Impact of corticotropin-releasing factor receptor 1 on alzheimer’s disease

, ,

(1.Department of Physiology,Zunyi Medical University,Zunyi Guizhou 563099，China；2.Department of Psychiatry and Behavioral Sciences,Northwestern University ,Chicago 60611，USA)

Impact of corticotropin-releasing factor receptor 1 on alzheimer’s disease

YanYan1,2,ChenYuanshou1,DongHongxin1,2

(1.Department of Physiology,Zunyi Medical University,Zunyi Guizhou 563099，China；2.Department of Psychiatry and Behavioral Sciences,Northwestern University ,Chicago 60611，USA)

Growing evidence suggests that stress-induced release of corticotrophin-releasing factor (CRF) and subsequent effects through CRF receptor 1(CRFR1),have been linked to Alzheimer's disease (AD) onset and progression.The main function of CRFR1 is to act as a key regulator of stress-induced changes of the hypothalamic-pituitary-adrenal (HPA) axis.However,given the fact that CRFR1 is widely located in brain areas that are associated with emotion and cognition,additional functions of CRF/CRFR1 signaling have been proposed,including the regulation of AD neuropathology.In particular,specific downstream signaling of CRFR1 can promote amyloid β-protein accumulation and tau phosphorylation,which can be blocked by CRFR1 antagonist.Thus,CRFR1 antagonists may have therapeutic potential for the prevention and treatment of AD.So we reviewed current literature and our studies to clarify the relationship between CRF/CRFR1 signaling and AD.

stress;corticotropin-releasing factor (CRF);corticotropin-releasing factor receptor 1(CRFR1); CRFR1 antagonist;alzheimer`s disease

1 Introduction

Increasing evidence suggests that,in addition to genetic,environmental factors also markedly affect the development of Alzheimer’s disease (AD).Chronic psychosocial stress is believed to be one of the environmental factors that can significantly accelerate the onset and progression of AD[1-5].Clinical and preclinical studies support this hypothesis as plasma cortisol levels and brain corticotrophin-releasing factor (CRF) and CRF receptor 1 (CRFR1) expression,the main markers and regulators of hypothalamic-pituitary-adrenal (HPA) axis activity,are elevated in AD patients[6-10].Results from preclinical studies also show that stress and stress hormones accelerate amyloid levels and aggregation[11-13],and increase hippocampal tau phosphorylation (tau-P)[14-16].CRFR1 antagonists reverse stress-induced these abnormalities[15,17-21].These studies indicate that CRF/CRFR1 and their downstream signaling cascades,which include targets such as the adenylyl cyclase-cAMP signaling pathway,PKA-dependent protein phosphorylation and CREB-dependent transcription regulation,may play a significant role in the development of Alzheimer`s disease.In this review we will examine the role of CRFR1 signaling in Alzheimer`s disease pathogenesis based on the current available literature,with a focus on preclinical studies.

2 Stress,stress hormones and AD

Stress is a physical reaction to an emotional or physical stimulus (i.e.the reaction to an unpleasant social interaction or escaping from a predator.Stress-mediated effects on brain function can be beneficial or detrimental,depending on the intensity and duration of the stressor,as well as the developmental stage of the organism.The hypothalamic-pituitary-adrenal (HPA) axis is one of the primary effectors of the stress response in mammals,and allows an organism to adapt to changes in its internal or external environment[22].Under ordinary circumstances,secretion of stress hormones,cortisol (in humans) and corticosterone (in rodents),enables the organism to adapt physically and mentally to the stressor.However,HPA axis dysregulation during chronic stress can result in a number of neuropsychological disorders such as anxiety and depression[23-24].While the impact of stress on neurodegeneration has been studied for many years[25-27],the impact of stress on the neuropathogenesis of AD has only recently been appreciated[15,19].Stress responses may be enhanced in AD patients because of decreased resilience due to age,other health problems,and/or limited resources.Increases in plasma cortisol levels have been reported in AD patients[7-8].Furthermore,there are significant correlations between increased 24h cortisol levels and the severity of cognitive decline in AD patients[28].These changes in the HPA axis do not appear to be secondary to depression as AD patients with and without depression have higher cerebrospinal fluid cortisol levels compared to controls[29].A reduction in CRF immunoreactive cells has been reported in the brain of AD patients[30].In addition,increases in the density of CRF receptors have been observed,possibly in response to disinhibition of the HPA axis triggered by AD-related hippocampal degeneration.HPA axis changes have been associated with hippocampal atrophy in AD patients[31]and dysregulation of the HPA axis may be responsible for the abnormal behaviors exhibited by AD patients[32].While one report suggests that stress plays a causal role in a preclinical model of AD[33],most literature supports the hypothesis that stress functions as an environmental factor that can accelerate the progression of the disease in vulnerable individuals[22,25,34-35].

