RESUMEN
OBJECTIVE: We investigated induction of α-secretase A disintegrin and metalloprotease 10 (ADAM10) by the synthetic retinoid acitretin (Neotigason; Actavis, München-Riem, Germany) in patients with mild to moderate Alzheimer disease (AD) via measurement of CSF content of α-secretase-derived amyloid precursor protein (APPs-α). METHODS: Twenty-one patients clinically diagnosed with mild to moderate AD received acitretin (30 mg per day) or placebo in a 4-week double-blind study. Primary endpoint was the difference of CSF APPs-α ratios calculated from the APPs-α levels after treatment and at baseline. We monitored safety and tolerability of the treatment. In addition, we assessed biomarkers such as ß-amyloid 42 (Aß42) under treatment conditions. RESULTS: The acitretin group showed a significant increase in CSF APPs-α levels compared with the placebo group (difference 0.38, 95% confidence interval 0.03-0.72, p = 0.035) within this rather short treatment period. The synthetic retinoid acitretin was overall safe and well tolerated. CONCLUSIONS: Our pilot study highlights that acitretin is able to enhance the nonamyloidogenic APP processing in human patients. Clinical consequences of this regulation should be investigated in larger and longer trials in patients with AD to evaluate acitretin's potential to serve as a novel therapeutic drug. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that in patients with AD, oral acitretin increases CSF APPs-α levels.
Asunto(s)
Acitretina/uso terapéutico , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquídeo , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: ADAM10 (a disintegrin and metalloproteinase 10) has been demonstrated to act as the main physiological α-secretase. Enzymatic activity of the α-secretase on the one hand prevents the formation of toxic Aß peptides and on the other hand promotes the secretion of a neurotrophic and neuroprotective amyloid precursor protein fragment (APPs-α) by cleaving the amyloid precursor protein within its Aß sequence. Enhancement of ADAM10's gene expression may therefore present a valuable therapeutic approach for the treatment of Alzheimer's disease (AD), where Aß peptides are severely involved in the pathogenesis. OBJECTIVE: In cell culture and in a transgenic mouse model of AD, retinoids led to increased ADAM10 expression and activity. We therefore endeavor to develop a clinical application of synthetic retinoids such as acitretin in AD. METHODS: The effect of synthetic retinoids on ADAM10 gene expression was analyzed by reporter gene assays in human neuroblastoma cell line SH-SY5Y. Penetrance of acitretin into the murine brain was analyzed by high-performance liquid chromatography. P-glycoprotein (P-gp) double-knockout mice with a deficiency in both isoforms, mdr1a and 1b, were used to analyze a possible role of P-gp-dependent efflux on acitretin distribution. RESULTS: Acitretin and tamibarotene are both potent activators of ADAM10 promoter activity. Acitretin crosses the murine blood-brain barrier and its level in the mouse brain is not reduced by P-gp. CONCLUSION: Synthetic retinoids and especially acitretin seem to be ideal candidates to establish an ADAM10-based AD treatment, and therefore have already entered first clinical trials.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Acitretina/metabolismo , Acitretina/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Acitretina/sangre , Secretasas de la Proteína Precursora del Amiloide/genética , Análisis de Varianza , Animales , Barrera Hematoencefálica/fisiología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Neuroblastoma/patología , Factores de Tiempo , TransfecciónRESUMEN
ADAM10 (A disintegrin and metalloproteinase 10) has been demonstrated as an enzyme with protective properties in Alzheimer's disease: in mouse models it not only lowered generation of toxic A-beta peptides and formation of senile plaques but also alleviated learning deficits and enhanced synaptic density. This is due to cleavage of the amyloid precursor protein (APP) within its A-beta stretch and to the release of the extracellular domain of APP with neuroprotective function. Aside from cleaving APP, ADAM10 has been linked to over 40 putative substrates at least in cell culture. These substrates are connected with important cellular functions such as cell migration, stress response and transport. For this contribution we focussed on ADAM10 acting on the APP-like proteins since for some of their representatives - in particular APLP2 and APP-L - a shedding by ADAM10 has been demonstrated in vivo. In addition, the importance of these proteins, especially of APLP2, has been repeatedly shown by intense analysis of double and triple transgenic mice which lack APP in combination with one or both APLPs: several phenotypes such as defects in migration of neuroblasts, in formation of synapses and synaptic transmission have been reported. However, the specific contribution of either the uncleaved full-length proteins or their extracellular domains secreted upon ADAM10 activity has not been elucidated. In this review, we report on recent findings concerning shedding of APP-like proteins and resulting functional consequences.
