Mechanisms regulating the expression,self-maintenance,and signaling-function of the bradykinin B2 and B1 receptors

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Galphai and Galphaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg(10)-kallidin or desArg(9)-BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-alpha. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self-maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980, Pharmacol Rev 32:1-46; Regoli et al., 1990, J Cardiovasc Pharmacol 15(Suppl 6):S30-S38; Regoli et al., 1993, Can J Physiol Pharmacol 71:556-557; Marceau, 1995, Immunopharmacology 30:1-26; Regoli et al., 1998, Eur J Pharmacol 348:1-10).     

Oxidative Stress in Synaptosomal Proteins from Mutant Presenilin-1 Knock-in Mice: Implications for Familial Alzheimer's Disease

Presenilin-1 (PS-1) is a transmembrane protein that may be involved in the processing of amyloid precursor protein (APP). Mutations in PS-1 are the major cause of familial Alzheimer's disease (AD). AD brain is under significant oxidative stress, including protein oxidation. In the present study, protein oxidation was compared in synaptosomes from knock-in mice expressing mutant human PS-1 (M146V mutation) and from wild-type mice expressing non-mutant human PS-1. Synaptosomal membrane protein conformational alterations associated with oxidative stress were measured using electron paramagnetic resonance (EPR) in conjunction with a protein-specific spin-label. Direct synaptosomal protein oxidation was assessed by a carbonyl detection assay. Synaptosomal proteins from PS-1 mutant mice displayed increased oxidative stress as measured by both techniques, compared with synaptosomal proteins from wild type mice. These data suggest that PS-1 mutations cause oxidative alterations in synaptosomal membrane protein structure and oxidative modification of synaptosomal proteins. Our findings suggest that familial AD may be associated with oxidative stress that may play a pivotal role in neuronal dysfunction and death.     

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells

Bradykinin (BK) and angiotensin II (AngII) often have opposite roles in cardiovascular diseases. Our aim here was to construct hybrid receptors which bind AngII but signal as BK. Various sequences of the intracellular face of the AngII type I receptor, AT1R, were replaced with corresponding sequences from the bradykinin B2 receptor (BKB2R). The hybrids demonstrated a number of signaling characteristics of the BKB2R. For example, the hybrids demonstrated BK as opposed to AngII like phosphorylation of Akt and JNK. The hybrids containing the BKB2R intracellular loop 2 (IC2) displayed minimal G-protein, Galphai/Galphaq, linked signaling. Computer based molecular models suggested that Ser-Met-Gly from the IC2 of the BKB2R is detrimental for the Galphai/Galphaq coupled functions of this hybrid. The return of Lys-Ser-Arg of the AT1R to this hybrid led to almost full recovery of Galphai and Galphaq activation. The design and production of AT1/BKB2 hybrid receptors is a potential approach in the treatment of hypertension related diseases where the presence of AngII, its AT1 receptor and the consequent signal transduction has proven detrimental.     

Differential alterations in antioxidant capacity in cells from Alzheimer patients

Oxidative stress occurs in brains of Alzheimer's disease (AD) patients. A major question in AD research is whether the oxidative stress is just secondary to neurodegeneration. To test whether oxidative stress is an inherent property of AD tissues, the ability of cultured fibroblasts bearing the AD Presenilin-1 246 Ala-->Glu mutation to handle reactive oxygen species (ROS) was compared to controls. Although ROS in cells from AD subjects were only slightly less than cells from controls under basal conditions (-10%) or after exposure to H(2)O(2) (-16%), treatment with antioxidants revealed clear differences. Pretreatment with DMSO, a hydroxyl radical scavenger, reduced basal and H(2)O(2)-induced ROS levels significantly more in cells from controls (-22%, -22%) than in those from AD subjects (-4%, +14%). On the other hand, pretreatment with Trolox diminished H(2)O(2)-induced ROS significantly more in cells from AD (-60%) than control subjects (-39%). In summary, cells from AD patients have greater Trolox sensitive ROS and less DMSO sensitive ROS than controls. The results demonstrate that fibroblasts bearing this PS-1 mutation have altered means of handling oxidative stress and appear useful for determining the mechanism underlying the altered redox metabolism.     