Post-mortem studies of AD patients have not yet been helpful in elucidating the effects of stress on the development of AD; in one report,ante-mortem distress was associated with late-life dementia,but not with any specific form of neuropathology[35].Advanced neuro-imaging with Aβ plaque labeling techniques may provide a more accurate way to reveal the impact of chronic stress on the pathogenesis of AD patients.However,genetically manipulated animal models that parallel some of the neuropathological changes of AD provide an excellent opportunity to investigate the impact of stress on AD pathogenesis and to discover the molecular mechanisms underlying these effects.The Tg2576 mouse model,which contains the Swedish AD mutant gene,is arguably the most well-established mouse model of AD.This mouse over-expresses the human amyloid precursor protein (APP) 695 and,at 9-10 months of age,shows Aβ plaques in the cortex and hippocampus that coincide with behavioral deficits associated with AD[36].Studies of Tg2576 and APPV717I-CT100 transgenic mice suggest that behavioral stressors accelerate the production of Aβ and its incorporation into Aβ plaques[11,13].Furthermore,treatment of triple transgenic APP/PS1/MAPT mice with dexamethasone increases brain APP and Aβ levels,as well as β-secretase and the β-CTF of APP[12].Our group and others have reported that behavioral stressors increase Aβ levels via the activation of CRF/CRFR1 and concomitant increases in neuronal activity[15,18-19,37].However,the underlying mechanisms linking CRF/CRFR1 to the pathogenesis of AD remain largely speculative.

3 The dual function of CRF in the HPA axis and brain

CRF and CRFR1 are critical regulators of HPA axis activity in response to stress and modulators of neuronal activity throughout the brain.Following stress inducing stimuli,neurons within the paraventricular nucleus of the hypothalamus (PVN) and release CRF into the pituitary portal system causing the release of adrenocorticotropic hormone (ACTH) from the pituitary,which in turn,causes the release of glucocorticoids (GCs) from the adrenal cortex.GCs act on the hypothalamus and pituitary to suppress CRF and ACTH production in a negative-feedback loop to terminate the stress response after a threat has passed,thereby preventing prolonged responses that could lead to pathological conditions[38](Fig 1).

CRF is synthesized and released in PVN via HPA release ACTH, which in turn cause GCs release. However, GCs can act like negative feedback on HPA or activation link to stress response in extra-hypothalamus, such as in hippocampus, cortex and amygdala. Fig 1 CRF associated stress regulation and neuronal modulation

CRF has a second,interrelated function in the central nervous system.CRF and CRFR1 are extensively distributed throughout the cortex,hippocampus and amygdala.At those extra-hypothalamic sites,CRF exerts a modulatory effect on the neuronal activity associated with stress-sensitive processes,such as memory and anxiety[39-40].Additionally,CRF has been shown to play a role in nerve cell survival under neuro-toxic conditions[41]and prevents kainate-induced neurodegeneration in the hippocampus[42].However,the dual roles of CRF as a critical stress regulator of the HPA axis and a neuromodulator in the CNS,as well as its actions on neuroprotection and neurodegeneration in the brain are not yet well understood.

4 CRF/CRFR1 downstream signal transduction pathways

CRF is synthesized in parvocellular neuroendocrine cells,stored in axon terminals,and released from neurosecretory terminals onto the cell bodies and axon initial segments of pyramidal cells.CRF exerts its cellular effects by activating one of two known G-protein-coupled receptors (GPCRs),CRFR1 and CRFR2[43].Since CRF binds to CRFR1 with a ～40-fold higher affinity,most of its activity in the CNS has been attributed to the activation of CRFR1[44].CRFR1 is expressed in hippocampus and prefrontal cortex,which specially expressed in the post-synaptic density of excitatory synapses on dendritic spines in pyramidal neurons[45].CRF can exert its action through several different classes of G-proteins,affecting downstream signaling cascades including cAMP/PKA and PLC/PKC pathways[46- 47].Subsequently,these effects can lead to a reduction in neuronal firing and affect brain function accordingly[48- 49].

4.1 CRFR1-PKA signaling pathway Evidence indicates that the dominant mode of endogenous CRFR1 signaling is activation of the adenylyl cyclase-protein kinase A (PKA) pathway.CRF binds to membrane-bound CRFR1,increasing the affinity of the α subunit to the Gs protein.The agonist-activated coupling of Gαs to the third intracellular loop of CRFR1 stimulates adenylyl cyclase activity,producing the second messenger cyclic AMP (cAMP).Activation of PKA by cAMP results in the phosphorylation of the transcription factor cAMP response element-binding protein (CREB),the main transcriptional regular for downstream PKA signaling involving gene expression[50- 51](Fig 2).

