Studying The Effect Of GHS-R1a Deficiency On Hippocampus-dependent Memory Impairment In AD Model Mice

Alzheimer's disease(AD)is the most common progressive and destructive neurodegenerative disease,characterized by ?-amyloid deposition and Tau hyperphosphorylation.Currently,there is no effective treatment for AD.Declarative memory and cognition impairment are characteristics of brain function decline in early stage of AD.The hippocampus close related to learning and memory,is one of the brain regions most susceptible to AD neurodegeneration.Structure and/or function abnormalities in hippocampal neural circuit are considered neurobiology basis of memory impairments.Recent studies have shown that neurodegenerative diseases such as AD are usually accompanied by metabolic disorders and chronic neuroinflammation,which may aggravate neuronal lesions and cause cognitive dysfunction.Microglia is not only the most important immune cells that mediate neuroinflammation in the central nervous system;it is also the key regulators of neuronal synaptic function.many studies have reported that microglia dysfunction during neurodegeneration contributed to memory impairment.Ghrelin is a brain-gut peptide hormone mainly secreted by gastric fundus cells and consists of 28 amino acids.There are two forms of ghrelin,acetylated and non-acetylated,only acetylated ghrelin is functional by binding to its receptor GHS-R1 a.GHS-R1 a is a typical G protein-coupled receptor,which is widely distributed in the brain.GHS-R1 a is enriched in the hypothalamus and other brain regions involved in memory and emotion regulation,such as the hippocampus,prefrontal cortex,amygdala etc.Studies have indicated that Ghrelin/GHS-R1 a signaling is involved in many important brain functions such as learning and memory,stress responses,motivation and rewards,anxiety and depression.Our previous study showed that ghrelin micro-infusion into dorsal CA1(d CA1)of the hippocampus or virus-mediated Ghsr1 a overexpression in d CA1 pyramidal neurons impaired,while Ghsr1 a knockout or interference promoted hippocampus-dependent memory formation.These findings suggest that ghrelin/GHS-R1 a signaling blocks learning and memory,and GHS-R1 a deficiency may enhance learning and memory.It is noteworthy that,a recent clinical study reported that GHS-R1 a expression was dramatically increased in the hippocampus of both AD patients and 5×FAD model mice,suggesting that the chronic upregulation of GHS-R1 a may be a molecular biomarker for AD neurodegeneration.To further clarify the effect and underlying mechanism of GHS-R1 a deficiency on AD-related memory impairment,we investigated the effect of GHS-R1 a deficiency or interference(Ghsr1a KO) on memory impairment in AD model mice.LPS-induced hippocampal neuroinflammation mice and APP/PS1 mutant mice were adopted in our study as two AD mice model.Our main findings were described as following:1.GHS-R1 a deficiency rescued memory deficit in LPS-induced hippocampal neuroinflammation mice.1.1 An open field(OF)test showed that hippocampal neuroinflammation mice,induced by either lateral ventricle injection or intraperitoneal injection of LPS,displayed similar locomotor activity as the control mice in the open filed arena.This result thus indicated that LPS-induced hippocampal neuroinflammation did not affect basal anxiety and locomotor activities(as shown in Fig.3 and Fig.7).1.2 A New object recognition(NOR)and a new object-place recognition(NPR)test showed that LPS-induced hippocampal neuroinflammatory mice displayed lower recognition memory index than control mice,indicating that LPS injection induced hippocampal neuroinflammation and cause both object memory and object-place memory deficits(as shown in Fig.4 and Fig.8).1.3 A Morris water maze(MWM)test showed that LPS pre-injection impaired spatial memory formation,but had no effect on spatial learning process(as shown in Fig.5 and Fig.9).In addition,LPS administrated after spatial memory formation impaired spatial memory retrieval(as shown in Fig.6).1.4 An OF test showed that Ghsr1 a KO mice and WT mice after LPS injection exhibited similar performance in open field arena,indicating that GHS-R1 a deficiency does not affect basal anxiety or locomotor activity in those mice(as shown in Fig.10 and Fig.14).1.5 A NOR and a NPR test showed that LPS-injected Ghsr1 a KO mice displayed higher recognition memory index than control,LPS-injected WT mice.This result thus indicated that GHS-R1 a deficiency alleviated recognition memory deficit caused by LPS-induced hippocampal neuroinflammation(as shown in Fig.11 and Fig.15).1.6 A MWM test showed that Ghsr1 a KO mice receiving LPS injection exhibited significantly higher percentage of exploration time in the target quadrant than other three quadrants,while wild-type control mice receiving LPS injection displayed even percentage of exploration time in four quadrants.This finding thus indicated that GHS-R1 a deficiency alleviated LPS-induced spatial memory deficit(as shown in Fig.12 and Fig.16).In addition,GHS-R1 a deficiency rescued spatial memory retrieval deficit induced by LPS injection before memory retrieval(as shown in Fig.13).2.Interfering with GHS-R1 a expression in hippocampal d CA1 partially improved memory impairment in APP/PS1 mice.2.1 APP/PS1 mice showed normal basal anxiety and normal locomotor activity in an OF test(as shown in Fig.17).2.2 APP/PS1 mice showed lower recognition memory index than WT control mice,suggesting that APP/PS1 mice had object-memory and object-place memory deficit(as shown in Fig.18).2.3 APP/PS1 mice displayed spatial memory impairment(as shown in Fig.19).2.4 Interfering with Ghsr1 a expression in the hippocampal dCA1 does not affect basal anxiety or locomotor activity in APP/PS1 mice(as shown in Fig.20).2.5 Interfering with Ghsr1 a expression in the hippocampal d CA1 rescued recognition memory deficit in APP/PS1 mice.APP/PS1 mice infected by Ghsr1a-sh RNA virus displayed higher recognition memory index than APP/PS1 mice infected by control virus(as shown in Fig.21).2.6 Interfering with Ghsr1 a expression in the hippocampal dCA1 could not rescue spatial memory impairment in APP/PS1 mice(as shown in Fig.22).3.GHS-R1 a deficiency suppressed LPS-induced neuroinflammation and microglia activation in the hippocampus.3.1 qRT-PCR analysis revealed that LPS injection led to elevated m RNA expression of microglia markers Iba-1 and Lgals3 in the hippocampus(as shown in Fig.23).3.2 q RT-PCR analysis revealed that the expressions of microglia markers Iba-1 and Lgals3 were reduced in the hippocampus of Ghsr1 a KO mice after LPS injection,in comparison to that in WT mice after LPS injection(as shown in Fig.24).In summary,our study demonstrated that GHS-R1 a deficiency ameliorates hippocampus-dependent recognition memory impairment and spatial memory deficit in both LPS-induced hippocampal neuroinflammation mice and APP/PS1 transgenic mice.The inhibitory effect of GHS-R1 a deficiency on hippocampal neuroinflammation and microglia proinflammatory activation may contribute to its memory-improving effect on AD model mice..Further studies are still required to elucidate the underlying molecular and cell mechanisms.