Down-regulation Of Astrocytic Nav1.6 Improves Cognitive Function In Alzheimer’s Disease And Its Mechanism Study

Background:Alzheimer’s disease（AD）is one of the most common neurodegenerative disorders.The two main pathological features of AD were amyloid plaques（Aβ）deposition and neurofibrillary tangles.Aβ（especially Aβ42）is the key molecule in the pathogenesis of AD,which has strong neurotoxicity and is also the main component of amyloid plaques in the brain of AD patients.The metabolic precursor of Aβis amyloid precursor protein（APP）,which also plays an important role in the prevention and treatment of AD.The etiology of AD is complex and unclear,but the Aβhypothesis still dominates so far.There were a large number of reactive astrocytes around the plaques,and the presence of reactive astrocytes also promoted the deposition of Aβin the early stage of AD.In addition,the astrocytes themselves expressed certain levels of APP,βsecretase andγsecretase.The development of AD may take decades,and the number of astrocytes is at least five times that of neurons.Therefore,astrocyte-derived APP may have a significant effect on the accumulation of Aβin AD.Voltage-gated sodium channel（Nav）is not only a key channel for the initiation of action potentials but also neuronal excitability.Nav is closely associated with neurological diseases.At least nine different sodium channel subtypes（NAV1.1-NAV1.3,NAV1.5-NAV1.9,and Nav X）have been detected in the peripheral and central nervous systems.Among them,Nav1.6 is the most densely expressed sodium channel in the adult central nervous system and is a key determinant of neuronal excitability in vivo.It is expressed in a variety of cells,including neurons and glial cells.Nav1.6 is closely related to APP.Nav1.6 and APP co-locate and interact with each other in mouse cortical neurons,and APP can enhance the expression and current of Nav1.6.In conclusion,Nav1.6 on astrocytes may play an important role in the occurrence and development of AD.This study mainly explored the effects of astrocytic Nav1.6 on pathological and functional phenotypes of APP/PS1 transgenic mice.Methods:1.The constructed target knockdown of astrocytic Nav1.6 or just the empty AAV virus targeting astrocytes was used to create an animal model by stereotaxic injection of the bilateral hippocampus.The names of the viral vectors are AAV-Gfa ABC1D-m Cherry-mi R30sh RNA-Nav1.6-WPRE and AAV-Gfa ABC1D-m Cherry-mi R30sh RNA-Nav1.6-WPRE,respectively.2.Behavioral experiments were used to test the learning and memory ability of mice.New object recognition（NOR）,Y maze（Y maze）,Morris water maze（MWM）and passive avoidance（PA）experiments were used to detect novelty-induced exploration ability,short-term memory,long-term memory and fear memory in mice,respectively.3.The brain slices patch clamp was used to detect the excitability of the brain for recording the action potentials.4.The pathological changes of mice were detected by immunohistochemistry,and the expression levels of two glial markers GFAP and Iba1 in mouse hippocampus were detected by Western blot.Confocal detects the morphological changes of activated glial cells around Aβplaques.5.Immunohistochemical was used to detect the Aβplaque deposition in APP/PS1 mice.The expressions of proteins related to amyloid processing（BACE,APP and s APPβ）were detected by Western blot.6 Scanning electron microscope was used to detect the autophagosome and autolysosome of APP/PS1 mice,and Western blot was used to detect the autophagy-lysosome-related proteins of APP/PS1 mice（ATG5,ATG7,LC3Ⅱ/Ⅰ,P62,Lamp1）.7.Western blot detection of APP/PS1 mice and primary cultured astrocytes upstream of autophagy-related signaling pathway proteins（p-ERK,ERK,p-AKT,AKT,p-m TOR,m TOR,p-ULK,ULK）.8.Nav1.6 knockout primary astrocytes were cultured in vitro,and Western blot was used to detect the total protein,the membrane protein of astrocytic Nav1.6.The expression and distribution of Nav1.6in astrocytes changed.The expression of autophagy-lysosome-related proteins（ATG5,ATG7,LC3II/I,P62,Lamp1）was also detected.9.HEK293 cells overexpressing Nav1.6 and primary astrocytes with Nav1.6 knockout were cultured in vitro,the whole-cell patch clamp was used to record calcium ion current and sodium-calcium exchange current,and FLUO-4AM fluorescence was used to detect intracellular calcium,and the fusion of autophagosomes and lysosomes were detected by confocal laser scanning.10.Immunofluorescence was used to observe the localization of APP and lysosome in astrocytes after knockdown of Nav1.6 in astrocytes.After adding the autophagy inhibitor 3-MA or the lysosome inhibitor CQ,the effect of the inhibition of the autophagy-lysosome on the expression of APP protein was detected.Results:Part 1.Effects of down-regulation of astrocytic Nav1.6 on cognitive function and neural network in APP/PS1 mice1.1.Down-regulation of astrocytic Nav1.6 improved cognitive function in APP/PS1 miceNovel object recognition experiments showed that down-regulation of astrocytic Nav1.6 could improve the exploratory ability of APP/PS1 mice;the Y-maze experiment showed that down-regulation of astrocytic Nav1.6 could improve the exploratory short-term memory ability of APP/PS1 mice.Water maze experiments showed that down-regulation of astrocytic Nav1.6 could improve long-term spatial learning and memory in APP/PS1 mice.Passive avoidance experiments showed that down-regulation of astrocytic Nav1.6 could improve the memory of fearful situations in APP/PS1 mice.1.2.Down-regulation of astrocytic Nav1.6 improves the abnormal excitation of neural network in