Posttraumatic epilepsy: Integrating clinical, inflammatory, and genetic profiles in traumatic brain injury patients.

OBJECTIVE: This study aims to assess the clinical, inflammatory, and genetic profiles of traumatic brain injury (TBI) patients over a 2-year follow-up period, focusing on the development of posttraumatic epilepsy (PTE). METHODS: Fifty-nine patients with acute TBI were recruited in the emergency unit of a hospital in Brazil. Clinical data and blood samples were collected after 10 days of hospitalization for posterior genetic profile (Apolipoprotein E- ApoE and Glutamic Acid Descarboxylase-GAD sequencing) analyses. A subset of 19 patients were assessed for cytokine markers (mRNA expression). The development of PTE was investigated for two years following TBI. Statistical analyses including univariate analysis, multiple correspondence analysis, and Mann-Whitney test were performed. RESULTS: Analysis revealed an association between severe TBI and requirement for neurosurgery and polytrauma (p<0.05), as well as the development of PTE over a two-year follow-up period (p<0.05). Multiple correspondence analysis identified two distinct profiles associated with PTE and Non-PTE outcomes. The PTE profile showed a higher prevalence of the ApoE genotype E3/E3 and GAD1 SNP (rs769391) genotype AA in our study, while the Non-PTE profile showed a higher presence of E3/E4. mRNA expression analysis demonstrated acute elevated levels of TNF-α in the PTE group as compared to Non-PTE patients (6.70±1.53 vs 5.31 ±0.33, p<0.01). SIGNIFICANCE: Our findings underscore the multifactorial nature of aspects potentially contributing to PTE. It is unlikely that any single factor might in isolation have a strong causative influence over the development of epilepsy after TBI. Our results provide a suggestion of potential clustering that might be relevant as prognostic factors for PTE.

Pathological remodeling of reactive astrocytes: Involvement of DNA methylation and downregulation of homeostatic genes.

Astrocytes provide metabolic support to neurons, maintain ionic and water homeostasis, and uptake and recycle neurotransmitters. After exposure to the prototypical PAMP lipopolysaccharide (LPS), reactive astrocytes increase the expression of pro-inflammatory genes, facilitating neurodegeneration. In this study, we analyzed the expression of homeostatic genes in astrocytes exposed to LPS and identified the epigenetic factors contributing to the suppression of homeostatic genes in reactive astrocytes. Primary astrocytic cultures were acutely exposed to LPS and allowed to recover for 24, 72 h, and 7 days. As expected, LPS exposure induced reactive astrogliosis and increased the expression of pro-inflammatory IL-1B and IL-6. Interestingly, the acute exposure resulted in persistent hypermethylation of astroglial DNA. Similar hypermethylation was observed in highly reactive astrocytes from the traumatic brain injury (TBI) penumbra in vivo. Hypermethylation was accompanied by decreased expression of homeostatic genes including LDHA and Scl16a1 (MCT1) both involved in the lactate shuttle to neurons; glutamine synthase (GS) responsible for glutamate processing; Kcnj10 (Kir4.1) important for K+ homeostasis, and the water channel aquaporin-4 (Aqp4). Furthermore, the master regulator of DNA methylation, MAFG-1, as well as DNA methyl transferases DNMT1 and DNMT3a were overexpressed. The downregulation of homeostatic genes correlated with increased methylation of CpG islands in their promoters, as assessed by methylation-sensitive PCR and increased DNMT3a binding to the GS promoter. Treatment with decitabine, a DNMT inhibitor, prevented the LPS- and the HMGB-1-induced downregulation of homeostatic genes. Decitabine treatment also prevented the neurotoxic effects of these astrocytes in primary cortical cultures. In summary, our findings reveal that the pathological remodeling of reactive astrocytes encompasses not only the pro-inflammatory response but, significantly, also entails a long-term suppression of homeostatic gene expression with methylation of crucial CpG islands within their promoters.

miR-9-5p is Downregulated in Serum Extracellular Vesicles of Patients Treated with Biperiden After Traumatic Brain Injury.

Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood-brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10 days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with three subjects developing PTE, and 19 received placebo, with two developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings indicate that biperiden may alter miR-9-5p expression in serum EVs, which may play a role in TBI resolution.

Astrocyte-derived exosomal lncRNA 4933431K23Rik modulates microglial phenotype and improves post-traumatic recovery via SMAD7 regulation.

