RESUMEN
Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80-90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson's disease and other synucleinopathies.
Asunto(s)
Antiparkinsonianos/farmacología , Conducta Animal/efectos de los fármacos , Descubrimiento de Drogas , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones TransgénicosRESUMEN
UNLABELLED: Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid ß (Aß) and microtubule associate protein tau, leading to the selective degeneration of neurons in the neocortex, limbic system, and nucleus basalis, among others. Recent studies have shown that α-synuclein (α-syn) also accumulates in the brains of patients with AD and interacts with Aß and tau, forming toxic hetero-oligomers. Although the involvement of α-syn has been investigated extensively in Lewy body disease, less is known about the role of this synaptic protein in AD. Here, we found that reducing endogenous α-syn in an APP transgenic mouse model of AD prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. Together, these results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of Aß. SIGNIFICANCE STATEMENT: Reducing endogenous α-synuclein (α-syn) in an APP transgenic mouse model of Alzheimer's disease (AD) prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. These results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of amyloid ß.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Neuronas/metabolismo , Neuronas/patología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/patología , Regulación hacia Abajo/genética , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , alfa-Sinucleína/genética , Proteína de Unión al GTP rab3A/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
INTRODUCTION: Loss of synapses best correlates to cognitive deficits in Alzheimer's disease (AD) in which oligomeric neurotoxic species of amyloid-ß appears to contribute synaptic pathology. Although a number of clinical pathologic studies have been performed with limited sample size, there are no systematic studies encompassing large samples. Therefore, we performed a meta-analysis study. METHODS: We identified 417 publications reporting postmortem synapse and synaptic marker loss from AD patients. Two meta-analyses were performed using a single database of subselected publications and calculating the standard mean differences. RESULTS: Meta-analysis confirmed synaptic loss in selected brain regions is an early event in AD pathogenesis. The second meta-analysis of 57 synaptic markers revealed that presynaptic makers were affected more than postsynaptic markers. DISCUSSION: The present meta-analysis study showed a consistent synaptic loss across brain regions and that molecular machinery including endosomal pathways, vesicular assembly mechanisms, glutamate receptors, and axonal transport are often affected.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/patología , Sinapsis/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Ensayos Clínicos como Asunto/estadística & datos numéricos , Diagnóstico , Humanos , MEDLINE/estadística & datos numéricos , Sinapsis/patologíaRESUMEN
Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R) Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer's disease, three-repeat (3R) Tau is the most abundant splice, in Pick's disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick's disease (L266V and G272V mutations). Two lines expressing high (Line 13) and low (Line 2) levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick's-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the natural history and progression of 3R tauopathies and their relationship with mitochondrial alterations and might be suitable for therapeutical testing.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Pick , Secuencias Repetitivas de Aminoácido , Tauopatías , Proteínas tau/química , Proteínas tau/genética , Animales , Axones/patología , Conducta Animal , Regulación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Filamentos Intermedios/patología , Memoria , Ratones , Ratones Transgénicos , Mitocondrias/patología , Neocórtex/metabolismo , Neocórtex/patología , Enfermedad de Pick/genética , Enfermedad de Pick/metabolismo , Enfermedad de Pick/patología , Enfermedad de Pick/fisiopatología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/patología , Tauopatías/fisiopatología , Factores de Tiempo , Proteínas tau/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. EXPERIMENTAL APPROACH: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. KEY RESULTS: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 µM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. CONCLUSIONS AND IMPLICATIONS: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders.
Asunto(s)
Complejo SIDA Demencia , Antineoplásicos/farmacología , Quinasa 5 Dependiente de la Ciclina/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/toxicidad , Indoles/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Animales , Dasatinib , Clorhidrato de Erlotinib , Flavonoides/farmacología , Proteína gp120 de Envoltorio del VIH/genética , Técnicas In Vitro , Lapatinib , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas , Purinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Ratas , Roscovitina , Sunitinib , Tiazoles/farmacologíaRESUMEN
BACKGROUND: In dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aß) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aß and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aß in the hippocampus. RESULTS: We generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aß, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aß oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aß oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aß in α-syn-expressing neuronal cells. CONCLUSIONS: Together, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aß might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , alfa-Sinucleína/metabolismo , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Transgénicos , Neuronas/patología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , alfa-Sinucleína/genéticaRESUMEN
In Parkinson's disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson's disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.
