RESUMEN
Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele â¼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Predisposición Genética a la Enfermedad/genética , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Proteínas tau/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endofenotipos , Expresión Génica/genética , Humanos , Ratones , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Proteínas Nucleares/biosíntesis , Placa Amiloide/patología , Polimorfismo de Nucleótido Simple/genética , Sinaptosomas/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Proteínas tau/antagonistas & inhibidoresRESUMEN
The two main histopathological hallmarks that characterize Alzheimer's Disease are the presence of amyloid plaques and neurofibrillary tangles. One of the current approaches to studying the consequences of amyloid pathology relies on the usage of transgenic animal models that incorporate the mutant humanized form of the amyloid precursor protein (hAPP), with animal models progressively developing amyloid pathology as they age. However, these mice models generally overexpress the hAPP protein to facilitate the development of amyloid pathology, which has been suggested to elicit pathological and neuropathological changes unrelated to amyloid pathology. In this current study, we characterized APP knock-in (APP-KI) animals, that do not overexpress hAPP but still develop amyloid pathology to understand the influence of protein overexpression. We also induced tau pathology via human-derived tau seeding material to understand the neurophysiological effects of amyloid and tau pathology. We report that tau-seeded APP-KI animals progressively develop tau pathology, exacerbated by the presence of amyloid pathology. Interestingly, older amyloid-bearing, tau-seeded animals exhibited more amyloid pathology in the entorhinal area, isocortex and hippocampus, but not thalamus, which appeared to correlate with impairments in gamma oscillations before seeding. Tau-seeded animals also featured immediate deficits in power spectra values and phase-amplitude indices in the hippocampus after seeding, with gamma power spectra deficits persisting in younger animals. Both deficits in hippocampal phase-amplitude coupling and gamma power differentiate tau-seeded, amyloid-positive animals from buffer controls. Based on our results, impairments in gamma oscillations appear to be strongly associated with the presence and development of amyloid and tau pathology, and may also be an indicator of neuropathology, network dysfunction, and even potential disposition to the future development of amyloid pathology.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Placa Amiloide/patología , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Alzheimer's Disease (AD) is a neurodegenerative disease characterized by two main pathological hallmarks: amyloid plaques and intracellular tau neurofibrillary tangles. However, a majority of studies focus on the individual pathologies and seldom on the interaction between the two pathologies. Herein, we present the longitudinal neuropathological and neurophysiological effects of a combined amyloid-tau model by hippocampal seeding of human-derived tau pathology in the APP.PS1/L166P amyloid animal model. We statistically assessed both neurophysiological and pathological changes using linear mixed modelling to determine if factors such as the age at which animals were seeded, genotype, seeding or buffer, brain region where pathology was quantified, and time-post injection differentially affect these outcomes. We report that AT8-positive tau pathology progressively develops and is facilitated by the amount of amyloid pathology present at the time of injection. The amount of AT8-positive tau pathology was influenced by the interaction of age at which the animal was injected, genotype, and time after injection. Baseline pathology-related power spectra and Higuchi Fractal Dimension (HFD) score alterations were noted in APP.PS1/L166P before any manipulations were performed, indicating a baseline difference associated with genotype. We also report immediate localized hippocampal dysfunction in the electroencephalography (EEG) power spectra associated with tau seeding which returned to comparable levels at 1 month-post-injection. Longitudinal effects of seeding indicated that tau-seeded wild-type mice showed an increase in gamma power earlier than buffer control comparisons which was influenced by the age at which the animal was injected. A reduction of hippocampal broadband power spectra was noted in tau-seeded wild-type mice, but absent in APP.PS1 animals. HFD scores appeared to detect subtle effects associated with tau seeding in APP.PS1 animals, which was differentially influenced by genotype. Notably, while tau histopathological changes were present, a lack of overt longitudinal electrophysiological alterations was noted, particularly in APP.PS1 animals that feature both pathologies after seeding, reiterating and underscoring the difficulty and complexity associated with elucidating physiologically relevant and translatable biomarkers of Alzheimer's Disease at the early stages of the disease.