**1. Introduction**

Mitochondria are essential organelles for cellular bioenergetics, which are responsible for producing nearly 95% of cellular ATP through oxidative phosphorylation. Under pathological conditions, a progressive decrease in the mitochondrial integrity abrogates respiratory capacities and increases production of free radicals, leading to aberrant structural and/or functional changes in mitochondria. Therefore, the maintenance of mitochondrial redox status is very important for cell viability [1–5].

Lon protease 1 (LONP1) belongs to the ATPases associated with diverse cellular activities (AAA+) protease family in the mitochondrial matrix that has a proteolytic activity of oxidized, dysfunctional, and misfolded proteins in ATP-dependent manner. Thus, LONP1 is rapidly up-regulated to prevent accumulation and aggregation of abnormal mitochondrial proteins under pathophysiological conditions [3–7]. LONP1 over-expression also activates extracellular signal regulated kinase 1/2 (ERK1/2), providing survival advantages and adaptation to cells [8]. Furthermore, ERK1/2 is

required for the up-regulation of LONP1 during epidermal growth factor (EGF)-induced tumorigenic transformation [9]. Therefore, it is likely that the reciprocal regulation between ERK1/2 and LONP1 may a ffect neuron viability against harmful stresses, although the underlying mechanisms have been elusive.

Transient receptor potential canonical channel-6 (TRPC6) is one of Ca2<sup>+</sup>-permeable non-selective cation channels, which protects neurons from ischemia [10], excitotoxicity [11], and status epilepticus (a prolonged seizure activity, SE) [12]. In the rat hippocampus, TRPC6 is highly expressed in the dentate granule cells (DGC), which are more resistant to various insults than other hippocampal neurons [13,14]. Furthermore, TRPC6 knockdown reduces ERK1/2 activity, and results in the massive DGC degeneration following SE [14–16]. Recently, we have reported that the abrogation of up-regulation of LONP1 expression by its siRNA evokes massive DGC death following SE [17]. Therefore, it is presumable that TRPC6-mediated ERK1/2 activation may be one of the up-stream signaling cascades that protect DGC from SE by regulating LONP1 expression, which is less defined.

Here, we show that TRPC6 knockdown led to mitochondrial elongation in DGC concomitant with decreases in LONP1 expression and ERK1/2 phosphorylation. Hyperforin (a TRPC6 activator) showed the reverse e ffects on ERK1/2 activity, LONP1 expression, and mitochondrial length. In addition, TRPC6 siRNA and U0126 (an ERK1/2 inhibitor) resulted in massive DGC degeneration following SE. However, LONP1 siRNA evoked SE-induced DGC degeneration without a ffecting TRPC6 expression, ERK1/2 phosphorylation, or mitochondrial morphologies. These findings for the first time demonstrate TRPC6-ERK1/2 activation may increase DGC invulnerability to SE by regulating LONP1 expression and mitochondrial dynamics.

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

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

Male Sprague–Dawley (SD) rats (7 weeks old) were used in the present study. Animals were kept under controlled environmental conditions (23–25 ◦C, 12 h light/dark cycle) with free access to water and standard laboratory food. All animal protocols were approved by the Administrative Panel on Laboratory Animal Care of Hallym University (Hallym 2018-2, April, 2018). All possible e fforts were taken to avoid animals' su ffering and to minimize the number of animals used during the experiment. All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

#### *2.2. siRNA and Drug Infusion*

Under Isoflurane anesthesia (3% induction, 1.5–2% for surgery, and 1.5% maintenance in a 65:35 mixture of N2O:O2), animals were stereotaxically implanted with a brain infusion kit 1 (Alzet, Cupertino, CA, USA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth to the bregma). The infusion kit was sealed with dental cement and connected to an osmotic pump (1007D, Alzet, Cupertino, CA, USA) containing (1) control siRNA, (2) rat TRPC6 siRNA, (3) rat LONP1 siRNA, (4) vehicle, (5) U0126 (a selective ERK1/2 inhibitor, 25 μM), (6) hyperforin (a TRPC6 activator, 6 μM), or (7) hyperforin + U0126 [12,15,16,18]. Rat TRPC6 siRNA and LONP1 siRNA sequences were 5'-GGAAUAUGCUUGACUUUGGAAUGUUUU-3' [14] and 5'-GAGACAAGUUGCGCAUGAUTT-3' [17], respectively. The non-targeting control siRNA sequence was 5'-GCAACUAACUUCGUUAGAAUCGUUAUU-3'. In a previous study and the present study, 50 μM of U0126 inhibited ERK1/2 phosphorylation in the hippocampus by ~50% after 7 days of over infusion [15]. An osmotic pump was placed in a subcutaneous pocket in the interscapular region. To measure the e ffect of each siRNA, U0126 or hyperforin on seizure susceptibility in response to pilocarpine, some animals were also implanted with a recording electrode (Plastics One, Roanoke, VA, USA) into the left dorsal hippocampus (−3.8 mm posterior; 2.0 mm lateral; −2.6 mm depth). Before an EEG recording, connecting wire and an electrode socket were inserted in an electrode pedestal (Plastics One, Roanoke, VA, USA).

