Dynamic miRNA changes during the process of epileptogenesis in an infantile and adult-onset model

Temporal lobe epilepsy (TLE) is the most common epilepsy type. TLE onset in infancy aggravates features like severity, drug responsiveness, or development of comorbidities. These aggravations may arise from altered micro RNA (miRNA) expression specific to the early onset of the disease. Although the miRNA involvement in TLE is widely studied, the relationship between the onset-age and miRNA expression has not been addressed. Here, we investigated the miRNA profile of infantile and adult-onset TLE in rats combining sequencing and PCR. Since miRNA expression changes with the disease progression, we scrutinized miRNA dynamics across three stages: acute, latent, and chronic. We report that infantile-onset TLE leads to changes in the expression of fewer miRNAs across these stages. Interestingly, the miRNA profile in the acute stage of infantile-onset TLE overlaps in dysregulation of miR-132-5p, -205, and -211-3p with the chronic stage of the disease starting in adulthood. The analysis of putative targets linked the majority of dysregulated miRNAs with pathways involved in epilepsy. Our profiling uncovered miRNA expression characteristic for infantile and adulthood-onset epileptogenesis, suggesting the distinct biology underlying TLE in the onset age-dependent matter. Our results indicate the necessity of addressing the onset age as an important parameter in future epilepsy research.

