Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage

Abstract

Background and purpose: Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage.

Methods: We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures.

Results: Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons.

Conclusions: Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.

Keywords: autophagy; carnosine; mitochondria.

Figure 1. Protective effect of carnosine against brain damage during ischemic stroke

Ischemic stroke was achieved by middle cerebral artery occlusion (MCAO) in rats. (A) Carnosine (1000 mg/kg) was administered 6 hr after onset of ischemia. Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining at 24 hr after MCAO. The representative photos are shown. N=13-15. *p<0.05 vs. saline-treated rats. (B and C) Carnosine (1000 mg/kg) was administered to rats at 6 hr after ischemic onset during transient MCAO (6 hr ischemia/18 hr reperfusion). Behavioral tests were performed at 24 hr before and after ischemia. (B) Somatosensory deficit was determined using the Adhesive Tape tests, where required time to remove adhesives on fore limbs were measured. (C) In the RotaRod test, motor-ambultatory function was determined. Latencies to fall off from the rotarod with accelerated speeds were measured. B: N=13-15, C: N=15-16. **p<0.01, #p<0.05 vs. the corresponding group. Data were expressed as mean ± SEM and analyzed by Student's t-test.

Figure 2. Inhibitory effect of carnosine on autophagy in ischemic brain

Brain homogenates were isolated from contralateral (Contra) or ipsilateral (Ipsi) hemispheres from saline- or carnosine (1000 mg/kg; 6 hr post treatment)-administered rats following pMCAO. (A) The extent of autophagy was examined using the conversion of LC3-II from LC3-I. (B) Autophagic signaling was examined by phosphorylation of mTOR, p70S6K and ERK. The representative bands are shown. Relative density of each band was analyzed by ImageJ. N=4. *p<0.05, **p<0.01 vs. contralateral hemisphere from saline-treated rats. #p<0.05 vs. ipsilateral hemisphere from saline-treated rats. Data were expressed as mean ± SEM and analyzed by Student's t-test.

Figure 3. Protective effect of carnosine on mitochondrial damage in ischemic brain

Brain mitochondria were isolated from contralateral (Contra) or ipsilateral (Ipsi) hemispheres from saline- or carnosine (1000 mg/kg; 6 hr post treatment)-administered rats following pMCAO. (A) Complex I activity was measured using colorimetric method. (B) The extent of mitochondrial fragmentation and mitophagy was examined using the level of p-Drp 1 and Parkin. Mitochondrial levels of apoptosis inducing factor (AIF) and cytochrome C were measured. Relative density of each band was analyzed by ImageJ. N=4. *p<0.05, **p<0.01 vs. contralateral hemisphere from saline-treated rats. #p<0.05 vs. ipsilateral hemisphere from saline-treated rats. Data were expressed as mean ± SEM and analyzed by Student's t-test.

Figure 4. Inhibitory effect of carnosine on neuronal autophagy following NMDA stimulation

Primary cortical neurons were pre-treated with carnosine 30 min prior to exposure to NMDA (N-methyl-d-aspartate; 25 μM). (A) Neuronal cell death was determined by extent of lactate dehydrogenase leakage at 24 hr after NMDA stimulation. N=5. (B and C) The conversion of LC3-II from LC3-I (B) and the phosphorylation of mTOR (C) in NMDA (25 μM)-stimulated primary neurons with or without carnosine (100 μM) pretreatment. The representative bands are shown. Relative density of each band was analyzed by ImageJ. N=3. *p<0.05, **p<0.01 vs. control group. #p<0.05 vs. NMDA-treated group. Data were expressed as mean ± SEM and analyzed by one way ANOVA followed by Tukey test (A) or by Student's t-test (B and C).