CRFR1 can via PKA/PKC cascades, ERK-MAPK cascades and β-arrestin cascades to regulate downstream signaling transduction. Fig 2 The intracellular signal transduction pathways of CRFR1

CRFR1-driven cAMP/PKA pathway can diverge through activation of other downstream signalling molecules,including membrane guanylyl cyclase,the NF-κB transcription factor,glycogen synthase kinase-3 and the Wnt/β-catenin pathway,K(+) current [I(sAHP)],ERK1/2 and tyrosine hydroxylase.In addition,the cAMP/PKA pathway may have a role in this mechanism by activating GRK2 through phosphorylation[52-53].CRF/CRFR1 also couple to the Gq protein,stimulating phospholipase C activity and intracellular calcium mobilization,in turn,leading to phosphorylation of phospholipase protein kinase C (PKC) which also can phosphorylate cytosolic proteins,modulates gene transcription,and influence cell excitability[54].

In stress response,cellular CRF-related cAMP/PKA cascade activation regulates neuronal activity.For instance,CRF can suppress Ca(2+)-activated K(+) current in CA1 hippocampal pyramidal neurons to affect calcium mobilization[43].CRF also involves gene transcriptional activation and expression by cAMP/PKA signaling pathway[55].Additionally,stress-induce response activation of CRFR1 in the The Bed Nucleus of the Stria Terminalis (BNST) induces startle hyperreactivity which is mediated by PKC pathway activation[56].

4.2 CRFR1- β-arrestin signaling pathway As a class B GPCR,CRFR1 can interact with the β-arrestin,predominately with β-arrestin2.The association of CRFR1 with β-arrestin2 promotes internalization into endosomes where it can be recycled back to the plasma membrane or degraded by lysosomes,resulting in receptor downregulation[57].Some phosphorylated GPCRs remain at the membrane and rapidly rebind β-arrestin2 when the agonist re-stimulate (Fig 2).Interaction of CRFR1- β-arrestin2 can result in aberrant trafficking and signaling in the β-arrestin2 pathway[58].Extracellular signal-regulated kinases1/2 (ERK1/2) plays an important role in stress-induce CRFR1-β-arrestin downstream signaling pathway[59].Phosphorylated on the carboxy tail serine/threonine residues by GRKs and third intracellular loop motif configured by agonist-induced changes in CRFR1 conformation are involved in β-arrestin2 recruitment[58].However,overexpressing GRK3 did not alter β-arrestin-2 recruitment or prevent dissociation of the CRFR1 receptor-arrestin complex[58].However,recruitment of β-arrestin 1 to the membrane following receptor activation is not a prerequisite for CRFR1 internalization[60].

4.3 CRFR1- ERK-MAPK Pathway Another important function of the CRF/CRFR1 is CRFR1 signaling via the extracellular signal-regulated kinases (ERK)- mitogen-activated protein kinase (MAPK) cascade regulates stress-induced synaptic function,possibly through disruption of the excitatory and inhibitory balance[39,61].This pathway has been shown to regulate synaptic plasticity,cognitive and emotional behaviors,and learning and memory processes[58]through regulation of BDNF signaling,dendritic stabilization,ion channel transmission,CREB transcription,scaffolding protein interactions,protein trafficking.ERK-MAPK signaling pathway is activated following restraint stress in the frontal cortex and hippocampal CA1 and CA3 pyramidal neurons,likely as a result of CRFR1 signaling through CREB activation of this pathway[62](Fig 2).One reported that ERK1/2 phosphorylation was increased in hypothalamic tissue under stress conditions[63].Another study identified that ERK1/2 may regulate CRHR1α overexpression through PKC pathway.Finally,CRF-ERK1/2 signaling selectivity and cellular response may be mediated by multiple phosphatidylinositol 3-kinase (PI3-K) subunits,a potentially,important mediator of CRF/CRFR1-ERK1/2 signaling pathway[52].

5 Regulation of CRFR1 in Alzheimer`s Disease

As we have addressed above,both clinic and preclinical evidence suggests that stress may affect AD through CRF/CRFR1 signaling.We and some other researchers demonstrate that CRFR1 signaling modulates AD neuropathogenesis,such as amyloid-beta and phosphorylation tau,although most of these results are most from the animal model studies.Specifically,we found that chronic isolation stress increases Aβ production in Tg2576 mice through increases in CRF transmission[13,19,37].Our work and others also suggest that CRFR1 signaling pathways such as cAMP/PKA,PLC/PKC and (GSK)-3β contribute to Aβ accumulation and tau phosphorylation[20].