monitoring and housed in standard cages in stable pairs for the whole study. The majority of animals assigned for a threemonth interval experienced highly similar seizures, comprising of bilateral forelimb clonus with or without rearing and falling. Seizure frequency per day in individual animals is summarized in Supplementary figure 1. One animal with multiple generalized tonic-clinic seizures (N12 Supplementary figure 1) and one without seizures (N6 not included in the figure) during the monitoring period were excluded from analysis prior to MPS as potential outliers. One animal was euthanized during the monitoring due to the tumor.
For experiments with rat pups, dams with litters were transferred from breeding facilities at P9-10. At P11 animals were randomly assigned a code that allowed for their individual history to be followed throughout the entire study and animals were randomly divided into two groups (control and SE; n=5 each) and injected with LiCl (for details see below). For each time point, two litters composed of 5 controls and 5 SE animals were used. On the day of the experiment, pups were separated from their dams and transferred into a silent room with controlled conditions. The mean weight at P12 was 29.6±4.1g. Experiments were always performed in the same period of the day, between 10 am and 2 pm (i.e. during the light period). Animals were placed individually into small containers made of transparent plastics and they were maintained at +33±1 o C with a Physiological-Biological Temperature Controller (TMP-5b; Supertech; Hungary) to compensate for the immature thermoregulation at this age (Conklin and Heggeness, 1971) during the entire period of separation from their mothers. SE was induced with a single injection of pilocarpine (for details see below). After 1.5 hours of convulsive SE, animals were given a single dose of paraldehyde (for details see below), and approximately 30 min later they were injected subcutaneously with 0.5 ml saline to restore the volume loss. After the brief recovery, pups were returned to their dams (the duration of isolation from mothers in the control and SE groups was the same approximately 4 hours). The body weight of pups was checked daily and animals that did not gain any weight within 24 hours were given 0.5ml of saline subcutaneously to prevent dehydration. SE induction -Animals in both age groups were injected intraperitoneally with LiCl (3 mmol/ml/kg; # L-0505, Sigma Chemical Co., St. Louis, MO) 24 hours prior to intraperitoneal injection pilocarpine (35 mg/ml/kg in P12 and 45 mg/ml/kg in adult animals; # P-6503, Sigma Chemical Co.) (Hirsch et al., 1992). The manifestation of the first clonic motor seizures was considered to be the beginning of SE. To decrease mortality, a single dose of paraldehyde (0.07 ml/kg for P12 rats and 0.6 ml/kg for adult animals; # 76260, Fluka Chemie AG, Buchs, Switzerland) was injected intraperitoneally 1.5 hours after the onset of SE. Control animals in both age groups were treated with equal doses of LiCl and paraldehyde, but the pilocarpine solution was replaced with saline.
The severity of motor SE was assessed using the following scoring system: 0normal behavior 1stereotypic behavior (face washing, scratching), isolated myoclonic jerks 2 -head bobbing, pivoting, swimming movements 3clonic seizures with preserved righting reflex 4repeated episodes of wild running 5generalized tonic clonic seizures with loss of righting reflex.
Animals were assigned a score for the most severe behavior observed. Latency to the onset of motor seizures was recorded. Mortality was recorded throughout the entire experimental period. Only rats that exhibited behavioral manifestations of seizures progressing to forelimbs clonus (i.e., score 3) for at least 1 h and without periods of wild running and generalized tonic clonic seizures (score [4][5] were used for further studies. In acute (24 hours) and latent period (7 days after SE), spontaneous motor seizures (Racine 3-5) were occasionally observed during animal care in all P60 and more than 60% of P12 rats. Most P12 rats exhibited seizures in the first 3-4 days after SE. This observation is concordant with previous reports of seizures occurring during the so-called latent stage of epileptogenesis, which is typically considered as a seizure-free period lasting 1-6 weeks in rats with pilocarpine-induced TLE 1 . Hence, the term "latent stage" (in our case 7 days after SE) should be conceived as the early stage of TLE accompanied by pathological transformations with the possibility of occasional seizure occurrence, rather than truly seizure-free period 2 .
Animals were sacrificed by decapitation under deep anesthesia 24 hours, 1 week and 3 months after SE, brains were quickly removed, both hippocampi were dissected and frozen in dry ice. Animals assigned to 3 months interval were video-monitored 24/7 for 1 week before scarifying with IP infrared Camera Edimax IC-3140W for wireless monitoring.
Synology Surveillance Station 7 software was used for both registration and evaluation. Recordings were evaluated manually by an experienced observer. The incidence of motor seizures (Racine stage 3-5) was registered. EEG monitoring was not included in this study due to technical limitation rendering long term EEG impossible in rat pups (maternal care, skull growth, and ossification) and to prevent bias arising from the effect of anesthetics on the brain (especially during development) and inflammation induced by electrode implantation 3,4 .
Table S1Asignificantly dysregulated miRNAs in miR-Seq -adults -miRNAs identified by DESeq2 or limma as significantly altered in animals after status epilepticus within each stage of epileptogenesis respectively. The list contains all miRNAs with fold-change above 1.4 and p-value <0.05 that reached the threshold of 500 reads in a given stage in adulthood epilepsy-onset group.  Table S1Bsignificantly dysregulated miRNAs in miR-Seq -infants -miRNAs identified by DESeq2 or limma as significantly altered in animals after status epilepticus within each stage of epileptogenesis respectively. List contains all miRNAs with fold-change above 1.4 and p-value <0.05 that reached the threshold of 500 reads in a given stage of infantile-onset epilepsy group. miRNA p-value p-adj FC p-value p-adj FC miRNA p-value p-adj FC p-value p-adj FC miRNA p-value p-adj FC p-value p-adj FC miR-1247-5p miR-365-3p miR-205 mir-451-5p miR-20a-5p miR-6215 miR-211-5p miR-7a-2-3p miR-212-3p miR-212-5p miR-21-5p miR-221-3p miR-221-5p miR-222-3p miR-223-3p miR-23a-3p miR-24-2-5p miR-27a-3p miR-298-5p miR-29b-5p miR-339-3p miR-344b-1-3p miR-3473 miR-34b-3p miR-34c-3p miR-34c-5p miR-361-3p miR-365-3p miR-376b-3p miR-504 miR-7b Table S3 predicted targets of miRNAs included in the validation group -Brain physiology, function and development-related predicted targets of miRNAs identified with altered regulation by miRNA sequencing. miRNA targets were selected from a list produced by the Target Search tool (http://mirdb.org/cgi-bin/search.cgi) with a target score above 90 for all miRNAs included in the qPCR validation group. Validated targets of these miRNAs based on strong experimental evidence listed in miRTarbase (http://mirtarbase.cuhk.edu.cn) are underlined. miRNAs validated by the qPCR are displayed in bold font.   The figure shows the number of motor seizures detected in individual animals with adult-onset TLE over 7 day period of video-monitoring 3 months after induction of status epilepticus (prior to brain tissue collection). Animals (n = 13) were allocated numbers 2 to 14 based on the camera channels used for their monitoring. One animal was euthanized due to the tumor and one animal did not have any motor seizures during the monitoring period (N10 and N6, not displayed). Animal N12 had repeated generalized tonic-clonic seizures (GTCS). Animals N6,10 and 12 were excluded from further analysis. Figure S2 interactome maps of predicted targets -brain physiology, function and development-related predicted targets of miRNAs identified with altered regulation by miRNA sequencing. Displayed genes linked with individual miRNAs were identified as putative targets by Target Search tool (http://mirdb.org/index.html) with a score over 90 or as validated brain-expressed targets listed in miRTarbase. Interactome maps display miRNA targets separately for each stage of epileptogenesis in adult-(A-acute; B -latent and C-chronic) and infantile-(D-acute; and E-chronic) onset TLE in rats. Line thickness represents miRNA expression fold change between TLE and control rats, while colours indicate up-(red) or downregulation (blue) in post-SE animals.

A-Acute stage of adult-onset TLE
TLE