**1. Introduction**

Status epilepticus (SE) is a condition which shows prolonged and uncontrolled seizure activity [1]. One of the most remarkable SE-induced consequences is a massive neuronal death, which triggers long-term and profound alterations in the neuronal network that lead to the development of temporal lobe epilepsy (TLE) [2–4]. The neuronal death pattern and susceptibility to SE shows the regional specific heterogeneity: Neurons in the hilus region of the dentate gyrus, such as mossy cells and hilus interneurons (in particular parvalbumin (PV) interneurons), are the most vulnerable to SE insults, while dentate granule cells are less vulnerable. Furthermore, SE induces programmed necrotic CA1 neuronal death, while it evokes apoptosis in the hilus region [5–10]. Therefore, information regarding the molecular events underlying neuron-specific vulnerability may be one of the therapeutic strategies for neuroprotection against SE, which inhibits undesirable output including epileptogenesis.

Mitochondria are dynamic organelles of eukaryotic cells responsible for generating ATP. In addition, mitochondria participate in the synthesis of reactive oxygen species (ROS), cell homeostasis and

calcium bu ffering. In the process of supplying ATP, mitochondria produce ROS through the electron transport chain, which induces oxidative stress. ROS production and oxidative damage induced by neuronal insults rapidly change mitochondrial morphologies, although mitochondria-derived ROS act as homeostatic signaling molecules in various physiological processes [11–15]. To exert their functions properly, mitochondria change their morphologies by two continuous antagonistic processes: fusion and fission (mitochondrial dynamics) [11]. As vital determinants of the fission-fusion balance, various proteins share reciprocal relationships [12]. Briefly, mitochondrial fusion (elongation) is regulated by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), while fission (fragmentation) is modulated by dynamin-related proteins 1 (DRP1) [11–13]. Thus, perturbation of mitochondrial dynamics and the altered expressions/activities of regulatory enzymes lead to neurodegeneration. In particular, the post-translational modification of DRP1 is focused on various neurodegenerative disorders. DRP1 activity is reversely regulated by phosphorylation of serine (S) 616 and 637 sites: DRP1-S616 phosphorylation facilitates mitochondrial fission. However, DRP1-S637 phosphorylation leads to DRP1 detaching from mitochondria, which subsequently inhibits mitochondrial fission. *S*-nitrosylation of cysteine 644 by nitric oxide (NO) also facilitates mitochondrial fragmentation by increasing S616 phosphorylation. Thus, the imbalance of DRP1 phosphorylation and *S*-nitrosylation evokes dysfunctions of mitochondrial dynamics, which are involved in pathological processes such as cancer and neurological diseases [14,15].

Interestingly, the impaired mitochondrial dynamics distinctly participate in heterogeneous neuronal death patterns in the hippocampus. Briefly, PV interneurons in the hilus regions show apoptotic degeneration following SE, accompanied by extensive mitochondrial fission [10]. In contrast, the abnormal elongation of swollen mitochondria contributes to CA1 neuronal necrosis, which is initiated by aberrant cell cycle entry in post-mitotic neurons [7,8,16–18]. Given these previous reports, the stoichiometric relationship between fission and fusion plays an important role in neuronal viability. Thus, insight into the molecular mechanism responsible for the impaired mitochondrial dynamics provides a deeper understanding of the distinct vulnerability of a neuronal subpopulation to SE.

2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid that exhibits promising anti-cancer, anti-inflammatory, antioxidant and neuroprotective activities. CDDO-Me a ffects cellular di fferentiation and cell cycle arrest [19,20]. CDDO-Me also irreversibly inhibits Lon protease-1 (LONP1) activity by forming covalent LONP1-CDDO adducts [21,22]. LONP1 is a highly conserved serine peptidase that contributes to protein quality control processes [23], which play a crucial role in maintaining mitochondrial morphology and function [24]. With respect to these previous studies, exploring the e ffects of CDDO-Me on mitochondrial dynamics is noteworthy in order to understand the mechanism of the distinct neuronal death that occurs in response to SE, which has been elusive.