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedades Neurodegenerativas , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Proteínas Amiloidogénicas , Amiloidosis/complicaciones , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas/complicaciones , Placa Amiloide , Presenilina-1/genética , Proteínas tau/genéticaRESUMEN
BACKGROUND AND PURPOSE: The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice. EXPERIMENTAL APPROACH: The effect of obestatin on 6-h cumulative food intake was studied in fasted wildtype (ghrelin+/+) and ghrelin knockout (ghrelin-/-) mice. In both genotypes, the effect of obestatin and/or ghrelin was studied in vivo on gastric emptying measured with the (14)C-octanoic acid breath test and in vitro on neural responses elicited by electrical field stimulation (EFS) of fundic smooth muscle strips. KEY RESULTS: Administration of obestatin did not influence fasting-induced hyperphagia or gastric emptying in both genotypes. Injection of ghrelin accelerated gastric emptying in ghrelin+/+ and ghrelin-/- mice but the effect was not reversed by co-injection with obestatin. In fundic strips from ghrelin+/+ and ghrelin-/- mice, ghrelin increased EFS-induced contractions, but obestatin was without effect. However, co-administration with obestatin tended to reduce the excitatory effect of ghrelin in both genotypes. CONCLUSIONS AND IMPLICATIONS: In ghrelin-/- mice, obestatin failed to affect food intake and gastric motility. These results suggest that endogenous ghrelin does not mask the effect of obestatin and confirm that obestatin administered peripherally is not a major regulator of satiety signalling or gut motility.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Vaciamiento Gástrico/efectos de los fármacos , Ghrelina/farmacología , Animales , Pruebas Respiratorias , Caprilatos , Estudios Cruzados , Estimulación Eléctrica , Fundus Gástrico/efectos de los fármacos , Fundus Gástrico/metabolismo , Genotipo , Ghrelina/administración & dosificación , Ghrelina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Respuesta de Saciedad/efectos de los fármacos , Respuesta de Saciedad/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Abstract Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild-type and CB2(-/-) mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (n = 32). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10 mg kg(-1)), however, AM1241 (10 mg kg(-)1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2(-/-) mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.
Asunto(s)
Mesenterio/inervación , Neuronas Aferentes/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Bradiquinina/farmacología , Cannabinoides/farmacología , Electrofisiología , Indoles/farmacología , Yeyuno/inervación , Yeyuno/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mesenterio/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Aferentes/efectos de los fármacosRESUMEN
Synaptic dysfunction and disconnectivity are core deficits in Alzheimer's disease (AD), preceding clear changes in histopathology and cognitive functioning. Here, the early and late effects of tau pathology induction on functional network connectivity were investigated in P301L mice. Multichannel EEG oscillations were used to compute (1) coherent activity between the prefrontal cortex (PFC) and hippocampus (HPC) CA1-CA3 networks; (2) phase-amplitude cross frequency coupling (PAC) between theta and gamma oscillations, which is instrumental in adequate cognitive functioning; (3) information processing as assessed by auditory evoked potentials and oscillations in the passive oddball mismatch negativity-like (MMN) paradigm. At the end, the density of tau aggregation and GABA parvalbumin (PV+) interneurons were quantified by immunohistochemistry. Early weakening of EEG theta oscillations and coherent activity were revealed between the PFC and HPC CA1 and drastic impairments in theta-gamma oscillations PAC from week 2 onwards, while PV+ interneurons count was not altered. Moreover, the tau pathology disrupted the MMN complex amplitude and evoked gamma oscillations to standard and deviant stimuli suggesting altered memory formation and recall. The induction of intracellular tau aggregation by tau seed injection results in early altered connectivity and strong theta-gamma oscillations uncoupling, which may be exploited as an early electrophysiological signature of dysfunctional neuronal networks.