteomic data sets can increase our knowledge about molecular alterations characterizing the different phases of epilepsy development following an initial epileptogenic insult. Here, we report findings from a focused analysis of proteomic data obtained for the hippocampus and parahippocampal cortex samples collected during the early post-insult phase, latency phase, and chronic phase of a rat model of epileptogenesis. The study focused on proteins functionally associated with cell stress, cell death, extracellular matrix (ECM) remodeling, cell-ECM interaction, cell-cell interaction, angiogenesis, and blood-brain barrier function. The analysis revealed prominent pathway enrichment providing information about the complex expression alterations of the respective protein groups. In the hippocampus, the number of differentially expressed proteins declined over time during the course of epileptogenesis. In contrast, a peak in the regulation of proteins linked with cell stress and death as well as ECM and cell-cell interaction became evident at later phases during epileptogenesis in the parahippocampal cortex. The data sets provide valuable information about the time course of protein expression patterns during epileptogenesis for a series of proteins. Moreover, the findings provide comprehensive novel information about expression alterations of proteins that have not been discussed yet in the context of epileptogenesis. These for instance include different members of the lamin protein family as well as the fermitin family member 2 (FERMT2). Induction of FERMT2 and other selected proteins, CD18 (ITGB2), CD44 and Nucleolin were confirmed by immunohistochemistry. Taken together, focused bioinformatic analysis of the proteomic data sets completes our knowledge about molecular alterations linked with cell death and cellular plasticity during epileptogenesis. The analysis provided can guide future selection of target and biomarker candidates.

A systems level analysis of epileptogenesis-associated proteome alterations.

Despite intense research efforts, the knowledge about the mechanisms of epileptogenesis and epilepsy is still considered incomplete and limited. However, an in-depth understanding of molecular pathophysiological processes is crucial for the rational selection of innovative biomarkers and target candidates. Here, we subjected proteomic data from different phases of a chronic rat epileptogenesis model to a comprehensive systems level analysis. Weighted Gene Co-expression Network analysis identified several modules of interconnected protein groups reflecting distinct molecular aspects of epileptogenesis in the hippocampus and the parahippocampal cortex. Characterization of these modules did not only further validate the data but also revealed regulation of molecular processes not described previously in the context of epilepsy development. The data sets also provide valuable information about temporal patterns, which should be taken into account for development of preventive strategies in particular when it comes to multi-targeting network pharmacology approaches. In addition, principal component analysis suggests candidate biomarkers, which might inform the design of novel molecular imaging approaches aiming to predict epileptogenesis during different phases or confirm epilepsy manifestation. Further studies are necessary to distinguish between molecular alterations, which correlate with epileptogenesis versus those reflecting a mere consequence of the status epilepticus.

miRNA-187-3p-Mediated Regulation of the KCNK10/TREK-2 Potassium Channel in a Rat Epilepsy Model.

Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.

Proteomic profiling of epileptogenesis in a rat model: Focus on inflammation.

Detailed knowledge about the patterns of molecular alterations during epileptogenesis is a presupposition for identifying targets for preventive or disease-modifying approaches, as well as biomarkers of the disease. Large-scale differential proteome analysis can provide unique and novel perspectives based on comprehensive data sets informing about the complex regulation patterns in the disease proteome. Thus, we have completed an elaborate differential proteome analysis based on label-free LC-MS/MS in a rat model of epileptogenesis. Hippocampus and parahippocampal cortex tissues were sampled and analyzed separately at three key time points chosen for monitoring disease development following electrically-induced status epilepticus, namely, the early post-insult phase, the latency phase, and the chronic phase with spontaneous recurrent seizures. We focused the bioinformatics analysis on proteins linked to immune and inflammatory responses, because of the emerging evidence of the specific pathogenic role of inflammatory signalings during epileptogenesis. In the early post-insult and the latency phases, pathway enrichment analysis revealed an extensive over-representation of Toll-like receptor signaling, pro-inflammatory cytokines, heat shock protein regulation, and transforming growth factor beta signaling and leukocyte transendothelial migration. The inflammatory response in the chronic phase proved to be more moderate with differential expression in the parahippocampal cortex exceeding that in the hippocampus. The data sets provide novel information about numerous differentially expressed proteins, which serve as interaction partners or modulators in key disease-associated inflammatory signaling events. Noteworthy, a set of proteins which act as modulators of the ictogenic Toll-like receptor signaling proved to be differentially expressed. In addition, we report novel data demonstrating the regulation of different Toll-like receptor ligands during epileptogenesis. Taken together, the findings deepen our understanding of modulation of inflammatory signaling during epileptogenesis providing an excellent and comprehensive basis for the identification of target and biomarker candidates.

Microglial ROS production in an electrical rat post-status epilepticus model of epileptogenesis.

Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.

Canine epilepsy as a translational model?

Dogs with spontaneous diseases can exhibit a striking similarity in etiology, clinical manifestation, and disease course when compared to human patients. Therefore, dogs are intensely discussed as a translational model of human disease. In particular, genetic studies in selected dog breeds serve as an excellent tool to identify epilepsy disease genes. In addition, canine epilepsy is discussed as a translational platform for drug testing. On one hand, epileptic dogs might serve as an interesting model by allowing the evaluation of drug efficacy and potency under clinical conditions with a focus on chronic seizures resistant to standard medication, preventive strategies, or status epilepticus. On the other hand, several limitations need to be considered including owner-based seizure monitoring, species differences in pharmacokinetics and drug interactions, as well as cost-intensiveness. The review gives an overview on the current state of knowledge regarding the etiology, clinical manifestation, pathology, and drug response of canine epilepsy, also pointing out the urgent need for further research on specific aspects. Moreover, the putative advantages, the disadvantages, and limitations of antiepileptic drug testing in canine epilepsy are critically discussed.

Comparative analytical utility of DNA derived from alternative human specimens for molecular autopsy and diagnostics.

Genetic testing and research have increased the demand for high-quality DNA that has traditionally been obtained by venipuncture. However, venous blood collection may prove difficult in special populations and when large-scale specimen collection or exchange is prerequisite for international collaborative investigations. Guthrie/FTA card-based blood spots, buccal scrapes, and finger nail clippings are DNA-containing specimens that are uniquely accessible and thus attractive as alternative tissue sources (ATS). The literature details a variety of protocols for extraction of nucleic acids from a singular ATS type, but their utility has not been systematically analyzed in comparison with conventional sources such as venous blood. Additionally, the efficacy of each protocol is often equated with the overall nucleic acid yield but not with the analytical performance of the DNA during mutation detection. Together with a critical in-depth literature review of published extraction methods, we developed and evaluated an all-inclusive approach for serial, systematic, and direct comparison of DNA utility from multiple biological samples. Our results point to the often underappreciated value of these alternative tissue sources and highlight ways to maximize the ATS-derived DNA for optimal quantity, quality, and utility as a function of extraction method. Our comparative analysis clarifies the value of ATS in genomic analysis projects for population-based screening, diagnostics, molecular autopsy, medico-legal investigations, or multi-organ surveys of suspected mosaicisms.