As mitochondria is the site of oxidative phosphorylation, it is likely to produce high amount of O2− radicals and thereby making this organelle the most susceptible one to undergo ROS led dysfunctions in the high energy demanding brain cells in particular. Thus, oxidative stress induced mitochondrial dysfunction is now considered the most common neurochemical aberration associated with the pathogenesis of many brain disorders. The neurotoxic effect of ammonia in CNS, symptomized mainly by the HE associated neuropsychiatric complications, is also argued to implicate enhanced mitochondrial ROS load mainly due to declined levels of antioxidant enzymes like; SOD, catalase etc. [34, 35]. The mitochondrial MnSOD is considered the committed enzyme to neutralize O2− radical and therefore, the declined level of this antioxidant enzyme has been reported associated with the pathogenesis of many brain disorders like AD, PD, ALS . Consequently, by using knocked in experimental models, many workers have tried to manipulate the expression level MnSOD to prevent neurodegeneration in different neuropathology models . However, to qualify Mn-SOD as a doable therapeutic target, there is need to define protein level reversible modification vs activity changes and the mechanism by which its expression is regulated during a neuropathogenesis.
There are reports suggesting activation of MnSOD by a number of compounds , however, the mode of action of most of them remain unexplained. In this respect, particularly in mitochondria, there is an evolving concept of activating this enzyme by a mitochondrial SIRT3 dependent deacetylation resulting into better cell survival . In case of the animal model of neurodegenerative brain disorders, such examples are limited. Moreover, in western diet fed SIRT3−/− mice model, an increased MnSOD acetylation could be correlated with the diminished activity of this enzyme . We have recently reported a cause-and-effect relationship between SIRT3 activation by a natural compound honokiol (HKL) and recovery in the compromised mitochondrial functions in hippocampus of the MoHE rats . In this respect, the findings of Fig. 1(a-c) clearly demonstrate another cause - effect relationship between activation of Mn-SOD vs deacetylation of this enzyme due to the treatment with HKL. Though fragmentary but there are some reports on SIRT3 dependent deacetylation of certain ETS enzymes vis a vis normalizing the declined levels of those enzymes . However, such reports are scanty in case of deacetylation of Mn-SOD vs recovery in ROS led mitochondrial dysfunction. Our previous report has demonstrated a correlation between the enhanced ROS level led compromised mPTP, declined ETS activity and redox ratio in the hippocampal mitochondria from the MoHE rats. However, all these parameters could be recovered back to the normal level due to SIRT3 activation by HKL . Herein, the finding of significantly declined level of the acetylated Mn-SOD (Fig. 1c) due to HKL treatment to the MoHE rats provides evidence to advocate Mn-SOD as another protein target of SIRT3 activation. Thus, suggesting about mechanistic aspect of how HKL could normalize ROS challenges in the hippocampal mitochondria of the MoHE rats. Additionally, this is a first report on HKL dependent alterations in the acetylation status MnSOD vs a similar pattern of its activity changes in mitochondria of a susceptible brain region of an MoHE animal model of excitotoxicity. The argument gets support from a report describing association of SIRT3 deletion with the enhanced acetylation led decreased activity of MnSOD which was found accountable for the increasing oxidative stress and decline of MPTP in an age-related loss of substantia nigra (SNc) dopaminergic neurons of the PD mouse model . A recent report in metal toxicity model has also shown that fluoride reduced mitochondrial antioxidant enzyme activities and elevated SOD2 acetylation by downregulating SIRT3 expression in the brain of mice and in the SH-SY5Y cells .
Another mechanism advocated accountable for maintaining MnSOD level in the brain cells is the regulation of its expression during pathogenesis and treatment. According to the findings from Fig. 2, a recovery in the MoHE associated declined expression of MnSOD, both at transcript and at protein levels, due to the treatment with HKL, clearly suggest about a significant role of HKL in the regulation of MnSOD expression in the hippocampus mitochondria. There are some reports, though on other neurodegenerative models, describing recovery in the Mn-SOD expression vis a vis normalization of the disease pathogenesis. In a Diabetic neuropathic pain (DNP) model in rats, upregulation of MnSOD in the spinal dorsal horn could be correlated with the pain reduction . Another study suggests that a trans sodium crocetinate (TSC) could exert protection against cerebral ischemia/reperfusion (I/R) injury by increasing SOD2 protein levels and decreasing its acetylation . However, to our knowledge, there is a little information on the HKL dependent modulation of Mn-SOD expression in an excitotoxic brain disorder condition. Therefore, our finding of Fig. 2 necessitated investigation of how HKL could modulate expression of MnSOD expression in the hippocampus of the MoHE rats.
