Calmodulin inhibitor ameliorates cognitive dysfunction via inhibiting nitrosative stress and NLRP3 signaling in mice with bilateral carotid artery stenosis

Abstract

Aims: Vascular dementia (VaD) is a heterogeneous brain disorder for which there are no effective approved pharmacological treatments available. We aimed to evaluate the effect of calmodulin inhibitor, DY-9836, and its loaded nanodrug carrier system on cognitive impairment and gain a better understanding of the protective mechanisms in mice with bilateral carotid artery stenosis (BCAS).

Results: DY-9836 (0.5 or 1 mg/kg) or DY-9836 (0.25 mg/kg)-encapsulated polysialic acid-octadecylamine (PSA-ODA) micelles (PSA-ODA/DY) were given to BCAS mice for 4 weeks. Administration of DY-9836 or PSA-ODA/DY reduced escape latency in space exploration and working memory test compared with vehicle group. Vehicle-treated mice showed reduced phospho-CaMKII (Thr286/287) levels in the hippocampus, whereas partially restored by DY-9836 (1 mg/kg) or PSA-ODA/DY (0.25 mg/kg) treatment. In accordance with the pharmacological profile of DY-9836 observed during behavioral studies, experimental molecular and biochemical markers induced by BCAS, such as protein tyrosine nitration, Nod-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1β, were reduced by DY-9836 and PSA-ODA/DY treatment.

Conclusions: These data disclose novel findings about the therapeutic potential of DY-9836, and its encapsulated nanodrug delivery system significantly enhanced the cognitive function via inhibitory effect on nitrosative stress and NLRP3 signaling in VaD mice.

Keywords: calmodulin inhibitor; cognitive impairment; inflammasome; nitrosative stress; vascular dementia.

Figure 1

DY‐9836 enhances the hippocampal‐based spatial learning and memory in mice model of vascular dementia. (A) A schematic illustration of experimental plan. (B) Effects of DY‐9836 on changes in escape latency to find the hidden platform. (C‐E) Spatial probe trial and test to evaluate the effect of DY‐9836. DY‐9836, PSA and PSA‐ODA/DY significantly attenuated the escape latency. The number of crossing platform decreased in bilateral carotid artery stenosis (BCAS) mice model group, whereas time of staying at platform was significantly enhanced in all treated groups. (F, G) Short‐term memory test was performed, and the escape latency in sample trial and in the test trials was noted. Data represent mean ± SEM (n = 10‐12 per group). ^### P< 0.001 vs sham‐operated group and *P < 0.05; **P < 0.01; ***P < 0.01 vs vehicle‐treated group. PSA, polysialic acid; ODA, octadecylamine

Figure 2

DY‐9836 prevents the dephosphorylation of CaMKII in bilateral carotid artery stenosis (BCAS) mice model. Immunohistochemical localization of phospho‐CaMKII and MAP‐2 was examined in the hippocampal CA1 (A). Markedly decreased phospho‐CaMKII (green) and MAP‐2 (red) staining were observed by confocal microscopy in the hippocampus of BCAS mice model. (B) Quantification of phospho‐CaMKII immunofluorescence expressed as mean intensity. DY‐9836, PSA, and PSA‐ODA/DY treatment restored the decrease in immunostaining for phospho‐CaMKII (Thr286/287) in hippocampal pyramidal neurons of BCAS mice. DAPI counterstaining indicates nuclear localization (blue). Data are representative of 3 independent experiments. *P < 0.05, vs sham operated mice. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001, vs saline‐treated BCAS operated mice. Scale bar = 50 μm. PSA, polysialic acid; ODA, octadecylamine

Figure 3

DY‐9836 inhibits the NLRP3 inflammasome activation in mice model of bilateral carotid artery stenosis (BCAS). (A) Temporal changes in expression of hippocampal NLRP3 in mice model of BCAS. Representative Western blots of NLRP3 in the hippocampal region in BCAS mice. Levels of β‐actin were used as the loading control. (B) Quantitative analysis of relative NLRP3 in BCAS mice was performed by intensity of expression with respect to sham‐operated group. Data are expressed as values of control animals (mean ± SEM, n = 5). ^## P < 0.01 vs sham‐operated group and *P < 0.05 vs vehicle‐treated group

Figure 4

DY‐9836 attenuates the caspase‐1 activation in bilateral carotid artery stenosis (BCAS) mice model. Representative image of hippocampal CA1 (A) pyramidal neurons stained with caspase‐1. (B) Quantification of caspase‐1 immunofluorescence expressed as mean intensity. Increased immunostaining of caspase‐1 was observed by confocal microscopy in the hippocampus of BCAS mice that was reversed by treatment of DY‐9836 (1 mg/kg) and PSA‐ODA/DY (DY, 0.25 mg/kg). DAPI counterstaining indicates nuclear localization (blue). Data are representative of 3 independent experiments. **P < 0.01, vs sham operated mice. ^## P < 0.01, vs saline‐treated BCAS operated mice. Scale bar = 20 μm. PSA, polysialic acid; ODA, octadecylamine

Figure 5

Nitrosative stress and proinflammatory IL‐1β activation in bilateral carotid artery stenosis (BCAS) mice are inhibited by DY‐9836 treatment. (A) Fluorescent immunohistochemical staining of IL‐1β (H‐153) and nitrotyrosine in the hippocampal CA1 region of BCAS mice. Antinitrotyrosine and IL‐1β (H‐153) staining were performed after BCAS. IL‐1β expression was disturbed with increased in nitrotyrosine‐positive staining in hippocampus of BCAS mice model. BCAS‐induced nitrotyrosine formation and IL‐1β expression colocalized in the CA1 region of hippocampus. Quantification of IL‐1β (H‐153) (B) and nitrotyrosine (C) immunofluorescence expressed as mean intensity. DY‐9836 (1 mg/kg) and PSA‐ODA/DY (DY, 0.25 mg/kg) significantly reduced nitrotyrosine immune reactivity and IL‐1β activation as compared to vehicle‐treated mice group. DAPI counterstaining indicates nuclear localization (blue). Data are representative of 3 independent experiments. ***P< 0.001, vs sham operated mice. ^## P < 0.01, ^### P < 0.001 vs saline‐treated BCAS operated mice. Scale bar = 20 μm. PSA, polysialic acid; ODA, octadecylamine

Figure 6

Schematic illustration of the mechanisms by which DY‐9836 inhibits NLRP3 activation and cognitive dysfunction in bilateral carotid artery stenosis mice model