#### *2.3. SE Induction and EEG Analysis*

Three days after surgery, 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 [14,15]. Before pilocarpine injection, animals were given atropine methylbromide (5 mg/kg i.p.) to block the peripheral e ffect of pilocarpine. As controls, rats were treated with saline instead of pilocarpine. After injection, animals were monitored continuously for 2 h to register the extent of behavioral seizure activity. Behavioral seizure severity was also evaluated according to Racine's scale [19]: (1) immobility, eye closure, twitching of vibrissae, sni ffing, or facial clonus; (2) head nodding associated with more severe facial clonus; (3) clonus of one forelimb; (4) rearing, often accompanied by bilateral forelimb clonus; and (5) rearing with loss of balance and falling accompanied by generalized clonic seizures. Within 20–45 min of treatment with pilocarpine, animals became catatonic and began staring, followed by myoclonic twitching and often frequent rearing and falling. The behavioral seizure score reached 4–5 in all groups. There was no di fference in the behavioral seizure score induced by pilocarpine among all the groups. In some animals, EEG signals were also recorded with a DAM 80 di fferential amplifier (0.1–3000 Hz bandpass, World Precision Instruments, Sarasota, TL, USA), digitized (sampling rates, 1000 Hz) and analyzed using LabChart Pro v7 (AD Instruments, Bella Vista, NSW, Australia). Total EEG power and spectrograms were automatically calculated in 2-hour recording session using a Hanning sliding window with 50% overlap [14,15]. Two hours after SE, animals received diazepam (Valium; Roche, France; 10 mg/kg, i.p.) to terminate SE.

## *2.4. Tissue Processing*

Seven days after surgery (non-SE induced animals) or three days after SE, rats were perfused transcardially first with phosphate-bu ffered saline (PBS) followed by a fixative solution (4% paraformaldehyde in 0.1 M phosphate bu ffer, pH 7.4) during 30 min under urethane anesthesia (1.5 g/kg, i.p.). The brains were removed and submerged in the same fixative solution for 4 h at 4 ◦C. Following postfixation, brains were cryoprotected overnight in 30% sucrose solution (in 0.1 M PBS), and coronally sectioned with a cryostat at 30 μm, and consecutive sections were contained in six-well plates containing PBS. For western blot, animals were decapitated under urethane anesthesia (1.5 g/kg, i.p.). The hippocampus was rapidly removed and homogenized in lysis bu ffer. The protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA).

## *2.5. Western Blot*

Western blotting was performed according to standard procedures. Briefly, tissue lysate proteins were blotted onto nitrocellulose transfer membranes (Schleicher and Schuell BioScience Inc., Keene, NH, USA), then incubated with primary antibodies in Table 1. Immunoreactive bands were detected and quantified on ImageQuant LAS4000 system (GE Healthcare, Piscataway, NJ, USA). The values of each sample were normalized with the corresponding amount of β-actin as internal reference.

#### *2.6. Immunohistochemistry and Fluoro-Jade B Staining*

As previously described [14–16], free-floating sections were first incubated with 10% normal goa<sup>t</sup> serum (Vector, Burlingame, CA, USA) in PBS for 30 min at room temperature. Sections were then incubated at room temperature for overnight in the mixture of primary antibodies (Table 1) in PBS containing 0.3% triton X-100 (Table 1). After three washes in PBS, sections were incubated for 1 h in fluorescein isothiocyanate (FITC)-, Cy3- or aminomethylcoumarin acetate (AMCA)-conjugated secondary antibodies (Vector, Burlingame, CA, USA). Sections (reacted with TRPC6 antibody only) were reacted with biotinylated secondary antiserum and avidin–biotin complex (Vector, Burlingame, CA, USA). Thereafter, immunoreactivity was developed by standard 3,3'-Diaminobenzidine reaction. The antibody that was preincubated with 1 μg of purified peptide (for TRPC6) or pre-immune serum

was used as for negative control. To analyze the neuronal damage, we applied Fluoro-Jade B (FJB) staining (Histo-Chem Inc., Jefferson, AR, USA), according to the manufacturer's instructions. Images were captured using an AxioImage M2 microscope or a confocal laser scanning microscope (LSM 710, Carl Zeiss Inc, Oberkocken, Germany) [14,15].


**Table 1.** Primary antibodies used in the present study.

IF, Immunofluorescence; IHC, immunohistochemistry; WB, Western blot.

#### *2.7. Cell Count and Measurement of Mitochondrial Length*

As previously described [14,15], coronal images of the dentate gyrus (3–4 mm posterior to the bregma) were captured (15 sections per each animal) using 20× objectives, and areas of interest (1 × 10<sup>5</sup> μm2) were selected from the dentate granule cell layer. Thereafter, FJB-positive neurons were counted on 20× images using AxioVision Rel. 4.8 Software. Individual mitochondrion in DGC (*n* = 20/section) was also captured using 63× or 100× objectives, and each length was measured by using AxioVision Rel. 4.8 Software or ZEN lite (Blue Edition, Carl Zeiss Inc., Oberkocken, Germany) software following 3D-reconstruction. Two different investigators who were blind to the classification of tissues performed cell counts and measurement of mitochondrial length.

#### *2.8. Quantification of Data and Statistical Analysis*

All data were analyzed using Student *t*-test, one-way ANOVA, or one-way repeated measure ANOVA to determine statistical significance. Bonferroni's test was used for post hoc comparisons. A *p*-value below 0.05 was considered statistically significant.