Asunto(s)
Proteínas ADAM/metabolismo , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas ADAM/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Humanos , Ratones , Ratones TransgénicosRESUMEN
The amyloid precursor protein (APP) has a pivotal role in pathogenesis of Alzheimer's disease (AD) via its beta- and gamma-secretase-derived cleavage products--the A-beta peptides. An alternative processing pathway provided by the alpha-secretase prevents formation of those toxic peptides and gives rise to the neurotrophic and neuroprotective cleavage product APPs-alpha. The molecular identity of the alpha-secretase has been confirmed recently, and there is consistency about ADAM10 being the most relevant and physiological enzyme of this class. It is not clear to what extent a deficiency in the catalytic activity of ADAM10 contributes to AD pathology and whether a decline occurs in aging humans. Nevertheless, ADAM10 has been suggested as a valuable target for prevention and/or for treatment of Alzheimer's disease. This review focuses on our knowledge about regulation of ADAM10 on different levels of cell physiology, such as transcription and translation, as well as protein-protein interactions and how this especially in the case of transcriptional regulation by retinoic acids might lead to the development of new therapeutic approaches.
Asunto(s)
Proteínas ADAM/biosíntesis , Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Activación Enzimática/genética , Humanos , Proteínas de la Membrana/genética , Procesamiento Proteico-Postraduccional/genética , Transcripción GenéticaRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent α-secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimer's disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long-term intranasal daily PACAP application stimulated the nonamyloidogenic processing of amyloid precursor protein (APP) and increased expression of the brain-derived neurotrophic factor and of the antiapoptotic Bcl-2 protein. In addition, it caused a strong reduction of the amyloid ß-peptide (Aß) transporter receptor for advanced glycation end products (RAGE) mRNA level. PACAP, by activation of the somatostatin-neprilysin cascade, also enhanced expression of the Aß-degrading enzyme neprilysin in the mouse brain. Furthermore, daily PAC1-receptor activation via PACAP resulted in an increased mRNA level of both the PAC1 receptor and its ligand PACAP. Our behavioral studies showed that long-term PACAP treatment of APP[V717I]-transgenic mice improved cognitive function in animals. Thus, nasal application of PACAP was effective, and our results indicate that PACAP could be of therapeutic value in treating AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Administración Intranasal , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Transgénicos , Neprilisina/genética , Neprilisina/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Somatostatina/genética , Somatostatina/metabolismoRESUMEN
Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-beta protein precursor (AbetaPP) and are reported to stimulate the activity of alpha-secretase, the major preventive secretase of Alzheimer's disease. Statins can increase the alpha-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in alpha-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates alpha-secretase activity. Treatment of human neuroblastoma cells with 50 microM zaragozic acid resulted in a approximately 3 fold increase of alpha-secretase activity and reduced cellular cholesterol by approximately 30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 microM). Zaragozic acid-stimulated secretion of alpha-secretase-cleaved soluble AbetaPP was dose dependent and saturable. Lovastatin- or zaragozic acid-stimulated increase of alpha-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the alpha-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AbetaPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for alpha-secretase activation.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Anticolesterolemiantes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Colesterol/biosíntesis , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Ácidos Tricarboxílicos/farmacología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Western Blotting , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Lovastatina/farmacología , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Terpenos/farmacologíaRESUMEN
