RESUMEN
BACE1 is the rate-limiting enzyme for the production of the Aß peptide that forms amyloid plaques in Alzheimer's disease (AD). Small molecule inhibitors of BACE1 are being tested in clinical trials for AD, but the safety and efficacy of BACE1 inhibition has yet to be fully explored. Knockout of the Bace1 gene in the germline of mice causes multiple neurological phenotypes, suggesting that BACE1 inhibition could be toxic. However, these phenotypes could be the result of BACE1 deficiency during development rather than due to the lack of BACE1 function in the adult. To address this problem, we generated tamoxifen-inducible conditional BACE1 knockout mice in which the Bace1 gene may be deleted in the whole body of the adult at will. Importantly, the adult conditional BACE1 knockout mice largely lack phenotypes, indicating that many BACE1 functions are not required in the adult organism. However, a germline phenotype was observed after BACE1 knockout in the adult: reduced length and disorganization of the hippocampal mossy fiber infrapyramidal bundle comprised of axons of dentate gyrus granule cells. The infrapyramidal bundle abnormality correlated with reduced proteolytic processing of the neural cell adhesion protein CHL1 that is involved in axonal guidance. We conclude that BACE1 inhibition in the adult mouse brain does not lead to the phenotypes associated with BACE1 deficiency during embryonic and postnatal development. However, adult conditional BACE1 knockout mice also suggest that BACE1 inhibitor drugs may disrupt the organization of an axonal pathway in the hippocampus, an important structure for learning and memory. Here, I review the adult conditional BACE1 knockout results and consider their implications for BACE1 inhibitor clinical trials.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/genética , Orientación del Axón/genética , Cognición , Giro Dentado/patología , Memoria , Fibras Musgosas del Hipocampo/patología , Animales , Axones/patología , Moléculas de Adhesión Celular/metabolismo , Ensayos Clínicos como Asunto , Giro Dentado/citología , Técnicas de Inactivación de Genes , Hipocampo/patología , Humanos , Aprendizaje , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , FenotipoRESUMEN
Frontotemporal lobar degeneration (FTLD) is among the most prevalent dementias of early-onset. Pathologically, FTLD presents with tauopathy or TAR DNA-binding protein 43 (TDP-43) proteinopathy. A biallelic mouse model of FTLD was produced on a mix FVB/129SVE background overexpressing wild-type human TDP-43 (hTDP-43) using tetracycline transactivator (tTA), a system widely used in mouse models of neurological disorders. tTA activates hTDP-43, which is placed downstream of the tetracycline response element. The original study on this transgenic mouse found hippocampal degeneration following hTDP-43 expression, but did not account for independent effects of tTA protein. Here, we initially analyzed the neurotoxic effects of tTA in postweaning age mice of either sex using immunostaining and area measurements of select brain regions. We observed tTA-dependent toxicity selectively in the hippocampus affecting the dentate gyrus significantly more than CA fields, whereas hTDP-43-dependent toxicity in bigenic mice occurred in most other cortical regions. Atrophy was associated with inflammation, activation of caspase-3, and loss of neurons. The atrophy associated with tTA expression was rescuable by the tetracycline analog, doxycycline, in the diet. MRI studies corroborated the patterns of atrophy. tTA-induced degeneration was strain-dependent and was rescued by moving the transgene onto a congenic C57BL/6 background. Despite significant hippocampal atrophy, behavioral tests in bigenic mice revealed no hippocampally mediated memory impairment. Significant atrophy in most cortical areas due solely to TDP-43 expression indicates that this mouse model remains useful for providing critical insight into co-occurrence of TDP-43 pathology, neurodegeneration, and behavioral deficits in FTLD.SIGNIFICANCE STATEMENT The tTA expression system has been widely used in mice to model neurological disorders. The technique allows investigators to reversibly turn on or off disease causing genes. Here, we report on a mouse model that overexpresses human TDP-43 using tTA and attempt to recapitulate features of TDP-43 pathology present in human FTLD. The tTA expression system is problematic, resulting in dramatic degeneration of the hippocampus. Thus, our study adds a note of caution for the use of the tTA system. However, because FTLD is primarily characterized by cortical degeneration and our mouse model shows significant atrophy in most cortical areas due to human TDP-43 overexpression, our animal model remains useful for providing critical insight on this human disease.
