Targeting Autophagy, Apoptosis, and SIRT1/Nrf2 Axis with Topiramate Underlies Its Neuroprotective Effect against Cadmium-Evoked Cognitive Deficits in Rats

Abstract

Cadmium is an environmental toxicant that instigates cognitive deficits with excessive glutamate excitatory neuroactivity in the brain. Topiramate, a glutamate receptor antagonist, has displayed favorable neuroprotection against epilepsy, cerebral ischemia, and Huntington's disease; however, its effect on cadmium neurotoxicity remains to be investigated. In this study, topiramate was tested for its potential to combat the cognitive deficits induced by cadmium in rats with an emphasis on hippocampal oxidative insult, apoptosis, and autophagy. After topiramate intake (50 mg/kg/day; p.o.) for 8 weeks, behavioral disturbances and molecular changes in the hippocampal area were explored. Herein, Morris water maze, Y-maze, and novel object recognition test revealed that topiramate rescued cadmium-induced memory/learning deficits. Moreover, topiramate significantly lowered hippocampal histopathological damage scores. Mechanistically, topiramate significantly replenished hippocampal GLP-1 and dampened Aβ42 and p-tau neurotoxic cues. Notably, it significantly diminished hippocampal glutamate content and enhanced acetylcholine and GABA neurotransmitters. The behavioral recovery was prompted by hippocampal suppression of the pro-oxidant events with notable activation of SIRT1/Nrf2/HO-1 axis. Moreover, topiramate inactivated GSK-3β and dampened the hippocampal apoptotic changes. In tandem, stimulation of hippocampal pro-autophagy events, including Beclin 1 upregulation, was triggered by topiramate that also activated AMPK/mTOR pathway. Together, the pro-autophagic, antioxidant, and anti-apoptotic features of topiramate contributed to its neuroprotective properties in rats intoxicated with cadmium. Therefore, it may be useful to mitigate cadmium-induced cognitive deficits.

Keywords: Alzheimer; apoptosis; autophagy; cadmium; glutamate; topiramate.

Figure 1

Topiramate counteracts the impairments in spatial learning/retention memory and recognition memory in cadmium-intoxicated rats. (A) Topiramate chemical structure. (B) The Morris water maze (MWM) test included 3 days of training (4 training sessions per day; 1 min each) where the hidden platform was placed in a fixed quadrant. Twenty-four h later, a probe test was executed where the hidden platform was removed. In the probe test, topiramate significantly increased the time spent in the target quadrant following platform removal, revealing an enhanced retention memory in animals. (C) As part of the Y-maze test (1 h after the training), animal’s short-term recognition memory was examined by measuring the ratio of the time spent in the new/old arm. In this test, the ratio was significantly increased by topiramate. (D) As part of the novel object recognition test (1 day after the training), animal’s long-term recognition memory was examined by measuring the discrimination ratio. In this test, the ratio was significantly increased by topiramate. In each group, n = 6 (graph presenting mean ± standard error of the mean). Statistical significance was denoted by ** p < 0.01 or *** p < 0.001, versus the control group. Statistical significance was denoted by ^# p < 0.05, ^### p < 0.001, or ^#### p < 0.0001, versus the cadmium group. TOP, topiramate; Cd, cadmium chloride.

Figure 2

Topiramate attenuates hippocampal pyknosis and microglial cell influx in cadmium-intoxicated animals. Hematoxylin-eosin (H-E) staining of hippocampal sagittal sections was examined by light microscopy. In both the vehicle-treated control (A) and topiramate-treated control (B), intact subcellular and nuclear structures of the pyramidal neurons were revealed in the hippocampal region. (C) Cadmium intoxication triggered marked degenerative changes, including the pyknosis of pyramidal neurons (red arrow) and infiltration of microglial cells (arrowhead). (D) Topiramate administration to cadmium-intoxicated rats improved the hippocampal histological picture, as seen by lowered neuronal pyknosis (red arrow) and microglial cell influx (arrowhead) alongside an enhanced picture of intact neurons. (E,F) Significant lowering of pyknosis and microglial cell influx scores were observed in response to topiramate administration in cadmium-intoxicated animals. In each group, n = 6 (graph presenting median and interquartile range). Statistical significance was denoted by ** p < 0.01, versus the control group. Statistical significance was denoted by ^# p < 0.05, versus the cadmium group. TOP, topiramate; Cd, cadmium chloride.

Figure 3

Topiramate increases hippocampal GLP-1 and lowers neurodegeneration signals in cadmium-intoxicated rats. Topiramate administration replenishes the protein expression levels of glucagon-like peptide-1 (GLP-1; (A)). Moreover, the neurodegeneration signals amyloid-beta 42 (Aβ42; (B)) and phosphorylated tau (p-tau; (C)) were diminished. In each group, n = 6 (graph presenting mean ± standard error of the mean). Statistical significance was denoted by * p < 0.05, or **** p < 0.0001, versus the control group. Statistical significance was denoted by ^## p < 0.01, or ^### p < 0.001, versus the cadmium group. TOP, topiramate; Cd, cadmium chloride.