The role of melatonin in the treatment of type 2 diabetes mellitus and Alzheimer's disease

In type 2 diabetes mellitus (T2DM) and its related disorders like obesity, the abnormal protein processing, oxidative stress and proinflammatory cytokines will drive the activation of inflammatory pathways, leading to low-grade chronic inflammation and insulin resistance (IR) in the periphery and impaired neuronal insulin signaling in the brain. Studies have shown that such inflammation and impaired insulin signaling contribute to the development of Alzheimer''s disease (AD). Therefore, new therapeutic strategies are needed for the treatment of T2DM and T2DM-linked AD. Melatonin is primarily known for its circadian role which conveys message of darkness and induces night-state physiological functions. Besides rhythm-related effects, melatonin has anti-inflammatory and antioxidant properties. Melatonin levels are downregulated in metabolic disorders with IR, and activation of melatonin signaling delays disease progression. The aim of this Review is to highlight the therapeutic potentials of melatonin in preventing the acceleration of AD in T2DM individuals through its therapeutic mechanisms, including antioxidative effects, anti-inflammatory effects, restoring mitochondrial function and insulin sensitivity.     

Apelin/APJ system: A novel promising target for neurodegenerative diseases

Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), are incurable diseases characterized by progressive loss of cognitive or motor function, which construct a serious threat to the life quality of aging populations and their life spans. Apelin is an endogenous ligand for the G protein-coupled receptor. Apelin is reported to be detected not only in the cardiovascular system but also in neurons of the central nervous system (CNS). In addition, alterations in the expression level of apelin appear to play a pivotal role in various physiological processes including loss of structure or function of neurons, inflammatory responses, oxidative stress, Ca²⁺ signaling, apoptosis, and autophagy. All of these processes are intimately related to the occurrence of neurodegenerative diseases. Recently, apelin is reported to improve cognitive impairment in PD by antioxidant and antiapoptotic properties. Hence, it is becoming increasingly appreciated that altering the level of apelin can change the course or dictate the outcome of neurodegenerative events such as AD, PD, and HD, suggesting that apelin could be a potential target for the treatment of neurodegenerative diseases possibly acting on a variety of signaling pathways such as suppression of inflammatory responses, inhibition of oxidative stress, reduction of Ca²⁺ signaling, induction of autophagy, and suppression of apoptosis.     

Antisense directed against PS-1 gene decreases brain oxidative markers in aged senescence accelerated mice (SAMP8) and reverses learning and memory impairment: A proteomics study

Amyloid β-peptide (Aβ) plays a central role in the pathophysiology of Alzheimer's disease (AD) through the induction of oxidative stress. This peptide is produced by proteolytic cleavage of amyloid precursor protein (APP) by the action of β- and γ-secretases. Previous studies demonstrated that reduction of Aβ, using an antisense oligonucleotide (AO) directed against the Aβ region of APP, reduced oxidative stress-mediated damage and prevented or reverted cognitive deficits in senescence-accelerated prone mice (SAMP8), a useful animal model for investigating the events related to Aβ pathology and possibly to the early phase of AD. In the current study, aged SAMP8 were treated by AO directed against PS-1, a component of the γ-secretase complex, and tested for learning and memory in T-maze foot shock avoidance and novel object recognition. Brain tissue was collected to identify the decrease of oxidative stress and to evaluate the proteins that are differently expressed and oxidized after the reduction in free radical levels induced by Aβ. We used both expression proteomics and redox proteomics approaches. In brain of AO-treated mice a decrease of oxidative stress markers was found, and the proteins identified by proteomics as expressed differently or nitrated are involved in processes known to be impaired in AD. Our results suggest that the treatment with AO directed against PS-1 in old SAMP8 mice reverses learning and memory deficits and reduces Aβ-mediated oxidative stress with restoration to the normal condition and identifies possible pharmacological targets to combat this devastating dementing disease.     

Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts

The mechanisms regulating the opposing physiological actions of bradykinin (BK) and angiotensin II (AngII) are not well understood. Here we investigate signaling interactions between these two effectors. Connective tissue growth factor (CTGF) expression in IMR-90, human lung fibroblasts, is used as the endpoint target. In these cells the BK B2 receptor (BKB2R) is expressed constitutively, while no binding of AngII is detected. An inducible expression system is used to insert AngII receptor 1 (AT1R) and to obtain a signal level in response to AngII at the magnitude of BK. AngII and BK activate G protein-coupled targets, arachidonate release from cellular phospholipid stores, and intracellular phosphatidylinositol turnover equally. Both activate ERK, JNK, and p38 equally. However, AngII activates, whereas BK inactivates, RhoA. AngII induces a rapid (1 h) CTGF mRNA expression. RhoA siRNA and RhoA activation inhibitor, Y-27632, markedly reduce the AngII effect. Simultaneous treatment with BK and AngII attenuates the AT1R action. Additionally, BK in the absence of AngII lowers CTGF mRNA expression below basal levels over a span of 4 h. An AT1R/BKB2R chimera lacking heterotrimeric G protein coupling continues to activate MAP kinases to the same extent as wild-type (WT) AT1R and BKB2R. However, the increase of CTGF mRNA expression by this mutant is low, almost identical with that obtained by the simultaneous treatment of the WT AT1R-expressing cells with BK and AngII. In this context the chimeric receptor displays the characteristics of both receptors. These data demonstrate that, in human lung fibroblasts, BK modulates the action of AngII through the small G protein RhoA, but in a Galphai/Galphaq-independent manner.     

Microarray and phosphokinase screenings leading to studies on ERK and JNK regulation of connective tissue growth factor expression by angiotensin II 1a and bradykinin B2 receptors in Rat1 fibroblasts

Rat1 fibroblasts stably transfected with the rat angiotensin II (AngII) AT1a and bradykinin (BK) B2 receptor cDNAs gained the ability to bind Ang II and BK. Wild-type Rat1 cells bound neither ligand. Exposure to either effector led to characteristic Galphai and Galphaq signal cascades, the release of arachidonic acid (ARA), and the intracellular accumulation of inositol phosphates (IP). Microarray analyses in response to BK or AngII showed that both receptors markedly induce the CCN family genes, CTGF (CCN2) and Cyr61 (CCN1), as well as the vasculature-related genes, Cnn1 and Egr1. Real time PCR confirmed the increased expression of connective tissue growth factor (CTGF) mRNA. Combined sequence-based analysis of gene promoter regions with statistical prevalence analyses identified CREB, SRF, and ATF-1, downstream targets of ERK, and JNK, as prominent products of genes that are regulated by ligand binding to the BK or AngII receptors. The binding of AngII or BK markedly stimulated the phosphorylation and thus the activation of ERK2, JNK, and p38MAPK. A BKB2R and an AT1aR chimera which displayed only negligible G-protein-related signaling were constructed. Both mutant receptors continued to activate these kinases and stimulate CTGF expression. Inhibitors of ERK1/2 and JNK but not p38MAPK inhibited the BK- and AngII-stimulated expression of CTGF in cells expressing either the WT or mutant receptors, illustrating that ERK and JNK participate in the control of CTGF expression in a manner that appears to be independent of G-protein. Conversely, addition of BK or AngII to the cell line expressing WT AT1aR and BKB2R downregulated the expression of collagen alpha1(I) (COL1A1) mRNA. However, these effectors did not have this effect in cells expressing the mutant receptors. Thus, a robust G-protein related response is necessary for BK or AngII to affect COL1A1 expression.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.     

Short-term treatment with dabigatran alters protein expression patterns in a late-stage tau-based Alzheimer's disease mouse model

Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.     