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Red Nerviosa/patología , Red Nerviosa/fisiopatología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Cognición , Modelos Animales de Enfermedad , Electroencefalografía , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Ratones , Neuronas/metabolismo , Neuronas/patología , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Corteza Prefrontal/fisiopatología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin(-/-)) and control wild-type (ghrelin(+/+)) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin(-/-) mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000-1200 h) in ghrelin(+/+) mice was abolished in mutant mice. Diabetic ghrelin(-/-) mice lost 12.4% more body weight than ghrelin(+/+) mice. In diabetic ghrelin(+/+) mice, but not in ghrelin(-/-) mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and alpha-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced alpha-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Hiperfagia/etiología , Hormonas Peptídicas/fisiología , Animales , Núcleo Arqueado del Hipotálamo/fisiología , Glucemia/análisis , Peso Corporal , Ghrelina , Glucagón/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptido Y/análisis , Neuropéptido Y/fisiología , Oligopéptidos/farmacología , Hormonas Peptídicas/sangre , Estreptozocina , alfa-MSH/análisisRESUMEN
Lithium is the prototype mood stabilizer but its mechanism is still unresolved. Two hypotheses dominate-the consequences of lithium's inhibition of inositol monophosphatase at therapeutically relevant concentrations (the 'inositol depletion' hypothesis), and of glycogen-synthase kinase-3. To further elaborate the inositol depletion hypothesis that did not decisively determine whether inositol depletion per se, or phosphoinositols accumulation induces the beneficial effects, we utilized knockout mice of either of two inositol metabolism-related genes-IMPA1 or SMIT1, both mimic several lithium's behavioral and biochemical effects. We assessed in vivo, under non-agonist-stimulated conditions, 3H-inositol incorporation into brain phosphoinositols and phosphoinositides in wild-type, lithium-treated, IMPA1 and SMIT1 knockout mice. Lithium treatment increased frontal cortex and hippocampal phosphoinositols labeling by several fold, but decreased phosphoinositides labeling in the frontal cortex of the wild-type mice of the IMPA1 colony strain by ~50%. Inositol metabolites were differently affected by IMPA1 and SMIT1 knockout. Inositoltrisphosphate administered intracerebroventricularly affected bipolar-related behaviors and autophagy markers in a lithium-like manner. Namely, IP3 but not IP1 reduced the immobility time of wild-type mice in the forced swim test model of antidepressant action by 30%, an effect that was reversed by an antagonist of all three IP3 receptors; amphetamine-induced hyperlocomotion of wild-type mice (distance traveled) was 35% reduced by IP3 administration; IP3 administration increased hippocampal messenger RNA levels of Beclin-1 (required for autophagy execution) and hippocampal and frontal cortex protein levels ratio of Beclin-1/p62 by about threefold (p62 is degraded by autophagy). To conclude, lithium affects the phosphatidylinositol signaling system in two ways: depleting inositol, consequently decreasing phosphoinositides; elevating inositol monophosphate levels followed by phosphoinositols accumulation. Each or both may mediate lithium-induced behavior.
Asunto(s)
Encéfalo/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Inositol/metabolismo , Cloruro de Litio/farmacología , Simportadores/genética , Animales , Antimaníacos/farmacología , Autofagia/genética , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/genética , ARN Mensajero/genéticaRESUMEN
Proteolytic cleavage of the amyloid precursor protein (APP) has previously been shown to release its extracellular domain into the medium. The identification of the responsible proteinase(s), termed secretase(s), is a high priority in ongoing Alzheimer research. This is hampered by the unusual characteristics of these enzyme(s) and by the fact that they cleave only membrane associated APP. We report here, using a vaccinia virus based expression system, that pig kidney PK(15) cells express full-length, membrane bound APP695, but that secretion of APP is low. This heterologous expression system allows to assay candidate secretases in a cellular context by simple co-transfection of the APP and candidate secretase cDNA containing plasmids. Eight different members of the mouse and human furin family of proprotein processing enzymes were tested in this assay, but none of them enhanced the secretion of APP. Secretion of von Willebrand's factor was used as a positive control.
