**3. Results**

#### *3.1. TRPC6 Knockdown and Hyperforin Reversely Regulate LONP1 Expression, ERK1*/*2 Phosphorylation, and Mitochondrial Length in DGC under Physiological Conditions*

Figure 1A shows that TRPC6 expression was apparently detected in the DGC layer and the molecular layer of the DG rather than other regions. TRPC6 siRNA reduced TRPC6 expression in the hippocampus (Figure 1A). TRPC6 knockdown elongated mitochondrial length (~2.86 μm), as compared to control siRNA (~1.26 μm) (*p* < 0.05 vs. control siRNA, n = 7; Figure 1B,C and Supplementary Figure S1). TRPC6 siRNA decreased LONP1 expressions in mitochondria (Figure 1C). Western blot data demonstrated that TRPC6 knockdown led to ~65% and ~30% reductions of TRPC6 and LONP1 protein levels, respectively (*p* < 0.05 vs. control siRNA, n = 7, respectively; Figure 1D,E). TRPC6 knockdown also declined ERK1/2 phosphorylation (*p* < 0.05 vs. control siRNA, n = 7; Figure 1D E and Supplementary Figure S2). In contrast to TRPC knockdown, hyperforin (a TRPC6 activator) [12,18] decreased mitochondrial length to ~0.54 μm (*p* < 0.05 vs. vehicle, n = 7; Figure 1B,C). Hyperforin increased ERK1/2 phosphorylation and LONP1 expression without altering TRPC6 expression (*p* < 0.05 vs. vehicle, n = 7; Figure 1D,E and Supplementary Figure S2). Since TRPC6 regulates ERK1/2 activity [20], our findings indicate that ERK1/2 may be involved in a potential relationship between TRPC6 and LONP1.

**Figure 1.** Effects of TRPC6 siRNA and hyperforin on mitochondrial dynamics, LONP1 expression, and ERK1/2 phosphorylation under certain physiological conditions. (**A**) Representative images of control- and TRPC6 siRNA-treated animals. TRPC6 expression is predominantly detected in the molecular layer of the dentate gyrus and DGC. TRPC6 siRNA effectively decreases TRPC6 expression. (**B**,**C**) Effects of TRPC6 siRNA and hyperforin on mitochondrial length. TRPC6 siRNA (T-siRNA) leads to mitochondrial elongation, while hyperforin (HF) facilitates mitochondrial fragmentation. (**B**) Quantification of mitochondrial length. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA and vehicle, respectively; Student *t*-test; n = 7, respectively). (**C**) Representative double immunofluorescent images for LONP1 and mitochondria (Mito). Inserts are high magnification images (insert bar = 1.25 μm). (**D**,**E**) Effects of TRPC6 siRNA and hyperforin on expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation. TRPC6 siRNA (T-siRNA) decreases protein levels of TRPC6 and LONP1, and ERK1/2 phosphorylation. Hyperforin (HF) increases LONP1 expression and ERK1/2 phosphorylation without changing TRPC6 expression. (**D**) Representative western blots of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation (M.W. marker, Molecular weight marker). (**E**) Quantification of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA and vehicle, respectively; Student *t*-test; n = 7, respectively).

#### *3.2. LONP1 siRNA Does Not Influence TRPC6 Expression, ERK1*/*2 Phosphorylation and Mitochondrial Length under Physiological Condition*

Next, we applied LONP1 siRNA to confirm whether LONP1 reciprocally influences TRPC6 expression and ERK1/2 phosphorylation. LONP1 knockdown significantly decreased LONP1 expression (*p* < 0.05 vs. control siRNA, n = 7; Figure 2A,B and Supplementary Figure S3). However, LONP1 knockdown did not affect the TRPC6 expression level and mitochondrial length (Figure 2A–D and Supplementary Figure S1). In addition, LONP1 siRNA did not influence ERK1/2 expression and its phosphorylation (Figure 2A,B and Supplementary Figure S3). Thus, these findings sugges<sup>t</sup> that the RPC6-ERK1/2 signaling pathway may be one of the up-steam regulators for LONP1 expression.

#### *3.3. U0126 Abrogates Mitochondrial LONP1 Expression under Physiological Condition and after Hyperforin Treatment*

Since hyperforin increases ERK1/2 phosphorylation and LONP1 expression in a previous [16] and the present study, we further investigated whether ERK1/2 activity affects LONP1 expression. U0126 (an ERK1/2 inhibitor) reduced ERK1/2 phosphorylation and LONP1 expression, and led to mitochondrial elongation under physiological condition without affecting TRPC6 expression (*p* < 0.05 vs. vehicle, n = 7; Figure 3A–D and Supplementary Figures S1 and S4). In addition, U0126 co-treatment abolished mitochondrial elongation and up-regulations of LONP1 expression as well as ERK1/2 phosphorylation induced by hyperforin (*p* < 0.05 vs. hyperforin, n = 7; Figure 3A–D and Supplementary Figures S1 and S4). Together with the data obtained from TRPC6 knockdown, these findings indicate that TRPC6 activity may regulate LONP1 expression and mitochondrial dynamics through ERK1/2 activation.

