Mechanisms Of Novel BACE1 Substrates In Cognitive Impairment

Alzheimer’s disease(AD)is the most typical disease that accounts for dementia.Excessive Aβ plaques,neurofibrillary tangles triggered by phosphorylated tau as well as reactive neuroinflammation are classical hallmarks of AD pathology.P-site APP cleaving enzyme 1(BACE1)is the rate-limiting enzyme for Aβ production by cleaving APP followed by y-secretase.Increased BACE1 protein levels and enzymatic activity for its classical substrate APP have been found in the brain parenchyma,cerebrospinal fluid and blood of patients with AD.Thus BACE1 is considered as a predictive biomarker for AD.Recently,novel substrates of BACE1 have been found in the central nervous system(CNS)and peripheral tissues.However,physiological and pathological roles of these substrates remain unclear.Imbalance of excitatory-inhibitory network is considered as a trigger of brain dysfunction in AD.Inhibitory function in the brain mainly depend on GABA binding to GABA receptors to induce the influx of chloride ions.Three GABA receptors have been found in the nervous system,particularly,GABAA receptor(GABAAR)is the major inhibitory ionotropic receptor in the CNS.GABAAR is widely distributed in various brain regions,especially in the hippocampus.The decreased expression of GABAAR is considered to be an important mechanism that impairs inhibitory function in AD brain.To study the underlying mechanism of the alteration of GABAA receptors in AD,we detected the expression of GABAAR in AD patients and AD mouse models(APP23)by Western blotting and immunofluorescence,and found that the expression of GABAARα1,α5 and γ2 subunits were relatively unaffected,while three β subunits(β1-3)were strongly reduced.Importantly,we found the abnormally increased N-terminal and C-terminal fragments of three β subunits in the AD brains as well as AD mouse brains.Additionally,the decrease of β subunits was tightly correlated with the abnormally elevated BACE1 proteins,suggesting that BACE1 regulates the expression of GABAARβ subunits.To further address this scientific question,BACE1 was overexpressed via AAV-BACE1 transfection or knockdown by RNAi technology.We also used BACE1 inhibitor C3 to inhibit BACE1 enzymatic activity in primary cultured neurons.We found full lengths of three GABAARβ subunits were strongly increased but C-terminal fragments were significantly reduced when BACE1 expression or enzymatic activity was inhibited.However,BACE1 overexpression did decrease the expression of three GABAARβ subunits.These results further indicated that BACE1 regulates the expression of β subunits but not α1 subunit.To determine the underlying mechanism of BACE1 in regulating β subunits,we co-transfected BACE1 with each of three β subunits in HEK293 cells,respectively.We found that elevation of BACE1 specifically reduced the full length of three β subunits,whereas all of three corresponding C-terminal fragments were increased.However,the full length of βsubunits were significantly restored and C-terminal fragments were decreased when BACE1 was knockout or knockdown or was inhibited by using BACE1 inhibitor C3 treatment in HEK293 cells,suggesting that BACE1 cleaves GABAARβ1-3 in vitro.Moreover,similar results were also confirmed in BACE1 transgenic mouse models(HUBC-BACE1)and BACE1 knockout mice(BACE1-/-).Critically,the cleavage site of BACE1 was determined between L234(235 for β1/3)and S235(236 for β1/3)by using in-vitro digestion combined with liquid chromatography-mass spectrometry(LC-MS).Furthermore,BACE1 cleavage capacity could be blocked by single point mutations of L234/235/A.Together,we found the decreased expression of GABAARβ 1-3 subunits in AD patients and AD mouse models.Then,we performed in vitro and in vivo studies and demonstrated that GABAARβ 1-3 subunits were novel substrates of BACE1.The specific cleavage site of BACE1 was identified by in-vitro digestion and LC-MS.Thus,abnormally increased BACE1 caused the loss of GABAA receptors in AD.Lastly,we found that the loss of β subunits mainly occurred in excitatory neurons.The result further suggests that the abnormal cleavage of GABAARβ1-3 subunits by BACE1 results in impaired inhibitory function,which contributes to neuronal hyperactivity,promoting the occurrence of AD.In the early stage of AD,abnormal rhythmic oscillations and seizure-like discharges were found,and these were considered to be the main causes of cognitive impairment.Thus,BACE1 mediated GABAARβ1-3 subunits cleavage may be a potential target for AD therapy.Type 2 diabetes mellitus(T2DM)is a chronic metabolic disorder with a high prevalence,which is characterized by poorly controlling blood glucose metabolism.Particularly,insulin resistance is considered as an important risk factor that contributes to many complications,including cognitive impairment.Studies reported that the expression of BACE1 was abnormally upregulated in diabetic mice.In the current study,we have found that typical diabetic phenotypes in BACE1 transgenic mice.Importantly,high insulin sensitivity in BACE1-/-mice,suggesting that BACE 1 regulates insulin signaling.To examine whether plasma BACE1 was involved in the development of T2DM,we recruited a clinical cohort including patients with T2DM alone,T2DM patients with cognitive impairment,patients with cognitive impairment alone as well as healthy controls.Interestingly,we found that plasma BACE1 protein levels were significantly elevated in patients with T2DM alone and T2DM patients with cognitive impairment.In further study,we found high BACE1 expression correlated with hyperglycemic status.These findings indicated that BACE1 plays an important pathological role in T2DM.Insulin resistance is a typical feature of T2DM.However,whether BACE1 is involved in the development of insulin resistance remain unclear.In the present study,we found that plasma BACE1 cleavage activity for insulin receptor(INSR)were only significantly increased in patients with T2DM alone and T2DM patients with cognitive impairment compared to healthy controls or patients with cognitive impairment alone.BACE1-mediated INSR-β cleavage can release soluble INSR(sINSR).To quantitively measure sINSR,we developed ELISA to detect plasma sINSR levels in different groups.Consistent with BACE 1 enzymatic activity to INSR,elevated sINSR levels were only found in patients with T2DM alone and T2DM patients with cognitive impairment.Additionally,positive correlations were found between BACE1-mediated INSR cleavage and insulin resistance indices,suggesting that BACE1 mediated INSR cleavage were involved in the development of insulin resistance.Moreover,sINSR could effectively discriminate T2DM with cognitive impairment from patients with cognitive impairment alone.It was well established that insulin resistance contributes to cognitive impairment through hyperphosphorylation of tau and neuroinflammation.Collectively,we propose that BACE1 may trigger insulin resistance via abnormally cleaving INSR,which plays an important role in T2DM and T2DM-related cognitive impairment.BACE1-mediated INSR cleavage could be considered as a biomarker for accurate diagnosis and monitoring therapeutic courses during treatment of cognitive impairment in T2DM patients...