Asunto(s)
Proteínas de Unión al ADN/toxicidad , Modelos Animales de Enfermedad , Degeneración Lobar Frontotemporal , Transactivadores/toxicidad , Animales , Proteínas de Unión al ADN/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Accumulations of ß-amyloid (Aß) contribute to neurological deficits associated with Alzheimer's disease (AD). The effects of Aß on basal neuronal excitability and learning-related AHP plasticity were examined using whole-cell recordings from hippocampal neurons in the 5XFAD mouse model of AD. A robust increase in Aß42 (and elevated levels of Aß38-40) in naïve 5XFAD mice was associated with decreased basal neuronal excitability, evidenced by a select increase in Ca(2+)-sensitive afterhyperpolarization (AHP). Moreover, trace fear deficits observed in a subset of 5XFAD weak-learner mice were associated with a greater enhancement of the AHP in neurons, as compared to age-matched 5XFAD learner and 5XFAD naïve mice. Importantly, learning-related plasticity of the AHP remained intact in a subset of 5XFAD mice that learned trace fear conditioning to a set criterion. We show that APP-PS1 mutations enhance Aß and disrupt basal excitability via a Ca(2+)-dependent enhancement of the AHP, and suggest disruption to learning-related modulation of intrinsic excitability resulted, in part, from altered cholinergic modulation of the AHP in the 5XFAD mouse model of AD (170 of 170).
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Aprendizaje/fisiología , Presenilina-1/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/psicología , Precursor de Proteína beta-Amiloide/fisiología , Animales , Calcio/metabolismo , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Presenilina-1/fisiologíaRESUMEN
Alzheimer's disease (AD) is an intractable, neurodegenerative disease that appears to be brought about by both genetic and non-genetic factors. The neuropathology associated with AD is complex, although amyloid plaques composed of the beta-amyloid peptide (Abeta) are hallmark neuropathological lesions of AD brain. Indeed, Abeta plays an early and central role in this disease. beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the initiating enzyme in Abeta genesis and BACE1 levels are elevated under a variety of conditions. Given the strong correlation between Abeta and AD, and the elevation of BACE1 in this disease, this enzyme is a prime drug target for inhibiting Abeta production in AD. However, nine years on from the initial identification of BACE1, and despite intense research, a number of key questions regarding BACE1 remain unanswered. Indeed, drug discovery and development for AD continues to be challenging. While current AD therapies temporarily slow cognitive decline, treatments that address the underlying pathologic mechanisms of AD are completely lacking. Here we review the basic biology of BACE1. We pay special attention to recent research that has provided some answers to questions such as those involving the identification of novel BACE1 substrates, the potential causes of BACE1 elevation and the putative function of BACE1 in health and disease. Our increasing understanding of BACE1 biology should aid the development of compounds that interfere with BACE1 expression and activity and may lead to the generation of novel therapeutics for AD.
RESUMEN
Cholesterol metabolism has been linked to Alzheimer's disease (AD) neuropathology, which is characterized by amyloid plaques, neurofibrillary tangles and neuroinflammation. Indeed, the use of statins, which inhibit cholesterol and isoprenoid biosynthesis, as potential AD therapeutics is under investigation. Whether statins offer benefit for AD will be determined by the outcome of large, placebo-controlled, randomized clinical trials. However, their use as pharmacological tools has delineated novel roles for isoprenoids in AD. Protein isoprenylation regulates multiple cellular and molecular events and here we review the complex roles of isoprenoids in AD-relevant processes and carefully evaluate isoprenoid pathways as potential AD therapeutic targets.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Colesterol/biosíntesis , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Prenilación de Proteína/efectos de los fármacos , Terpenos/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/metabolismo , Placa Amiloide/efectos de los fármacos , Placa Amiloide/metabolismo , Prenilación de Proteína/fisiologíaAsunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/deficiencia , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Sistema Nervioso Central/enzimología , Endopeptidasas , Ratones , Ratones Noqueados/genética , Ratones Noqueados/metabolismo , Sistema Nervioso Periférico/enzimología , Fenotipo , Placa Amiloide/enzimologíaRESUMEN