Analysis of lipophilic fluorescent products in blood of Alzheimer's disease patients

Alzheimer's disease (AD) is a severe neurodegenerative disorder characterized by cognitive decline. Prodromal stage of AD, also called mild cognitive impairment (MCI), especially its amnestic type (aMCI), precedes dementia stage of AD. There are currently no reliable diagnostic biomarkers of AD in the blood. Alzheimer's disease is accompanied by increased oxidative stress in brain, which leads to oxidative damage and accumulation of free radical reaction end‐products. In our study, specific products of lipid peroxidation in the blood of AD patients were studied. Lipophilic extracts of erythrocytes (AD dementia = 19, aMCI = 27, controls = 16) and plasma (AD dementia = 11, aMCI = 17, controls = 16) were analysed by fluorescence spectroscopy. The level of these products is significantly increased in erythrocytes and plasma of AD dementia and aMCI patients versus controls. We concluded that oxidative stress end‐products are promising new biomarkers of AD, but further detailed characterisation of these products is needed.     

Insulin resistance and amyloidogenesis as common molecular foundation for type 2 diabetes and Alzheimer's disease

Characterized as a peripheral metabolic disorder and a degenerative disease of the central nervous system respectively, it is now widely recognized that type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share several common abnormalities including impaired glucose metabolism, increased oxidative stress, insulin resistance and amyloidogenesis. Several recent studies suggest that this is not an epiphenomenon, but rather these two diseases disrupt common molecular pathways and each disease compounds the progression of the other. For instance, in AD the accumulation of the amyloid-beta peptide (Aβ), which characterizes the disease and is thought to participate in the neurodegenerative process, may also induce neuronal insulin resistance. Conversely, disrupting normal glucose metabolism in transgenic animal models of AD that over-express the human amyloid precursor protein (hAPP) promotes amyloid-peptide aggregation and accelerates the disease progression. Studying these processes at a cellular level suggests that insulin resistance and Aβ aggregation may not only be the consequence of excitotoxicity, aberrant Ca²⁺ signals, and proinflammatory cytokines such as TNF-α, but may also promote these pathological effectors. At the molecular level, insulin resistance and Aβ disrupt common signal transduction cascades including the insulin receptor family/PI3 kinase/Akt/GSK3 pathway. Thus both disease processes contribute to overlapping pathology, thereby compounding disease symptoms and progression.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

Coulombic and hydrophobic interactions in the first intracellular loop are vital for bradykinin B2 receptor ligand binding and consequent signal transduction

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.     

Roles of humanin and derivatives on the pathology of neurodegenerative diseases and cognition

BackgroundAlzheimer's disease (AD), Parkinson's disease (PD), and age-related macular degeneration (AMD) are common among neurodegenerative diseases, but investigations into novel therapeutic approaches are currently limited. Humanin (HN) is a mitochondrial-derived peptide found in brain tissues of patients with familial AD and has been increasingly investigated in AD and other neurodegenerative diseases.Scope of reviewIn this review, we summarize and discuss the effects of HN on the pathology of neurodegenerative diseases and cognition based on several studies from preclinical to clinical models. The association between cardiac ischemia-reperfusion (I/R) injury and brain are also included. Findings from in vitro studies and those involving mice provide the most fundamental information on the impact of HN and its potential association with clinical studies.Major conclusionsHN plays a considerable role in countering the progression and neuropathology of AD. Inhibition and reduction of oxidative stress and neuroinflammation of the original amyloid hypothesis is the mainstay mechanism. Multiple intracellular mechanisms will be elucidated, including those involved in the anti-apoptotic signaling cascades, the insulin signaling pathway, and mitochondrial function, and especially autophagic activity. These beneficial roles are also found following cardiac I/R injury. Cognitive improvement was found to be related to maintenance of synaptic integrity and neurotransmitter modulation. Small humanin-like peptide 2 demonstrates the neuroprotective effects in PD and AMD via prevention of mitochondrial loss.General significanceComprehensive knowledge of HN effects on cognition and neurodegenerative diseases emphasizes its potential to treat a viable disease, as it ameliorates the pathogenesis of the disease.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.