In most of the reports describing modulation of the expression of antioxidant enzymes, under the condition of physiological stresses, by a number of modulators, it was observed that in case of MnSOD, there appears concordant interplay of SIRT3-FoXO3a-PGC1α axis in transactivating this mitochondrial isoform of SOD. It has been suggested that FoxO3a forms a feedback loop with PGC1α to regulate antioxidant genes and SIRT3 dependent deacetylation of these transcription factors has been argued as one of the mechanisms to regulate their expression and nuclear translocation to ultimately activate the ARE (the antioxidant response element) of the antioxidant enzymes genes . Independently also, PGC1α is known to regulate mitochondrial function by stimulating antioxidant enzymes . Also, the PGC1α is reported to trigger SIRT3 expression which by deacetylating MnSOD, normalizes the increased mitochondrial ROS .
In view of our previous report, describing HKL dependent recovery in SIRT3 activity in the hippocampal mitochondria of the MoHE rats, it is argued that SIRT3 activation could be accountable for not only deacetylating Mn-SOD (Fig. 1c) but also for the modulation of FoxO3a-PGC1a axis as well. Indeed, we observed a remarkable increase in the transcript levels of both, the Foxo3a and PGC1α, in the hippocampal fraction of the MoHE rats treated with HKL (Fig. 3). Importantly, such a pattern of the enhanced expression of both these critical factors was consistent with a similar recovery in the abundance of both these proteins in all the three major regions of hippocampus; DG, CA1 & CA3, accountable for memory formation and consolidation, in the HKL treated MoHE rats (Fig. 4, 5 &6).
HKL is known to display strong anti-inflammatory and antioxidant properties in a variety of diseases including certain neuropathology as well. However, the mechanism by which it imparts its neuroprotective action remains largely unexplored. During recent past, SIRT3 is emerging as a master regulator of mitochondrial integrity and HKL is suggested as an activator of SIRT3 [25, 29] both in vitro and in vivo. Therefore, in the present context, it is argued that HKL dependent SIRT3 activation could be accountable to upregulate Mn-SOD mainly by modulating the levels of Foxo3a and PGC1α in the hippocampus of the MoHE rats. This is supported, though indirectly, by the similar findings on the multimodal modulations of SIRT3-FoxO3a-PGC1α axis including SIRT3 dependent deacetylation of both the transcription factors due to the treatment with various other compounds.
For example, Chronic fluoride exposure has been found to induce mitochondrial dysfunction through inhibition of Sirt3/FoxO3a signaling in the SH-SY5Y cell lines . The overexpression of spinal cord SIRT3 in DNP rat model has been demonstrated to increase the expression and deacetylation of FoxO3a to ultimately upregulate MnSOD . Another study suggests that neuroprotective effect of trans sodium crocetinate (TSC) against cerebral ischemia/reperfusion (I/R) injury is mediated via increasing SIRT3 activity vs decreased acetylation of Foxo3a and SOD2 . SIRT3 dependent increased expression and nuclear translocation of FoxO3a have also been reported accountable to transactivate antioxidant enzymes like SOD in the activated microglia of the adult rats subjected to the traumatic brain injury . Similarly, PGC-1α, is a multifunctional critical regulator of various cellular functions including mitochondrial quality by suppressing oxidative damage . Importantly, FoxO3a has been found to protect cells from oxidative stress through direct interaction with PGC-1α in the promoter regions of the antioxidant enzymes . In MPTP PD model, PGC1a-ERRα/SIRT3 pathway has been demonstrated to play critical roles in protecting DAergic neurons against oxidative damage and ATP depletion mainly by deacetylating SOD2 and ATP synthase β . Also, PGC-1α expression is reported to decline in the AD brain as a function of dementia severity . Taking together, SIRT3-Foxo3a-PGC1a axis act as a master regulator of maintaining the levels of the antioxidant enzymes and the mitochondrial Mn-SOD, in particular, however, evidently this axis seems to be a modifiable target in many ways and therefore, deserve special merit to be categorized as a targetable hot spot in neurological disorders.
There is no previous report on HKL dependent modulation of SIRT3-Foxo3a-PGC1a loop in modulating the antioxidant potential of the brain cells in a neurological disorder. Thus, our findings of Fig-3-6 are first of its kind to provide HKL dependent positive modulation of this axis that could be involved in normalizing ROS challenge emerged during MoHE pathogenesis.