Evidence suggests that the beta-amyloid peptide (Abeta) is central to the pathophysiology of Alzheimer's disease (AD). Amyloid plaques, primarily composed of Abeta, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Abeta42 peptide. Given the strong association between Abeta and AD, therapeutic strategies to lower the concentration of Abeta in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Abeta. Abeta is a product of catabolism of the large Typel membrane protein, amyloid precursor protein (APP). Two proteases, called beta- and gamma-secretase, mediate the endoproteolysis of APP to liberate the Abeta peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the gamma-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the beta-secretase has been shown to be the novel transmembrane aspartic protease, beta-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the beta-secretase, and as the key rate-limiting enzyme that initiates the formation of Abeta, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/genética , Endopeptidasas , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia MolecularAsunto(s)
Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/biosíntesis , Ácido Aspártico Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/genética , Humanos , Sustancias Macromoleculares , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Presenilina-1 , Presenilina-2 , Procesamiento Proteico-Postraduccional , Receptores Notch , Transducción de SeñalRESUMEN
The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Ácido Aspártico Endopeptidasas/metabolismo , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/química , Secuencia de Bases , Catálisis , Línea Celular , Cartilla de ADN , Endopeptidasas , Furina , Aparato de Golgi/enzimología , Humanos , Hidrólisis , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismoRESUMEN
The cerebral deposition of amyloid beta-peptide is an early and critical feature of Alzheimer's disease. Amyloid beta-peptide is released from the amyloid precursor protein by the sequential action of two proteases, beta-secretase and gamma-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of beta-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu(46) and contains four N-glycosylation sites (Asn(153), Asn(172), Asn(223), and Asn(354)). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys(216)-Cys(420), Cys(278)-Cys(443), and Cys(330)-Cys(380)). Despite the conservation of the active site residues and the 30-37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Línea Celular , Endopeptidasas , Glicopéptidos/química , Glicósido Hidrolasas , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neuraminidasa , Pepsina A/metabolismo , Fragmentos de Péptidos/química , Conformación Proteica , Procesamiento Postranscripcional del ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase.
Asunto(s)
Encéfalo/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Glicoproteínas/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/anatomía & histología , Endopeptidasas/química , Glicoproteínas/metabolismo , Humanos , Hibridación in Situ , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Alineación de SecuenciaRESUMEN
Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Ácido Aspártico Endopeptidasas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Clonación Molecular , Endopeptidasas , Endosomas/enzimología , Expresión Génica , Biblioteca de Genes , Aparato de Golgi/enzimología , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/fisiología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas , Células CHO , Células Clonales , Cricetinae , Endopeptidasas/deficiencia , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , ARN Mensajero/metabolismoRESUMEN
We have cloned an additional member (GC-D) of the membrane receptor guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] family that is specifically expressed in a subpopulation of olfactory sensory neurons. The extracellular, putative ligand-binding domain of the olfactory cyclase is similar in primary structure to two guanylyl cyclases expressed in the retina but diverges considerably from other known guanylyl cyclases. The expression of GC-D RNA is restricted to a small, randomly dispersed population of neurons that is within a single topographic zone in the olfactory neuroepithelium and resembles the pattern of the more diverse seven-transmembrane-domain odorant receptors. These observations suggest that GC-D may function directly in odor recognition or in modulating the sensitivity of a subpopulation of sensory neurons to specific odors.
Asunto(s)
Guanilato Ciclasa/genética , Neuronas Receptoras Olfatorias/enzimología , Receptores de Superficie Celular , Receptores de Péptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Guanilato Ciclasa/biosíntesis , Hibridación in Situ , Datos de Secuencia Molecular , Ratas , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
The detection of odorant receptor mRNAs within the axon terminals of sensory neurons has permitted us to ask whether neurons expressing a given receptor project their axons to common glomeruli within the olfactory bulb. In situ hybridization with five different receptor probes demonstrates that axons from neurons expressing a given receptor converge on one, or at most, a few glomeruli within the olfactory bulb. Moreover, the position of specific glomeruli is bilaterally symmetric and is constant in different individuals within a species. These data support a model in which exposure to a given odorant may result in the stimulation of a spatially restricted set of glomeruli, such that the individual odorants would be associated with specific topographic patterns of activity within the olfactory bulb.