5.1 CRFR1 and Aβ deposition The amyloid β protein (Aβ) is the most important element in the development of AD and Aβ deposition in the brain is one of the hallmarks of AD neuropathology.It is believed that the neurotoxicity of Aβ is one of the causes underlying neuronal degeneration and memory loss.Aβ is generated from amyloid- precursor protein (APP).APP can be cleaved by three different secretases:(1) α-secretase cleaves APP within its Aβ domain to produce sAPP α,(2) β-secretase(BACE) cleaves the β-C-terminal fragment (β-CTF) of APP,producing Aβ40/Aβ42 peptides[64],the main components of Aβ plaques,and (3) γ-secretase cleaves β-CTF and 38,40,or 42 amino acid Aβ is released[12,65- 66].

Using the Tg2576 mouse model of AD,we observed accelerated accumulation of Aβ plaques throughout the cortex and hippocampus along with impaired memory-related behavior after exposure to chronic isolation from weaning until 7 months of age[13,37,67].In a follow-up microdialysis study,we found significant increases in interstitial fluid (ISF) levels of Aβ following exposure to acute restraint stress,as well as chronic isolation stress in Tg2576 mice.Moreover,administration of exogenous CRF,but not corticosterone,through a microdialysis probe to Tg2576 mice mimicked the effects of behavioral stressors[19].Chronic administration of CRFR1 antagonists decreased tissue levels of Aβ and plaque deposition in Tg2576 mice[37].

To investigate whether endogenous increases in CRF can recapitulate the effects of stress and exogenous administration of CRF on Aβ production and plaque formation,we created a novel triple transgenic mouse model that conditionally overexpresses CRF in the forebrain via a “tet-on” system,while concomitantly overexpressing a mutant version of human APP (Tg2576 mouse strain).These triple transgenic mice are referred to as APP+/CRF+/tTA+,or TT mice[68].Although TT mice develop normally,they display significant anxiety and memory-related deficits compared to Tg2576 and wild type mice[68].ELISA analysis showed that TT mice had higher levels of both soluble and insoluble Aβ at 7 months of age and immunohistochemistry revealed substantially increased Aβ plaque deposition in the cortex and hippocampus,which was reversed when CRF overexpression was turned off via doxycycline.

Specific GPCR activation has been associated with multiple stages of APP proteolysis,regulation of Aβ generation,and Aβ-mediated toxicity[69-70].GPCRs have been suggested as novel targets for AD drug development[71-72].Gs-PKA signaling pathways play an important role in the pathophysiology of AD[69,73-79]and PKA can influence the proteolysis of APP and the amyloid production cascade through modulation of α-,β- and γ secretases[69-70,80-81].While transient activation of these signaling cascades shifts APP metabolism towards the α secretase-mediated pathway that results in non-pathogenic amyloids,chronic activation shifts APP metabolism to the β- and γ secretase,perhaps also γ-secretase mediated pathways,that results in increased pathogenic Aβ generation[79,82].Additionally,PKA signaling is associated with tau phosphorylation,another molecular pathway that is highly implicated in AD pathogenesis[83-84].

Since Gs signaling is central to the formation of AD-causative amyloids and phospho-tau,CRFR1-Gs-PKA signaling could be a key pathway involved in AD pathogenesis by accelerating the rate of disease progression and cognitive decline in AD.Further understanding of this pathway could lead to novel therapeutic targets for the prevention and treatment of AD.Our previous studies support this hypothesis.We found that in neurons isolated from the hippocampus of Tg2576 mice and exposed to different concentrations of CRF,there was a significant increase in Aβ levels.Co-treatment with the CRFR1 antagonist,antalarmin,blocked the CRF-induced increase in Aβ levels.Since PKA is activated by CRFR1 in neuronal systems[85-86],and the PKAIIβ isoform is a major downstream target of CRFR1,we selected this isoform to investigate the involvement of PKA-signaling in CRF/CRFR1 activity and Aβ production.As predicted,co-treatment of Tg2576 hippocampal neurons with CRF and PKA inhibitors H-89 reduced the CRF-mediated rise in Aβ levels.In addition,48 hours of CRF stimulation resulted in a significant increase in PKAIIβ expression in hippocampal neurons,which was blocked by antalarmin or H-89 co-treatment,and levels of Aβ42 were strongly correlated with PKA expression(Fig 3).