BACKGROUND: In the past, we demonstrated that the disintegrin metalloproteinase ADAM10 has alpha-secretase activity in vitro and in cultured cells. We also found out that moderate overexpression of this proteinase inhibits Abeta peptide production and prevents the formation of amyloid plaques in an Alzheimer's disease (AD) mouse model. Moreover, it corrects early hippocampal defects like LTP impairment and increases cortical synaptogenesis. OBJECTIVE: Upregulation of ADAM10 might be an alternative approach concerning AD therapy. Our current research therefore focuses on substances and/or pathways which regulate ADAM10 gene expression. METHODS: Analyses of ADAM10 promoter activity were performed in human and murine neuroblastoma cell lines and important findings were validated in an AD mouse model. RESULTS: The outcome of our investigations indicates that a heterodimeric retinoid receptor mediates the activation of the ADAM10 promoter by all-trans-retinoic acid. This then results in enhanced ADAM10 expression, pronounced APPs-alpha secretion and diminished proteolytic processing products of the amyloidogenic pathway (APPs-beta, Abeta). CONCLUSION: Nontoxic synthetic retinoids - like acitretin - offer a valuable therapeutic approach in treatment of AD by moderately enhancing ADAM10 gene expression.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Retinoides/farmacología , Regulación hacia Arriba/efectos de los fármacos , Vitaminas/farmacología , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Línea Celular Tumoral , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de la Membrana/genética , Ratones , Morfolinas/farmacología , Neuroblastoma/patología , Retinoides/química , Transducción de Señal/efectos de los fármacos , Terpenos/farmacología , Vitaminas/químicaRESUMEN
Proteolytic processing of the amyloid precursor protein by alpha-secretase prevents formation of the amyloid beta-peptide (Abeta), which is the main constituent of amyloid plaques in brains of Alzheimer disease (AD) patients. alpha-Secretase activity is decreased in AD, and overexpression of the alpha-secretase ADAM10 (a disintegrin and metalloprotease 10) in an AD animal model prevents amyloid pathology. ADAM10 has a 444-nucleotide-long, very GC-rich 5'-untranslated region (5'-UTR) with two upstream open reading frames. Because similar properties of 5'-UTRs are found in transcripts of many genes, which are regulated by translational control mechanisms, we asked whether ADAM10 expression is translationally controlled by its 5'-UTR. We demonstrate that the 5'-UTR of ADAM10 represses the rate of ADAM10 translation. In the absence of the 5'-UTR, we observed a significant increase of ADAM10 protein levels in HEK293 cells, whereas mRNA levels were not changed. Moreover, the 5'-UTR of ADAM10 inhibits translation of a luciferase reporter in an in vitro transcription/translation assay. Successive deletion of the first half of the ADAM10 5'-UTR revealed a striking increase in ADAM10 protein expression in HEK293 cells, suggesting that this part of the 5'-UTR contains inhibitory elements for translation. Moreover, we detect an enhanced alpha-secretase activity and consequently reduced Abeta levels in the conditioned medium of HEK293 cells expressing both amyloid precursor protein and a 5'-UTR-ADAM10 deletion construct lacking the first half of the 5'-UTR. Thus, we provide evidence that the 5'-UTR of ADAM10 may have an important role for post-transcriptional regulation of ADAM10 expression and consequently Abeta production.
Asunto(s)
Regiones no Traducidas 5' , Proteínas ADAM/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Péptidos beta-Amiloides/biosíntesis , Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Biosíntesis de Proteínas , Proteínas ADAM/genética , Proteína ADAM10 , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/genética , Animales , Secuencia de Bases , Encéfalo/enzimología , Células COS , Chlorocebus aethiops , Humanos , Proteínas de la Membrana/genética , Eliminación de SecuenciaRESUMEN
A decade ago, a disintegrin and metalloproteinase 10 (ADAM10) was identified as an alpha-secretase and as a key proteinase in the processing of the amyloid precursor protein. Accordingly, the important role that it plays in Alzheimer's disease was manifested. Animal models with an overexpression of ADAM10 revealed a beneficial profile of the metalloproteinase with respect to learning and memory, plaque load and synaptogenesis. Therefore, ADAM10 presents a worthwhile target with respect to the treatment of a neurodegenerative disease such as Morbus Alzheimer. Initially, ADAM10 was suggested to be an enzyme, shaping the extracellular matrix by cleavage of collagen type IV, or to be a tumour necrosis factor alpha convertase. In a relatively short time, a wide variety of additional substrates (with amyloid precursor protein probably being the most prominent) has been identified and the search is still ongoing. Hence, any side effects concerning the therapeutic enhancement of ADAM10 alpha-secretase activity have to be considered. The present review summarizes our knowledge about the structure and function of ADAM10 and highlights the opportunities for enhancing the expression and/or activity of the alpha-secretase as a therapeutic target.