Asunto(s)
Amiloide/metabolismo , Riñón/enzimología , Proteínas de la Membrana , Precursores de Proteínas/metabolismo , Subtilisinas/metabolismo , Amiloide/biosíntesis , Animales , Células Cultivadas , Endopeptidasas/metabolismo , Furina , Humanos , Ratones , Plásmidos , Proteínas Priónicas , Priones , Precursores de Proteínas/biosíntesis , Porcinos , Transfección , Virus Vaccinia/enzimologíaRESUMEN
Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson's disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3ß because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3ß. GSK-3ß-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3ß to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3ß-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3ß-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3ß directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3ß. Together, these data establish a novel upstream role for GSK-3ß as one of several kinases associated with PTMs of key proteins known to be causal in PD.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/fisiopatología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animales , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Ratones Transgénicos , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMEN
We have reported transgenic mice with neuronal overexpression of the clinical mutant beta-amyloid precursor protein (APP) known as London, which develop an AD-related phenotype [Moechers, Dewachter, Lorent, Reversé, Baekelandt, Nadiu, Tesseur, Spittaels, Van den Haute, Checler, et al. (1999) J. Biol. Chem. 274, 6483-6492]. Characterized early symptoms (3-9 months) include disturbed behaviour, neophobia, aggression, hypersensitivity to kainic acid, hyposensitivity to N-methyl-D-aspartate, defective cognition and memory, and decreased long-term potentiation. Late in life, at 12-15 months, amyloid plaques develop in the brain and correlate with increased levels of beta-amyloid (A beta)40/42 (the 40- and 42-amino-acid forms of A beta). The formation of amyloid plaques is dissociated in time from and not involved in the early phenotype. Hyperphosphorylated protein tau is present but no tangle pathology is observed. In double-transgenic mice, i.e. APP/London x Presenilin 1, the increased production of A beta 42 results in amyloid plaques developing by the age of 6 months. Transgenic mice with overexpression of either human apolipoprotein E4 (ApoE4) or human protein tau in central neurons develop severe axonopathy in the brain and spinal cord. Progressive degeneration of nerves and muscles is demonstrated by motor problems, wasting and premature death. Tau is hyperphosphorylated but there is no formation of filaments or neurofibrillary tangles. The tangle aspect of AD pathology is still missing from all current transgenic amyloid models. Its implementation will require insight into the cellular signalling pathways which regulate the microtubule-stabilizing function by phosphorylation of neuronal tau.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/etiología , Precursor de Proteína beta-Amiloide/genética , Animales , Apolipoproteína E4 , Apolipoproteínas E/genética , Modelos Animales de Enfermedad , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Mutación , Degeneración Nerviosa/etiología , Degeneración Nerviosa/genética , Presenilina-1 , Proteínas tau/genéticaRESUMEN
A mutant amyloid precursor protein (APP/RK) designed to interfere with processing by alpha-secretase caused a severe phenotype in transgenic mice, including behavioural abnormalities, i.e. neophobia, aggression, hypersensitivity to kainic acid, hyposensitivity to N-methyl-D-aspartate, and premature death [Moechars D. et al. (1996) Eur. molec. Biol. Org. J. 15, 1265-1274]. We now demonstrated that the APP/RK transgene did not disturb the expression of several other genes, i.e. endogenous amyloid precursor protein and amyloid precursor protein-like proteins, members of the low density lipoprotein receptor lipoprotein receptor family and several of their ligands, including apolipoprotein E, but expression of alpha-2-macroglobulin was never detected. Neither amyloid deposits nor neurofibrillary tangles were detected in the brain of APP/RK transgenic mice, even when 15-months-old. The tendency for seizures and hyposensitivity for N-methyl-D-aspartate was not due to or reflected in the distribution of the three major types of glutamate receptors. The major and consistent finding in transgenic APP/RK mice that died prematurely was extensive neurodegeneration and apoptosis, mainly in hippocampus and cortex, and accompanied by astrocytosis throughout the brain. Reduced synaptic density and dendritic damage was only observed in three transgenic mice that were killed shortly after positive observation of seizures. In addition, the distribution of cathepsin D and ubiquitin was abnormal in these mice.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Apoptosis/fisiología , Longevidad/fisiología , Mutación/fisiología , Degeneración Nerviosa/fisiopatología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/patología , Catepsina D/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Transgénicos/genética , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Receptores de Glutamato/metabolismo , Receptores de LDL/metabolismo , Distribución Tisular/fisiología , Ubiquitinas/metabolismoRESUMEN