Asunto(s)
Neuronas Aferentes/citología , Bulbo Olfatorio/anatomía & histología , ARN Mensajero/aislamiento & purificación , Receptores Odorantes/genética , Animales , Axones/patología , Axones/ultraestructura , Femenino , Hibridación in Situ , Modelos Neurológicos , Proteínas del Tejido Nervioso/genética , Odorantes , Proteína Marcadora Olfativa , Percepción/fisiología , Ratas , Ratas Sprague-Dawley , SinapsisRESUMEN
Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10-nm filaments have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases. In this report, we review the cell biology and human genetics of these diseases, epidermolysis bullosa simplex and epidermolytic hyperkeratosis. Both of these diseases are epidermal disorders of keratin, typified by cell fragility as a consequence of defects in the mechanical strength of basal epidermolysis bullosa simplex or suprabasal epidermolytic hyperkeratosis cells.
Asunto(s)
Epidermólisis Ampollosa Simple/genética , Hiperqueratosis Epidermolítica/genética , Diferenciación Celular , Células Epidérmicas , Epidermis/metabolismo , Epidermis/ultraestructura , Humanos , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Queratinas/metabolismo , Queratinas/ultraestructuraRESUMEN
The signal elicited by the interaction of odorous ligands with receptors on olfactory sensory neurons must be decoded by the brain to determine which of the numerous receptors have been activated. We have examined the patterns of odorant receptor expression in the rat olfactory epithelium to determine whether the mammalian olfactory system employs spatial segregation of sensory input to encode the identity of an odorant stimulus. In situ hybridization experiments with probes for 11 different odorant receptors demonstrate that sensory neurons expressing distinct receptors are topologically segregated into a small number of broad, yet circumscribed, zones within the olfactory epithelium. Within a given zone, however, olfactory neurons expressing a specific receptor appear to be randomly distributed, rather than spatially localized. The complex mammalian olfactory system may therefore compartmentalize the epithelium into anatomically and functionally discrete units, such that each zone expresses only a subset of the entire receptor repertoire.
Asunto(s)
Células Quimiorreceptoras/anatomía & histología , Mucosa Olfatoria/anatomía & histología , ARN Mensajero/aislamiento & purificación , Animales , Animales Recién Nacidos , Hibridación in Situ , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.
Asunto(s)
Genes ras , Mutación , Papiloma/genética , Neoplasias Cutáneas/genética , Factor de Crecimiento Transformador alfa/genética , Animales , Secuencia de Bases , ADN de Neoplasias , Modelos Animales de Enfermedad , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Papiloma/etiología , Papiloma/patología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador alfa/metabolismo , Heridas y Lesiones/complicacionesRESUMEN
Previously we demonstrated that transgenic mice expressing a mutant keratin in the basal layer of their stratified squamous epithelia exhibited a phenotype bearing resemblance to a subclass (Dowling Meara) of a heterogeneous group of human skin disorders known as epidermolysis bullosa simplex (EBS) (Vassar, R., P. A. Coulombe, L. Degenstein, K. Albers, E. Fuchs. 1991. Cell. 64:365-380.). The extent to which subtypes of EBS diseases might be genetically related is unknown, although they all exhibit skin blistering as a consequence of basal cell cytolysis. We have now examined transgenic mice expressing a range of keratin mutants which perturb keratin filament assembly to varying degrees. We have generated phenotypes which include most subtypes of EBS, demonstrating for the first time that at least in mice, these diseases can be generated by different mutations within a single gene. A strong correlation existed between the severity of the disease and the extent to which the keratin filament network was disrupted, implicating perturbations in keratin networks as an essential component of these diseases. Some keratin mutants elicited subtle perturbations, with no signs of the tonofilament clumping typical of Dowling-Meara EBS and our previous transgenic mice. Importantly, basal cell cytolysis still occurred, thereby uncoupling cytolysis from the generation of large, insoluble cytoplasmic protein aggregates. Moreover, cell rupture occurred in a narrowly defined subnuclear zone, and seemed to involve three factors: (a) filament perturbation, (b) the columnar shape of the basal cell, and (c) physical trauma. This work provides the best evidence to date for a structural function of a cytoplasmic intermediate filament network, namely to impart mechanical integrity to the cell in the context of its tissue.