Astrocyte-microglial interaction plays a crucial role in brain injury-associated neuroinflammation. Our previous data illustrated that astrocytes secrete microRNA, leading to anti-inflammatory effects on microglia. Long non-coding RNAs participate in neuroinflammation regulation after traumatic brain injury. However, the effect of astrocytes on microglial phenotype via long non-coding RNAs and the underlying molecular mechanisms remain elusive. We used long non-coding RNA sequencing on murine astrocytes and found that exosomal long non-coding RNA 4933431K23Rik attenuated traumatic brain injury-induced microglial activation in vitro and in vivo and ameliorated cognitive function deficiency. Furthermore, microRNA and messenger RNA sequencing together with binding prediction illustrated that exosomal long non-coding RNA 4933431K23Rik up-regulates E2F7 and TFAP2C expression by sponging miR-10a-5p. Additionally, E2F7 and TFAP2C, as transcription factors, regulated microglial Smad7 expression. Using Cx3cr1-Smad7 overexpression of adeno-associated virus, microglia specifically overexpressed Smad7 in the attenuation of neuroinflammation, resulting in less cognitive deficiency after traumatic brain injury. Mechanically, overexpressed Smad7 physically binds to IκBα and inhibits its ubiquitination, preventing NF-κB signaling activation. The Smad7 activator asiaticoside alleviates neuroinflammation and protects neuronal function in traumatic brain injury mice. This study revealed that an exosomal long non-coding RNA from astrocytes attenuates microglial activation after traumatic brain injury by up-regulating Smad7, providing a potential therapeutic target.

Safe and neuroprotective vectors for long-term traumatic brain injury gene therapy.

Traumatic brain injury (TBI) is a complex and progressive brain injury with no approved treatments that needs both short- and long-term therapeutic strategies to cope with the variety of physiopathological mechanisms involved. In particular, neuroinflammation is a key process modulating TBI outcome, and the potentiation of these mechanisms by pro-inflammatory gene therapy vectors could contribute to the injury progression. Here, we evaluate in the controlled cortical impact model of TBI, the safety of integrative-deficient lentiviral vectors (IDLVs) or the non-viral HNRK recombinant modular protein/DNA nanovector. These two promising vectors display different tropisms, transduction efficiencies, short- or long-term transduction or inflammatory activation profile. We show that the brain intraparenchymal injection of these vectors overexpressing green fluorescent protein after a CCI is not neurotoxic, and interestingly, can decrease the short-term sensory neurological deficits, and diminish the brain tissue loss at 90 days post lesion (dpl). Moreover, only IDLVs were able to mitigate the memory deficits elicited by a CCI. These vectors did not alter the microglial or astroglial reactivity at 90 dpl, suggesting that they do not potentiate the on-going neuroinflammation. Taken together, these data suggest that both types of vectors could be interesting tools for the design of gene therapy strategies targeting immediate or long-term neuropathological mechanisms of TBI.

COMT and ANKK1 Genetics Interact With Depression to Influence Behavior Following Severe TBI: An Initial Assessment.

OBJECTIVE: Genetic variations in the dopamine (DA) system are associated with cortical-striatal behavior in multiple populations. This study assessed associations of functional polymorphisms in the ankyrin repeat and kinase domain (ANKK1; Taq1a) and catechol-O-methyltransferase (COMT; Val158Met) genes with behavioral dysfunction following traumatic brain injury (TBI). PARTICIPANTS: This was a prospective study of 90 survivors of severe TBI recruited from a level 1 trauma center. MAIN MEASURES: The Frontal Systems Behavior Scale, a self- or family report questionnaire evaluating behavior associated with frontal lobe dysfunction, was completed 6 and 12 months postinjury. Depression was measured concurrently with the Patient Health Questionnaire-9. Study participants were genotyped for Val158Met and Taq1a polymorphisms. RESULTS: No statistically significant behavioral differences were observed by Taq1a or Val158Met genotype alone. At 12 months, among those with depression, Met homozygotes (Val158Met) self-reported worse behavior than Val carriers (P = .015), and A2 homozygotes (Taq1a) self-reported worse behavior than A1 carriers (P = .028) in bivariable analysis. Multivariable models suggest an interaction between depression and genetic variation with behavior at 12 months post-TBI, and descriptive analysis suggests that carriage of both risk alleles may contribute to worse behavioral performance than carriage of either risk allele alone. CONCLUSION: In the context of depression, Val158Met and Taq1a polymorphisms are individually associated with behavioral dysfunction 12 months following severe TBI, with preliminary evidence suggesting cumulative, or perhaps epistatic, effects of COMT and ANKK1 on behavioral dysfunction.