We have analysed by northern blotting and by in situ hybridization the expression patterns of eight different genes during the second half of mouse embryonic development and in adult mouse brain: we compared the messenger RNA levels of amyloid precursor protein and of the two amyloid precursor protein-like proteins 1 and 2 and we have analysed expression of apolipoprotein E and of its main receptor in brain, the alpha-2-macroglobulin/low density lipoprotein receptor-related protein and three other ligands: the proteinase inhibitor alpha-2-macroglobulin, the modifying enzyme lipoprotein lipase and the 44,000 molecular weight heparin binding protein, a ligand of unknown function. During embryogenesis the temporal expression pattern differs considerably for the three members of the amyloid precursor proteins. Total embryo messenger RNA levels of amyloid precursor protein and amyloid precursor protein-like protein 2 increased progressively, while amyloid precursor protein-like protein 1 messenger RNA showed a burst of synthesis between days 10 and 13 post-coitum. Significantly, expression of the alpha-2-macroglobulin/low density lipoprotein receptor-related protein and of its associated protein, the 44,000 molecular weight heparin binding protein, exhibited their most important increase very similar to that of amyloid precursor protein-like protein 1, between days 10 and 13 post-coitum. Apolipoprotein E, lipoprotein lipase and alpha-2-macroglobulin messenger RNA levels in total embryos increased progressively, beginning most pronounced at days 13, 15 and 17, respectively. In mouse embryos, in situ hybridization established amyloid precursor protein, amyloid precursor protein-like protein 2 and alpha-2-macroglobulin/low density lipoprotein receptor-related protein messenger RNA to be expressed in most organs, with the notable exception of the liver, while expression of the other studied proteins was much more restricted. Among adult mouse tissues, the genes investigated were expressed very prominently in brain, except for lipoprotein lipase and for the complete absence of alpha-2-macroglobulin messenger RNA. In adult mouse brain, the cortex and hippocampus exhibited strong signals for most genes analysed. Exceptions are lipoprotein lipase and apolipoprotein E messenger RNAs, and the absent alpha-2-macroglobulin messenger RNA. Several interesting features, similarities as well as differences, between brain tissue sections hybridized with probes for amyloid precursor protein, amyloid precursor protein-like proteins 1 and 2 and between alpha-2-macroglobulin/low density lipoprotein receptor-related protein and heparin binding protein-44 were observed and are described. The results are further discussed in view of the known or anticipated physiological functions of the proteins examined and of their possible role in the etiology of Alzheimer's disease.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , Apolipoproteínas E/biosíntesis , Química Encefálica/fisiología , Proteínas Portadoras/biosíntesis , Glicoproteínas/biosíntesis , Receptores Inmunológicos/biosíntesis , Receptores de LDL/biosíntesis , alfa-Macroglobulinas/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Animales , Northern Blotting , Encéfalo/embriología , Proteínas Portadoras/genética , Femenino , Expresión Génica , Glicoproteínas/genética , Hibridación in Situ , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Lipoproteína Lipasa/biosíntesis , Lipoproteína Lipasa/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Peso Molecular , Embarazo , ARN/aislamiento & purificación , Receptores Inmunológicos/genética , Receptores de LDL/genética , alfa-Macroglobulinas/genéticaRESUMEN
The role of the receptor-associated protein in controlling the expression of the low-density lipoprotein receptor-related protein was analysed in brain and in cultured neurons of receptor-associated protein - / - mice. In addition, the effect of two important ligands of lipoprotein receptor-related protein in brain, i.e. apolipoprotein E and amyloid precursor protein, was examined by crossing the receptor-associated protein - / - mice with transgenic mice overexpressing these proteins specifically in neurons. The immunohistochemical localization of lipoprotein receptor-related protein and receptor-associated protein in wild-type mouse brain was demonstrated to be congruent over all structures, including the cortex and hippocampus. In primary hippocampal neurons, lipoprotein receptor-related protein was distributed somatodendritically and receptor-associated protein was concentrated perinuclearly. In hippocampal