Figure 4

Topiramate improves the hippocampal neurotransmitter changes in cadmium-intoxicated rats. Topiramate administration replenishes the levels of acetylcholine (A) and γ-aminobutyric acid (GABA; (C)) and diminishes acetylcholine esterase activity (B) and glutamate levels (D). In each group, n = 6 (graph presenting mean ± standard error of the mean). Statistical significance was denoted by ** p < 0.01, *** p < 0.001, or **** p < 0.0001, versus the control group. Statistical significance was denoted by ^## p < 0.01, or ^### p < 0.001, versus the cadmium group. TOP, topiramate; Cd, cadmium chloride.

Figure 5

Topiramate curtails hippocampal redox aberrations in cadmium-intoxicated rats. Topiramate replenishes the antioxidant levels of SIRT1 (A), nuclear Nrf2 (B), HO-1 (C), and GPx (D) and diminishes the levels of the pro-oxidant malondialdehyde (MDA) (E) and in the hippocampi of cadmium-intoxicated rats. In each group, n = 6 (graph presenting mean ± standard error of the mean). Statistical significance was denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001, versus the control group. Statistical significance was denoted by ^# p < 0.05, ^## p < 0.01, or ^### p < 0.001, versus the cadmium group. Cd, cadmium chloride; TOP, topiramate; HO-1, heme oxygenase-1; GPx, glutathione peroxidase; Nrf2, nuclear factor erythroid 2-related factor-2; SIRT1, silent information-regulated transcription factor 1.

Figure 6

Topiramate ameliorates impaired hippocampal autophagy events in cadmium-intoxicated rats. This was evidenced by reduced SQSTM-1/p62 levels (A) and elevated Beclin1 (B). Moreover, topiramate administration to cadmium-intoxicated animals stimulated the AMPK/mTOR pathway, with an elevated p-AMPK/AMPK ratio (C) and lowered p-mTOR/mTOR ratio (D). In each group, n = 6 (graph presenting mean ± standard error of the mean). Statistical significance was denoted by *** p < 0.001, or **** p < 0.0001, versus the control group. Statistical significance was denoted by ^# p < 0.05, ^## p < 0.01, or ^### p < 0.001, versus the cadmium group. AMPK, 5′adenosine monophosphate-activated protein kinase; Cd, cadmium chloride; mTOR, mammalian target of rapamycin; TOP, topiramate; SQSTM-1/p62, sequestosome-1/protein 62.

Figure 7

Topiramate lowers hippocampal apoptosis in cadmium-intoxicated animals. (A) The protein expression of p-GSK-3β(Ser9)/total GSK-3β. (B) Immunohistochemical staining of hippocampal Bax in rats (brown staining of the target protein is shown by red arrow; original magnification, 400×). (C) The graph displays Bax quantitative analysis (area %). (D) The activity of caspase 3. In each group, n = 6 non-overlapping fields (graph presenting mean ± standard error of the mean). Statistical significance was denoted by * p < 0.05, ** p < 0.01, or **** p < 0.0001, versus the control group. Statistical significance was denoted by ^# p < 0.05, or ^#### p < 0.0001, versus the cadmium group. Bax, Bcl-2 associated x protein; GSK-3β, glycogen synthase kinase—3 beta; Cd, cadmium chloride; TOP, topiramate.

Figure 8

Topiramate increases hippocampal Bcl-2 protein expression in cadmium-intoxicated animals. (A) Hippocampal Bcl-2 immunohistochemical staining (brown staining of the target protein is shown by a red arrow; original magnification, 400×). (B) The graph displays Bcl-2 quantitative analysis (area %). In each group, n = 6 non-overlapping fields (graph presenting mean ± standard error of the mean). Statistical significance was denoted by * p < 0.05 versus the control group. Statistical significance was denoted by ^# p < 0.05 versus the cadmium group. Cd, cadmium chloride; Bcl-2, B-cell lymphoma-2 protein; TOP, topiramate.

Figure 9

The mechanisms by which topiramate protected against cadmium-induced cognitive deficits. Herein, topiramate ameliorated cadmium-evoked memory and learning deficits by dampening hippocampal neurotoxic signals, including Aβ₄₂ and p-tau and counteracting the neurtranmitter aberrations. These events were mediated by (A) activation of the hippocampal SIRT1/Nrf2/HO-1 cascade and attenuation of neuronal pro-oxidative events. (B) AMPK/mTOR pathway stimulation with an enhancement of the autophagy response. (C) Dampening of hippocampal apoptotic cell death. In the figure, solid arrows represent activation, whereas blunt arrows represent inhibition.