Asunto(s)
Proteínas ADAM/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Regulación de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteína ADAM10 , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Animales , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Biológicos , Enfermedades Neurodegenerativas/metabolismo , Estructura Terciaria de Proteína , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Humanos , Proteínas de la Membrana/genética , Ratones , Péptidos/genética , Nexinas de Proteasas , Receptores de Superficie Celular/genéticaRESUMEN
Both the cellular prion protein (PrP(c)) and the amyloid precursor protein (APP) are physiologically subjected to complex proteolytic processing events. While for APP the proteinases involved--alpha-, beta- and gamma-secretase--have been identified in vitro and in vivo, the cleavage of PrP(c) by now has been linked only to the shedding activity of the metalloproteinase ADAM10 and/or ADAM17 in cell culture. Here we show that neuronal overexpression of the alpha-secretase ADAM10 in mice reduces all PrP(c) species detected in the brain instead of leading to enhanced amounts of specific cleavage products of PrP(c). Additionally, the incubation time of mice after scrapie infection is significantly increased in mice moderately overexpressing ADAM10. This indicates that overexpression of ADAM10 rather influences the amount of the cellular prion protein than its processing in vivo.
Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Proteínas de la Membrana/fisiología , Priones/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Scrapie/enzimología , Scrapie/transmisión , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAM10 , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Encéfalo/enzimología , Bovinos , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Priones/antagonistas & inhibidores , Scrapie/prevención & controlRESUMEN
BACKGROUND: In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the alpha-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. RESULTS: To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the alpha-secretase ADAM10, or a dominant-negative mutant (dn) of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant.Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and beta-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice.Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. CONCLUSION: In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of inflammation markers was observed. These results are further supportive for the strategy to treat AD by increasing the alpha-secretase activity.
Asunto(s)
Proteínas ADAM/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Encéfalo/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Proteína ADAM10 , Enfermedad de Alzheimer/genética , Animales , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genéticaRESUMEN
Although ADAM10 is a major alpha-secretase involved in non-amyloidogenic processing of the amyloid precursor protein, several additional substrates have been identified, most of them in vitro. Thus, therapeutical approaches for the prevention of Alzheimer's disease by upregulation of this metalloproteinase may have severe side effects. In the present study, we examined whether the ErbB receptor ligand neuregulin-1, which is essential for myelination and other important neuronal functions, is cleaved by ADAM10. Studies with beta- and gamma-secretase inhibitors, as well as with the metalloproteinase inhibitor GM6001, revealed an inhibition of neuregulin-1 processing in human astroglioma cell line U373; however, specific RNA interference-induced knockdown of ADAM10 remained without effect. In vivo investigations of mice overexpressing either ADAM10 or dominant negative ADAM10 showed unaltered cleavage of neuregulin-1 compared to wild-type animals. As a consequence, the myelin sheath thickness of peripheral nerves was unaffected in mice with altered ADAM10 activity. Thus, although the beta-secretase BACE-1 acts as a neuregulin-1 sheddase, ADAM10 does not lead to altered neuregulin-1 processing either in cell culture or in vivo. Adverse reactions of an ADAM10-based therapy of Alzheimer's disease due to neuregulin-1 cleavage are therefore unlikely.
Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Proteínas de la Membrana/fisiología , Neurregulina-1/metabolismo , Proteína ADAM10 , Secuencia de Aminoácidos , Animales , Astrocitoma , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Humanos , Hidrólisis , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The alpha-secretase cleaves in the non-amyloidogenic pathway the amyloid-beta protein precursor (AbetaPP) within the region of the amyloid-beta peptides to prevent their formation and aggregation in the brain. Members of the ADAM family (a disintegrin and metalloprotease) are the main candidates for physiologically relevant alpha-secretases. We recently demonstrated that overexpression of ADAM10 in mice transgenic for human AbetaPP (ADAM10 x APP[V717I]) alleviated functional deficits related to Alzheimer's disease. To further demonstrate that this is due to the specific activity of alpha-secretase, we characterized mice overexpressing an inactive form of ADAM10 (ADAM10[E384A]; ADAM10-dn). Three lines of mice (controls (C57Bl/6 x FVB), APP[V717I[ transgenics and ADAM10-dn x APP[V717I] double-transgenics) were investigated with respect to learning and memory in the Morris water maze. Double-transgenic mice overexpressing ADAM10-dn behaved similar to APP[V717I] overexpressing mice. This provides further evidence that ADAM10 in vivo by its enzymatic activity is able to counteract cognitive deficits. Stimulation of alpha-secretase activity might thus be a suitable approach to study treatment strategies of Alzheimer's disease.
Asunto(s)
Proteínas ADAM/metabolismo , Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Factores de Edad , Alanina/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Conducta Animal , Modelos Animales de Enfermedad , Ácido Glutámico/genética , Humanos , Isoleucina/genética , Aprendizaje por Laberinto/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tiempo de Reacción/genética , Natación , Valina/genéticaRESUMEN
Late-onset Alzheimer's disease is often connected with nutritional misbalance, such as enhanced cholesterol intake, deficiency in polyunsaturated fatty acids, or hypovitaminosis. The alpha-secretase ADAM10 has been found to be regulated by retinoic acid, the bioreactive metabolite of vitamin A. Here we show that retinoids induce gene expression of ADAM10 and alpha-secretase activity by nonpermissive retinoid acid receptor/retinoid X receptor (RAR/RXR) heterodimers, whereby alpha- and beta-isotypes of RAR play a major role. However, ligands of other RXR binding partners, such as the vitamin D receptor, do not stimulate alpha-secretase activity. On the basis of these findings, we examined the effect of synthetic retinoids and found a strong enhancement of nonamyloidogenic processing of the amyloid precursor protein by the vitamin A analog acitretin: it stimulated ADAM10 promoter activity with an EC(50) of 1.5 microM and led to an increase of mature ADAM10 protein that resulted in a two- to three-fold increase of the ratio between alpha- and beta-secretase activity in neuroblastoma cells. The alpha-secretase stimulation by acitretin was completely inhibited by the ADAM10-specific inhibitor GI254023X. Intracerebral injection of acitretin in APP/PS1-21 transgenic mice led to a reduction of Abeta(40) and Abeta(42). The results of this study may have clinical relevance because acitretin has been approved for the treatment of psoriasis since 1997 and found generally safe for long-term use in humans.