neurons from receptor-associated protein - / - mice, lipoprotein receptor-related protein was redistributed over the cell body at the expense of the dendrites. In the absence of receptor-associated protein, maturation of lipoprotein receptor-related protein is slow, resulting in accumulation of the uncleaved 600,000 mol. wt precursor. Neither the added expression of apolipoprotein E4 nor that of amyloid precursor protein in cultured neurons influenced the maturation of lipoprotein receptor-related protein, in either the presence or absence of receptor-associated protein. This result shows that receptor-associated protein is not needed to allow co-expression of lipoprotein receptor-related protein with these ligands in neurons. Furthermore, the typical ramified neuronal morphology of cultured primary neurons and the histology and architecture of the brain were normal in receptor-associated protein - / - mice and in all of the double transgenic mice. Finally, we demonstrated that the survival of receptor-associated protein - /- hippocampal neurons was normal and unaffected by the genotype of the glial feeder cells, whether they were derived from wild-type mice or from mice deficient in receptor-associated protein or apolipoprotein E. These results show that, despite the dramatic effect on maturation and cellular localization of lipoprotein receptor-related protein, the absence of receptor-associated protein did not result in any notable physiological, functional or morphological effects.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/genética , Proteínas Portadoras/genética , Glicoproteínas/genética , Hipocampo/química , Neuronas/química , Receptores de LDL/genética , Animales , Apolipoproteína E4 , Química Encefálica/genética , Proteínas Portadoras/análisis , Supervivencia Celular/fisiología , Células Cultivadas , Expresión Génica/fisiología , Genotipo , Glicoproteínas/análisis , Hipocampo/citología , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/citología , Neuroglía/fisiología , Neuronas/fisiología , Receptores de LDL/análisisRESUMEN
In transgenic mice that overexpress mutant Amyloid Precursor Protein [V717I], or APP/London (APP/Lo) (1999a. Early phenotypic changes in transgenic mice that overexpress different mutants of Amyloid Precursor Protein in brain. J. Biol. Chem. 274, 6483-6492; 1999b. Premature death in transgenic mice that overexpress mutant Amyloid precursor protein is preceded by severe neurodegeneration and apoptosis. Neuroscience 91, 819-830) the AD related phenotype of plaque and vascular amyloid pathology is late (12-15 months). This typical and diagnostic pathology is thereby dissociated in time from early symptoms (3-9 months) that include disturbed behavior, neophobia, aggression, glutamate excitotoxicity, defective cognition and decreased LTP. The APP/Lo transgenic mice are therefore a very interesting model to study early as well as late pathology, including the effect of age. In ageing APP*Lo mice, brain soluble and especially "insoluble" amyloid peptides dramatically increased, while normalized levels of secreted APPsalpha and APPsbeta, as well as cell-bound beta-C-stubs, remained remarkably constant, indicating normal alpha- and beta-secretase processing of APP. In double transgenic mice, i.e. APP/LoxPS1, clinical mutant PS1[A246E] but not wild-type human PS1 increased Abeta, and plaques and vascular amyloid developed at age 6-9 months. The PS1 mutant caused increasing Abeta42 production, while ageing did not. Amyloid deposits are thus formed, not by overproduction of Abeta, but by lack of clearance and/or degradation in the brain of ageing APP/Lo transgenic mice. The clearance pathways of the cerebral amyloid peptides are therefore valuable targets for fundamental research and for therapeutic potential. Although hyper-phosphorylated protein tau was evident in swollen neurites around the amyloid plaques, neurofibrillary pathology is not observed and the "tangle" aspect of AD pathology is therefore still missing from all current transgenic "amyloid" models. Also the "ApoE4" risk for late onset AD remains a problem for modeling in transgenic mice. We have generated transgenic mice that overexpress human ApoE4 (2000. Expression of Human Apolipoprotein E4 in neurons causes hyperphosphorylation of Protein tau in the brains of transgenic mice. Am. J. Pathol. 156 (3) 951-964) or human protein tau (1999. Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein. Am. J. Pathol. 155, 2153-2165) in their neurons. Both develop a similar although not identical axonopathy, with progressive degeneration of nerves and with muscle wasting resulting in motoric problems. Remarkably, ApoE4 transgenic mice are, like the tau transgenic mice, characterized by progressive hyper-phosphorylation of protein tau also in motor neurons which explains the motoric defects. Further crossing with the APP/Lo transgenic mice is ongoing to yield "multiple" transgenic mouse strains to study new aspects of amyloid and tau pathology.