Here, we demonstrate previously unsuspected e ffects of CDDO-Me on DRP1-mediated mitochondrial fissions, which selectively attenuated SE-induced CA1 neuronal death via activating extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun *N*-terminal kinase (JNK). In contrast to CDDO-Me, LONP1 knockdown aggravated SE-induced neuronal death without changing ERK1/2 and JNK activities. Co-treatment of CDDO-Me with LONP1 siRNA ameliorated only CA1 neuronal death, concomitant with abrogation of mitochondrial elongation induced by SE. Thus, our findings sugges<sup>t</sup> CDDO-Me may abrogate abnormal mitochondrial machinery-mediated neuronal death induced by SE, independent of LONP1 activity.

#### **2. Materials and Methods**

#### *2.1. Experimental Animals and Chemicals*

Male Sprague–Dawley (SD) rats (7 weeks old, Daehan Biolink, South Korea) were used in the present study. Animals were given a commercial diet and water ad libitum under controlled conditions (22 ± 2 ◦C, 55 ± 5% and a 12:12 light/dark cycle with lights). Animal protocols were approved by the Institutional Animal Care and Use Committee of Hallym University (Chuncheon, Republic of Korea). The number of animals used and their su ffering were minimized in all cases. All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

#### *2.2. Surgery, CDDO-Me Infusion and LONP1 Knockdown*

Under Isoflurane anesthesia (3% induction, 1.5–2% for surgery and 1.5% maintenance in a 65:35 mixture of N2O:O2), animals were infused with each chemical or siRNA into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth to the bregma) with a brain infusion kit 1 and an Alzet 1007D osmotic pump (Alzet, Cupertino, CA, USA). The osmotic pump contained (1) vehicle, (2) CDDO-Me (10 μM), (3) a non-targeting control siRNA (5-GCAACUAACUUCGUUAGAAUCGUUAUU-3), (4) LONP1 siRNA (5-GAGACAAGUUGCGCAUGAUTT-3) and (5) CDDO-Me + LONP1 siRNA. The pump was placed in a subcutaneous pocket in the dorsal region. Some animals were also implanted with a monopolar stainless steel electrode (Plastics One, USA) into the left dorsal hippocampus (−3.8 mm posterior; 2.0 mm lateral; −2.6 mm depth). The connecting wire and electrode socket were then inserted in an electrode pedestal (Plastics One, USA) and secured to the exposed skull with dental acrylic [8,16–18]. Three days after surgery, rats were induced with SE by lithium chloride (LiCl)-pilocarpine.

#### *2.3. SE Induction and Electroencephalogram (EEG) Analysis*

SE was induced by a single dose (30 mg/kg) of pilocarpine in rats pretreated (24 h before pilocarpine injection) with 127 mg/kg LiCl, as previously described [8,16–18]. Before pilocarpine injection, animals were given atropine methylbromide (5 mg/kg i.p.) to block the peripheral e ffect of pilocarpine. Two hours after SE, animals received diazepam (10 mg/kg, i.p.) to terminate SE. As controls, rats were treated with saline instead of pilocarpine. For evaluation of the e ffects of CDDO-Me infusion and LONP1 siRNA knockdown on seizure susceptibility in response to pilocarpine, some animals' EEG signals were recorded with a DAM 80 di fferential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA). EEG activity was measured during the2hrecording session from each animal. The data were digitized (400 Hz) and analyzed using LabChart Pro v7 (AD Instruments, New South Wales, Australia). Time of seizure onset was defined as the time point showing paroxysmal depolarizing shift, which lasted more than 3 seconds and consisted of a rhythmic discharge between 4 and 10 Hz with an amplitude of at least two times higher than the baseline EEG. EEG activity was measured during the2hrecording session from each animal. Spectrograms were also automatically calculated using a Hanning sliding window with 50% overlap. Two hours after SE onset, diazepam (Valium; Roche, France; 10 mg/kg, i.p.) was administered and repeated, as needed [18].