APP/PS1 miceBrain slice patch-clamp recording of excitatory postsynaptic potentials and neuronal action potentials showed that the frequency of s EPSCs in APP/PS1 mice was significantly reduced after down-regulation of astrocytic Nav1.6,and the magnitude of the amplitude did not change significantly but the distribution narrow.And after down-regulation of Nav1.6 in astrocytes,the number of APs in APP/PS1 mice decreased and the threshold of AP increased,indicating that the abnormal excitation of the neural network was alleviated.Part 2.Effects of down-regulation of astrocytic Nav1.6 on glial cells and Aβdeposition in APP/PS1 mice2.1.The effect of down-regulation of astrocytic Nav1.6 on glial cells of APP/PS1 miceAfter down-regulation of Nav1.6 in hippocampal astrocytes of APP/PS1 mice,the areas of GFAP-positive and Iba1-positive cells in the hippocampus and cortex were less than those in the APP/PS1group,and the protein expressions of GFAP and Iba1 were compared with those in APP/PS1 group was significantly reduced.In addition,the total area and neurite length of peri-plaque astrocytes were significantly reduced,and the total area of microglia was decreased with an increase in total neurite length.This indicates that down-regulation of Nav1.6 in hippocampal astrocytes reduces the overall level of APP/PS1 mice and the activation of glial cells and cell morphology around plaques.2.2.The effect of down-regulation of astrocytic Nav1.6 on Aβplaque deposition in APP/PS1micDown-regulation of astrocytic Nav1.6 significantly reduced the number and area of Aβplaques in the hippocampus of APP/PS1 mice,as well as the area of cortical plaques,but had no significant change in the number of cortical plaques.After down-regulation of astrocytic Nav1.6,the protein expression levels of APP,BACE1 and s APPβin APP/PS1 mice were significantly decreased.It suggested that the decreased deposition of Aβafter down-regulation of Nav1.6 in astrocytes may be caused by the decreased expression of APP and its related splicing proteins.Part 3.The mechanism of down-regulation astrocytic Nav1.6 to inhibit the production of Aβin APP/PS1 mice3.1.Astrocytic Nav1.6 regulates autophagy-lysosome via the m TOR-like pathway3.1.1 Down-regulation of Nav1.6 in astrocytes promotes autophagy-lysosome fusion and the expression of autophagy-related proteinsDown-regulation of astrocytic Nav.6 promoted the autophagy-lysosome formation and reversed the abnormal expression of autophagy-lysosome-related proteins in APP/PS1 mice.In vitro experiments also confirmed that knockdown of astrocytic Nav1.6 promoted the normalization of autophagy-lysosome-related protein expression in primary cultured astrocytes exposed to Aβ,and also promoted autophagosome and lysosomal fusion.3.1.2 Down-regulation of Nav1.6 in astrocytes reduces activation of AKT/m TOR/ULK signaling pathwayDown-regulation of astrocyte Nav.6 regulates the autophagy-lysosomal pathway through the AKT/m TOR/ULK signaling pathway but not through the Ras/Raf/MEK/ERK/m TORC1 signaling pathway in APP/PS1 mice.In vitro experiments with primary cultured astrocytes also yielded the same results.3.2.Astrocytic Nav1.6 regulates autophagy-lysosome via a non-m TOR-like pathwa3.2.1Aβpromotes the increased expression of Nav1.6 on the membrane of astrocytes and the enhancement of currentThe total protein expression of Nav1.6 on astrocytes did not change significantly after Aβstimulation,but the expression of it in membrane protein increased.The results of immunofluorescence also showed that Nav1.6 had a tendency to migrate to the membrane.The Nav1.6 current was significantly increased by Aβtreatment of the Nav1.6-overexpressing cell lines.In primary cultured astrocytes,astrocyte sodium current was reduced after Nav1.6 knockdown,indicating that Nav1.6 is an important component of sodium current on astrocytes.3.2.2.Nav1.6 regulates autophagy by regulating intracellular calcium through sodium-calcium convertorAβ-stimulated overexpressing Nav1.6 cell line significantly increased the level of intracellular calcium.In primary cultured astrocytes,Aβenhanced the level of intracellular calcium,while knockdown of Nav1.6 reduced the level of intracellular calcium.The expression of intracellular calcium was reduced after the addition of sodium-calcium exchange inhibitors.Aβ-stimulated overexpressing Nav1.6 cell line significantly increased the level of intracellular calcium.In primary cultured astrocytes,Aβenhanced the level of intracellular calcium,while knockdown of Nav1.6reduced the level of intracellular calcium.The expression of intracellular calcium was reduced after the addition of sodium-calcium exchange inhibitors.3.3.Down-regulation of astrocytic Nav1.6 can promote astrocyte-lysosomal fusion of APPAfter knocking out Nav1.6,the number of binding of APP and lamp1 in astrocytes was significantly increased,and there was a significant co-localization of the two.Knockdown of Nav1.6in astrocytes can significantly reduce the expression of APP in cells.After adding autophagosome inhibitor 3-MA（5 m M）or lysosome inhibitor CQ（40μM）,the expression of APP increased,indicating that knockdown of Nav1.6 in astrocytes can reduce the expression of endogenous APP by inhibiting the autophagy-lysosomal pathway.Conclusion:Astrocytic Nav1.6 improves cognitive function and Aβdeposition in APP/PS1 mice,which may be related to the extensive regulation of autophagy by knockdown of astrocytic Nav1.6 through AKT/m TOR/ULK signaling pathway and NCX/Ca²⁺.The lysosomal pathway promotes the degradation of APP,which in turn affects the production of Aβ.