Asunto(s)
Envejecimiento , Enfermedad de Alzheimer/etiología , Precursor de Proteína beta-Amiloide/fisiología , Modelos Animales de Enfermedad , Proteínas de la Membrana/fisiología , Animales , Apolipoproteína E4 , Apolipoproteínas E/fisiología , Humanos , Ratones , Ratones Transgénicos , Presenilina-1 , Proteínas tau/fisiologíaRESUMEN
Transgenic mouse strains were generated that overexpress human APP or clinical mutants of APP. All transgenic mouse strains that over-express APP displayed essentially the same phenotype of disturbed behaviour, differential glutamatergic responses, deficits in maintenance of long-term potentiation and premature death, but formation of amyloid plaques was seen in the highest expressing APP/London transgenic mice only. Apart from cognitive deficits, the APP transgenic mice were characterized by aggressive behaviour, which was pharmacologically alleviated with 8-OH-DPAT and buspirone, two serotonergic agonists. The atypical neuroleptic drug risperidone was equally active in this regard. The data establish an important aspect of the transgenic mice as experimental models for behavioural aspects of Alzheimer's disease, in addition to other early and late symptoms.
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Agresión , Precursor de Proteína beta-Amiloide/genética , Química Encefálica/efectos de los fármacos , Buspirona/farmacología , Agonistas de Receptores de Serotonina/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Química Encefálica/genética , Masculino , Ratones , Ratones Transgénicos , Risperidona/farmacología , Serotonina/farmacología , Antagonistas de la Serotonina/farmacologíaRESUMEN
Expression of alpha-secretion mutant APP/RK in mouse brain results in a progressive disorganization of the central nervous system, exemplified by behavioral deficits, premature death and neuropathology. Here we report on the progressive nature of this CNS disorder as indicated by the age dependency of the neophobic reaction in the open-field test. The earlier reported NMDA hypo-sensitivity in the transgenic APP/RK mice is likely to represent a subtle functional disturbance, since no changes in NMDA receptor density or distribution could be detected. None of the typical neuropathological hallmarks of Alzheimer's Disease, i.e. amyloid deposits and neurofibrillary tangles are detected in the brain of these transgenic mice. Nevertheless, the progressive CNS disorder elicited in the transgenic APP/RK mice recapitulates certain features and symptoms of patients with Alzheimer's disease as discussed.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Química Encefálica/genética , Enfermedades del Sistema Nervioso Central/genética , Enfermedades del Sistema Nervioso Central/metabolismo , Mutación/fisiología , Animales , Autorradiografía , Western Blotting , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/fisiología , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Tinción con Nitrato de PlataRESUMEN
Ghrelin is an important orexigenic peptide that not only exerts gastroprokinetic but also immunoregulatory effects. This study aimed to assess the role of endogenous and exogenous ghrelin in the pathogenesis of colitis and in the disturbances of gastric emptying and colonic contractility during this process. Dextran sodium sulphate colitis was induced for 5 days in (i) ghrelin(+/+) and ghrelin(-/-) mice and clinical and histological parameters were monitored at days 5, 10 and 26 and (ii) in Naval Medical Research Institute non-inbred Swiss (NMRI) mice treated with ghrelin (100 nmol kg(-1)) twice daily for 5 or 10 days. Neural contractility changes were measured in colonic smooth muscle strips, whereas gastric emptying was measured with the (14)C octanoic acid breath test. Inflammation increased ghrelin plasma levels. Body weight loss, histological damage, myeloperoxidase activity and IL-1beta levels were attenuated in ghrelin(-/-) mice. Whereas absence of ghrelin did not affect changes in colonic contractility, gastric emptying in the acute phase was accelerated in ghrelin(+/+) but not in ghrelin(-/-) mice. In agreement with the studies in ghrelin knockout mice, 10 days treatment of NMRI mice with exogenous ghrelin enhanced the clinical disease activity and promoted infiltration of neutrophils and colonic IL-1beta levels. Unexpectedly, ghrelin treatment decreased excitatory and inhibitory neural responses in the colon of healthy but not of inflamed NMRI mice. Endogenous ghrelin enhances the course of the inflammatory process and is involved in the disturbances of gastric emptying associated with colitis. Treatment with exogenous ghrelin aggravates colitis, thereby limiting the potential therapeutic properties of ghrelin during intestinal inflammation.