Growing evidence indicate that CRF/CRFR1-Gs-PKA directly affect amyloid related neuropathogenesis. CRF regulates tau-P through BNDF -GSK-3-CREB signaling pathways. Moreover, CRF can also via CRFR1-PKA/PKC pathways induce tau-P. Fig 3 CRF/CRFR1 signaling pathways are involved in the AD neuropathology

Stress-induced Aβ formation in cells can be modulated by CRFR1 associated γ-secretase activity through both β-arrestin1 and β-arrestin2 binding motifs[81].Reports suggest that decrease translocation of β-arrestin2 to CRFR1 exhibited a reduced interaction with γ-secretase[58].Additionally,G protein-coupled receptor 3 (GPR3) and the β2-adrenergic receptor regulate γ-secretase activity and Aβ through recruitment and binding of β-arrestin2[81].

5.2 CRFR1 and tau phosphorylation Tau,a microtubule associated protein,stabilization of microtubules contributes to the maintenance of neuronal structure,polarity,and axon transport[87].Intracellular neurofibrillary tangles (NFTs),another important factor in AD,are composed of hyperphosphorylated and aggregated forms of tau.Growing studies indicate that stresses not only induce Aβ deposition,but also affects tau phosphorylation (tau-P) and CRF signaling contributes to these effects[88-91].It has been shown in hippocampus,CRFR1 overexpression can induce tau-P accumulation[89].Stress-induced tau-P is an essential process required for stress adaption that becomes dysfunctional with advancing age or chronic over-simulation[14,18].Glycogen synthase kinase (GSK)-3β is one of CRFR1 downstream targets mediated by brain-derived neurotrophic factor (BDNF),which regulates CREB phosphorylation and modifies several sites of the tau protein present in neurofibrillary tangles[92].Another study indicates that chronic noise stress exposure elevates the expression of the CRF system,which may contribute to AD-like changes[88].Using a phosphoproteomic approach in CRF-overexpressing mice,we demonstrated that CRF overexpression in females was associated with increased tau phosphorylation and phosphorylation of β-secretase,the enzyme involved in the formation of Aβ[91](Fig 3).

6 CRFR1 antagonists and AD

Given the l utility of targeting GPCR for therapeutic benefit,efforts have been underway to develop CRFR1 antagonists for the potential treatment of stress related disorders,including mood disorders[93-98].Preclinical studies and clinical trials with CRFR1 antagonists have been assessed for the treatment of depression,anxiety,and irritable bowel disorders.However,CRFR1 antagonists have had a poor record of success as anti-depressants,perhaps because of low bioavailability[93-97].Nonetheless,new CRFR1 antagonists are being developed with better pharmacodynamics and pharmacokinetic properties and may be available for clinical testing soon[94].The therapeutic potential of CRFR1 antagonists as treatments for neurodegenerative disorders,such as AD,as evaluated by both our group and others indicates that CRFR1 antagonists block stress induced Aβ accumulation[17,19-20].Additionally,there have been reports of the beneficial effects of CRFR1 antagonists on tau phosphorylation[15,18].In our previous work,we have evaluated the acute and long-term effects of CRFR1 antagonist administration on Aβ production,plaque deposition,and behavior in an animal model of AD[19-20].CRFR1 antagonists widely used in AD animal models,such as R-121919,can reduce cellular and synaptic deficits,A β and C-terminal fragment-β levels[99].Also,R-121919 can reduce S-glutathionylated proteins (Pr-SSG) and increase glutathione peroxide activity to combat oxidative stress in AD[100].Furthermore,studies show that CRFR1 antagonist can effect amyloid plaque deposition in Tg2576 mice under stressed conditions and antalarmin can affect Aβ levels via cAMP/PKA signaling pathways in hippocampal neurons[20].Another study suggested that CRFR1 antagonist may regulate AD neuropathology and synaptic function in a sex biased manner[21].These results suggested that CRFR1 antagonists may have therapeutic potential for treatment of AD.

7 Conclusion

CRFR1 signaling plays significant role in regulation of HPA axis to respond stress and to modulate neuronal activity in brain.CRFR1 signaling may contribute to the neuropathogenesis of AD,at least in individuals that are more susceptible to chronic stress.This review integrates convergent findings supporting the novel concept of CRFR1 signaling and trafficking as contributing factors to AD pathology.Identifying specific consequences of CRFR1 signaling has revealed molecular mechanisms by which this signaling pathway may contribute to stress-related disease and AD.This knowledge can further be used to elucidate the pathophysiology underlying these disease states,leading to more efficacious therapeutic strategies aimed at earlier clinical intervention and treatment in patients.

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收稿2016-10-18;修回2016-11-21〗

(編輯：譚秀榮)