#### *3.4. The TRPC6-ERK1*/*2-LONP1 Signaling Pathway Inhibits SE-Induced DGC Degeneration, Independent of Seizure Severity*

TRPC6 knockdown provokes massive DGC degeneration following pilocarpine-induced SE, although DGC is remarkably resistant to neuronal damage induced by various insults [14,15]. Since seizure severity correlates to neuronal damage [21,22], we explored whether the modulations of the TRPC6-ERK1/2-LONP1 signaling pathway alter seizure susceptibility to pilocarpine. In control siRNA-treated animals, the seizure susceptibility to pilocarpine was similar to that in vehicle-treated animals (Figure 4A,B). TRPC6 siRNA reduced the latency of seizure on-set, and increased total EEG power during SE (*p* < 0.05 vs. control siRNA, n = 7; Figure 4A,B). These findings indicate that TRPC6 knockdown may increase seizure susceptibility. LONP1 siRNA and hyperforin could not affect the seizure susceptibility to pilocarpine (Figure 4A,B). Consistent with our previous study [15], U0126 delayed the seizure on-set, and reduced total EEG power in response to pilocarpine (*p* < 0.05 vs. vehicle, n = 7; Figure 4A,B). Co-treatment of U0126 with hyperforin also reduced seizure activity after pilocarpine injection (*p* < 0.05 vs. vehicle, n = 7; Figure 4A,B). However, TRPC6 siRNA and LONP1 siRNA evoked massive DGC degeneration (*p* < 0.05 vs. control siRNA, n = 7; Figure 5A,B). As compared to vehicle, U0126 aggravated DGC death induced by SE (*p* < 0.05 vs. vehicle, n = 7; Figure 5A,B). Hyperforin attenuated SE-induced DGC degeneration (*p* < 0.05 vs. vehicle, n = 7; Figure 5A,B), which in turn caused deterioration by U0126 co-treatment (*p* < 0.05 vs. hyperforin, n = 7; Figure 5A,B). Therefore, the severity of SE-induced DGC degeneration in each siRNA or compound-treated animals was LONP1 siRNA > U0126 > TRPC6 siRNA > hyperforin + U0126 > control siRNA = vehicle > hyperforin. These findings sugges<sup>t</sup> that the blockade of TRPC6-ERK1/2-LONP1 signaling pathway may increase SE-induced DGC degeneration, independent of seizure susceptibility or its severity.

**Figure 2.** Effects of LONP1 siRNA on expression levels of LONP1 and TRPC6, ERK1/2 phosphorylation and mitochondrial dynamics under physiological condition. (**A**,**B**) Effects of LONP1 siRNA on expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation. LONP1 siRNA decreases LONP1 expression without affecting TRPC6 expression and ERK1/2 phosphorylation. (**A**) Representative western blots of expressions of LONP1 and TRPC6, and ERK1/2 phosphorylation (M.W. marker, Molecular weight marker). (**B**) Quantification of expressions of LONP1 and TRPC6, and ERK1/2 phosphorylation based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA; Student *t*-test; n = 7, respectively). (**C**,**D**) Effects of LONP1 siRNA on mitochondrial length. LONP1 siRNA does not affect mitochondrial length. (**C**) Representative double immunofluorescent images for LONP1 and mitochondria (Mito). Inserts are high magnification images (insert bar = 1.25 μm). (**D**) Quantification of mitochondrial length. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA; Student *t*-test; n = 7, respectively).

**Figure 3.** Effects of U0126, hyperforin, and co-treatment of hyperforin and U0126 on expression levels of LONP1 and TRPC6, ERK1/2 phosphorylation and mitochondrial dynamics under physiological condition. (**A**,**B**) Effects of U0126, hyperforin (HF) and co-treatment of hyperforin and U0126 (HF + U0126) on expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation. U0126 decreases LONP1 expression and ERK1/2 phosphorylation without affecting TRPC6 expression. Hyperforin increases LONP1 expression and ERK1/2 phosphorylation, which are abrogated by U0126 co-treatment. (**A**) Representative western blots of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation (M.W. marker, Molecular weight marker). (**B**) Quantification of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA; one-way ANOVA; n = 7, respectively). (**C**,**D**) Effects of U0126, hyperforin (HF) and co-treatment of hyperforin and U0126 (HF + U0126) on mitochondrial length. U0126 increases mitochondrial length. In contrast, hyperforin diminishes it, which is abrogated by U0126 co-treatment. (**C**) Representative double immunofluorescent images for LONP1 and mitochondria (Mito). Inserts are high magnification images (insert bar = 1.25 μm). (**D**) Quantification of mitochondrial length. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA; one-way ANOVA; n = 7, respectively).

**Figure 4.** Effects of control siRNA, TRPC6 siRNA, LONP1 siRNA, U0126, hyperforin and co-treatment of hyperforin and U0126 on seizure activity in response to pilocarpine. As compared to control siRNA (C-siRNA), LONP1 siRNA (L-siRNA) does not affect seizure activity induced by pilocarpine. However, TRPC6 siRNA (T-siRNA) reduces seizure latency, and increases seizure severity in response to pilocarpine. No difference in seizure activity is observed between control siRNA and Vehicle (Veh)-treated animals. As compared to vehicle, hyperforin (HF) does not affect seizure activity induced by pilocarpine. However, U0126 and co-treatment of hyperforin and U0126 (HF + U0126) attenuate seizure activity in response to pilocarpine. (**A**) Representative EEG traces and frequency-power spectral temporal maps in response to pilocarpine. (**B**) Quantification of total EEG power (seizure intensity) in response to pilocarpine (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA or vehicle; one-way repeated measure ANOVA; n = 7, respectively).

**Figure 5.** Effects of control siRNA, TRPC6 siRNA, LONP1 siRNA, U0126, hyperforin, and co-treatment of hyperforin and U0126 on SE-induced DGC degeneration. As compared to control siRNA (C-siRNA), TRPC6 siRNA (T-siRNA), LONP1 siRNA (L-siRNA) and U0126 induce massive DGC degeneration induced by SE. Hyperforin (HF) ameliorates SE-induced DGC damage, which is reversed by U0126 co-treatment (HF + U0126). (**A**) Representative images for FJB-positive degenerating DGC. (**B**) Quantification of the number of FJB-positive DGC. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; \* *p* < 0.05 vs. control siRNA or vehicle; one-way ANOVA; n = 7, respectively).