Asunto(s)
Proteínas ADAM/metabolismo , Acitretina/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Queratolíticos/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Acitretina/química , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Receptores X del Hígado , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Estructura Molecular , Receptores Nucleares Huérfanos , PPAR gamma/genética , PPAR gamma/metabolismo , Regiones Promotoras Genéticas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Receptores X Retinoide/genética , Tretinoina/química , Tretinoina/metabolismo , Regulación hacia ArribaRESUMEN
We exploratively measured APPs alpha, a secreted fragment of the non-amyloidogenic cleavage of amyloid precursor protein via a-secretase, and tau protein phosphorylated at threonine 181 (p tau) in the cerebrospinal fluid of 10 patients with mild cognitive impairment, 20 patients with dementia of Alzheimer's type, and 10 controls. Cerebrospinal fluid APPs alpha and p tau levels were correlated with cognitive performance. P tau levels were significantly elevated in mild cognitive impairment and in patients with dementia of Alzheimer's type, APPs alpha levels were significantly reduced in patients with dementia of Alzheimer's type compared to the controls. APPs alpha levels were associated with Mini Mental State Examination total scores but not with Delayed Verbal Recall Test performance. Vice versa, pt au levels correlated only with Delayed Verbal Recall Test in patients with dementia of Alzheimer's type or mild cognitive impairment. Both, an increase in p tau levels and a decrease in cerebrospinal fluid APPs alpha, seem to refer to relevant but functionally different processes in the development of mild cognitive impairment and dementia of Alzheimer's type.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Secretasas de la Proteína Precursora del Amiloide/líquido cefalorraquídeo , Trastornos del Conocimiento/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/psicología , Análisis de Varianza , Biomarcadores/líquido cefalorraquídeo , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/psicología , Femenino , Humanos , Masculino , Memoria a Corto Plazo , Recuerdo Mental , Pruebas Neuropsicológicas/estadística & datos numéricos , Índice de Severidad de la Enfermedad , Distribución por Sexo , Análisis y Desempeño de TareasRESUMEN
The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteína ADAM10 , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Calcimicina/farmacología , Carcinógenos/farmacología , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Ionóforos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Inhibidores de Proteasas/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Cortical cholinergic, glutamatergic and GABAergic terminals become upregulated during early stages of the transgenic amyloid pathology. Abundant evidence suggests that sAPP alpha, the product of the non-amyloidogenic alpha-secretase pathway, is neurotrophic both in vitro and when exogenously applied in vivo. The disintegrin metalloprotease ADAM-10 has been shown to have alpha-secretase activity in vivo. To determine whether sAPP alpha has an endogenous biological influence on cortical presynaptic boutons in vivo, we quantified cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities in either ADAM-10 moderate expressing (ADAM-10 mo) transgenic mice, which moderately overexpress ADAM-10, or age-matched non-transgenic controls. Both early and late ontogenic time points were investigated. ADAM-10 mo transgenic mice display significantly elevated cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities at the early time point (8 months). Only the cholinergic presynaptic bouton density remains significantly elevated in late-staged ADAM-10 mo transgenic animals (18 months). To confirm that the observed elevations were due to increased levels of endogenous murine sAPP alpha, exogenous human sAPP alpha was infused into the cortex of non-transgenic control animals for 1 week. Exogenous infusion of sAPP alpha led to significant elevations in the cholinergic, glutamatergic and GABAergic cortical presynaptic bouton populations. These results are the first to demonstrate an in vivo influence of ADAM-10 on neurotransmitter-specific cortical synaptic plasticity and further confirm the neurotrophic influence of sAPP alpha on cortical synaptogenesis.
Asunto(s)
Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/genética , Corteza Cerebral/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Sinapsis/fisiología , Proteínas ADAM/fisiología , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/fisiología , Animales , Bovinos , Corteza Cerebral/citología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Ratones Transgénicos , Terminales Presinápticos/fisiología , Receptores Colinérgicos/fisiología , Receptores de GABA/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
In the non-amyloidogenic pathway the alpha-secretase cleaves the amyloid precursor protein (APP) within the sequence of Abeta-peptides and precludes their formation. In addition, alpha-secretase cleavage releases an N-terminal extracellular domain with neurotrophic and neuroprotective properties. The disintegrin metalloproteinase ADAM10 has been shown to act as alpha-secretase in vivo, to prevent amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. An increase in alpha-secretase activity therefore is an attractive strategy for treatment of AD and may be achieved by modulating selective signalling pathways. Functional characterization of the human ADAM10 promoter showed that it contains several binding elements for transcription factors which are regulated by extracellular ligands. Retinoic acid (RA) was identified as an inducer of human ADAM10 promoter activity. In human neuroblastoma cell lines RA treatment upregulated the expression of both the alpha-secretase ADAM10 and its substrates APP and the related APP-like-protein 2 (APLP2), and led to an enhanced secretion of their extracellular domains. Furthermore, G protein-coupled receptors (GPCRs) localized in brain areas affected by AD were investigated. Activation of the PAC1 receptor by the neuropeptide PACAP was identified as an approach for upregulation of the alpha-secretase pathway.