Asunto(s)
Colitis/fisiopatología , Ghrelina/metabolismo , Animales , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran/toxicidad , Vaciamiento Gástrico/efectos de los fármacos , Vaciamiento Gástrico/fisiología , Ghrelina/farmacología , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiologíaRESUMEN
To elucidate the role of endogenous ghrelin in the regulation of energy homeostasis and gastric emptying, ghrelin knockout mice (ghrelin(-/-)) were generated. Body weight, food intake, respiratory quotient, and heat production (indirect calorimetry), and gastric emptying ((14)C breath test) were compared between ghrelin(+/+) and ghrelin(-/-) mice. In both strains, the effect of exogenous ghrelin on gastric emptying and food intake was determined. Ghrelin(-/-) mice showed some subtle phenotypic changes. Body weight gain and 24-h food intake were not affected, but interruption of the normal light/dark cycle triggered additional food intake in old ghrelin(+/+) but not in ghrelin(-/-) mice. Exogenous ghrelin increased food intake in both genotypes with a bell-shaped dose-response curve that was shifted to the left in ghrelin(-/-) mice. During the dark period, young ghrelin(-/-) mice had a lower respiratory quotient, whereas their heat production was higher than that of the wild-type littermates, inferring a leaner body composition of the ghrelin(-/-) mice. Absence of ghrelin did not affect gastric emptying, and the bell-shaped dose-response curves of the acceleration of gastric emptying by exogenous ghrelin were not shifted between both strains. In conclusion, ghrelin is not an essential regulator of food intake and gastric emptying, but its loss may be compensated by other redundant inputs. In old mice, meal initiation triggered by the light/dark cue may be related to ghrelin. In young animals, ghrelin seems to be involved in the selection of energy stores and in the partitioning of metabolizable energy between storage and dissipation as heat.
Asunto(s)
Metabolismo Energético/genética , Vaciamiento Gástrico/genética , Homeostasis/genética , Hormonas Peptídicas/genética , Hormonas Peptídicas/fisiología , Envejecimiento/fisiología , Animales , Regulación de la Temperatura Corporal/genética , Regulación de la Temperatura Corporal/fisiología , Peso Corporal/genética , Peso Corporal/fisiología , Pruebas Respiratorias , Rastreo Diferencial de Calorimetría , Caprilatos , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/genética , Ingestión de Alimentos/fisiología , Metabolismo Energético/fisiología , Vaciamiento Gástrico/fisiología , Motilidad Gastrointestinal/genética , Motilidad Gastrointestinal/fisiología , Ghrelina , Homeostasis/fisiología , Inmunohistoquímica , Ratones , Ratones Noqueados , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Hormonas Peptídicas/farmacología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de GhrelinaRESUMEN
Several lines of evidence implicate a cholinergic deficit in Alzheimer's disease (AD). Transgenic mice that overexpress clinical mutants of the human amyloid precursor protein (APP) have been generated that recapitulate many aspects of AD. We now analyzed the cholinergic system in aged APP/London transgenic mice. The major finding was the reorganization of acetylcholinesterase-positive fibers within the hippocampus and the reduced size of cholinergic cells in the medial septum. The reduction of acetylcholinesterase-positive fibers in the subiculum together with increased fiber density in the CA1 and in the dentate gyrus suggests a synaptic sprouting compensatory mechanism within the hippocampus. In the cortex, amyloid plaques were associated with intense acetylcholinesterase activity and surrounded by dystrophic acetylcholinesterase-positive fibers. Nevertheless, the overall pattern of cholinergic innervation was unchanged. These results demonstrate that overexpression of APP/London caused, besides amyloid plaques in aged mouse brain, also cholinergic deafferentation and cholinergic cell shrinkage.