Asunto(s)
Encéfalo/patología , Degeneración Nerviosa/patología , Neuronas/metabolismo , Sinapsis/patología , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Ácido Glutámico/genética , Humanos , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Lisina/genética , Trastornos de la Memoria/etiología , Trastornos de la Memoria/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Considerable progress has been made in the past few years in the fight against Alzheimer's disease (AD) and Parkinson's disease (PD). Neuropathological studies in human brains and experimental in vivo and in vitro models support the notion that synapses are affected even at the earliest stages of the neurodegenerative process. The objective of this manuscript is to review some of the mechanisms of synaptic damage in AD and PD. Some lines of evidence support the notion that oligomeric neurotoxic species of amyloid ß, α-synuclein, and Tau might contribute to the pathogenesis of synaptic failure at early stages of the diseases. The mechanisms leading to synaptic damage by oligomers might involve dysregulation of glutamate receptors and scaffold molecules that results in alterations in the axonal transport of synaptic vesicles and mitochondria that later on lead to dendritic and spine alterations, axonal dystrophy, and eventually neuronal loss. However, while some studies support a role of oligomers, there is an ongoing debate as to the exact nature of the toxic species. Given the efforts toward earlier clinical and preclinical diagnosis of these disorders, understanding the molecular and cellular mechanisms of synaptic degeneration is crucial toward developing specific biomarkers and new therapies targeting the synaptic apparatus of vulnerable neurons.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedad por Cuerpos de Lewy/etiología , Sinapsis/patología , Enfermedad de Alzheimer/patología , Humanos , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Sinucleínas/metabolismoRESUMEN
Alzheimer's disease (AD) is associated with the formation of toxic amyloid-ß (Aß)42 oligomers, and recent evidence supports a role for Aß dimers as building blocks for oligomers. Molecular dynamics simulation studies have identified clans for the dominant conformations of Aß42 forming dimers; however, it is unclear if a larger spectrum of dimers is involved and which set(s) of dimers might evolve to oligomers verse fibrils. Therefore, for this study we generated multiple structural conformations of Aß42, using explicit all-atom molecular dynamics, and then clustering the different structures based on key conformational similarities. Those matching a selection threshold were then used to model a process of oligomerization. Remarkably, we showed a greater diversity in Aß dimers than previously described. Depending on the clan family, different types of Aß dimers were obtained. While some had the tendency to evolve into oligomeric rings, others formed fibrils of diverse characteristics. Then we selected the dimers that would evolve to membranephilic annular oligomers. Nearly one third of the 28 evaluated annular oligomers had the dimer interfaces between the neighboring Aß42 monomers with possible salt bridges between the residue K28 from one side and either residue E22 or D23 on the other. Based on these results, key amino acids were identified for point mutations that either enhanced or suppressed the formation and toxicity of oligomer rings. Our studies suggest a greater diversity of Aß dimers. Understanding the structure of Aß dimers might be important for the rationale design of small molecules that block formation of toxic oligomers.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Modelos Moleculares , Multimerización de Proteína/fisiología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/ultraestructura , Análisis de Varianza , Animales , Células Cultivadas , Dimerización , Hipocampo/citología , Humanos , Microscopía Electrónica , Simulación de Dinámica Molecular , Células-Madre Neurales/metabolismo , Mutación Puntual/genética , Conformación Proteica , Estructura Secundaria de Proteína , RatasRESUMEN
Decreases in testosterone and 17ß-oestradiol (E(2)) are associated with an increased risk for Alzheimer's disease (AD), which has been attributed to an increase in ß-amyloid and tau pathological lesions. Although recent studies have used transgenic animal models to test the effects of sex steroid manipulations on AD-like pathology, almost none have systematically characterised the associations between AD lesions and sex steroid levels in the blood or brain in any mutant model. The present study evaluated age-related changes in testosterone and E(2) concentrations, as well as androgen receptor (AR) and oestrogen receptor (ER) α and ß expression, in brain regions displaying AD pathology in intact male and female 3xTgAD and nontransgenic (ntg) mice. We report for the first time that circulating and brain testosterone levels significantly increase in male 3xTgAD mice with age, but without changes in AR-immunoreactive (IR) cell number in the hippocampal CA1 or medial amygdala. The age-related increase in hippocampal testosterone levels correlated positively with increases in the conformational tau isoform, Alz50. These data suggest that the over-expression of human tau up-regulate the hypothalamic-pituitary-gonadal axis in these mice. Although circulating and brain E(2) levels remained stable with age in both male and female 3xTgAD and ntg mice, ER-IR cell number in the hippocampus and medial amygdala decreased with age in female transgenic mice. Furthermore, E(2) levels were significantly higher in the hippocampus than in serum, suggesting local production of E(2). Although triple transgenic mice mimic AD-like pathology, they do not fully replicate changes in human sex steroid levels, and may not be the best model for studying the effects of sex steroids on AD lesions.
Asunto(s)
Enfermedad de Alzheimer/sangre , Estradiol/sangre , Testosterona/sangre , Factores de Edad , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Disorders characterized by α-synuclein (α-syn) accumulation, Lewy body formation and parkinsonism (and in some cases dementia) are collectively known as Lewy body diseases. The molecular mechanism (or mechanisms) through which α-syn abnormally accumulates and contributes to neurodegeneration in these disorders remains unknown. Here, we provide an overview of current knowledge and prevailing hypotheses regarding the conformational, oligomerization and aggregation states of α-syn and their role in regulating α-syn function in health and disease. Understanding the nature of the various α-syn structures, how they are formed and their relative contributions to α-syn-mediated toxicity may inform future studies aiming to develop therapeutic prevention and intervention.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/terapia , alfa-Sinucleína/química , alfa-Sinucleína/uso terapéutico , Animales , Humanos , Modelos Biológicos , Enfermedades Neurodegenerativas/genética , Conformación Proteica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Alzheimer's disease (AD) is the most common form of dementia among the elderly with women exhibiting a higher risk than men for the disease. Due to these gender differences, there is great interest in the role that estrogens play in cognitive impairment and the onset of the classic amyloid and tau lesions in AD. Human and rodent studies indicate a strong association between low brain aromatase, sex hormone levels, and beta amyloid deposition. Therefore, the effects of depleting both circulating and brain estrogen levels, through gonadectomy and/or treatment with the aromatase inhibitor, anastrozole, upon hippocampal AD-like pathology in male and female 3xTgAD mice were evaluated. Liquid chromatography-mass spectrometry revealed anastrozole serum levels of 10.19 ng/mL and for the first time brain levels were detected at 4.7 pg/mL. Densitometric analysis of the hippocampus revealed that anastrozole significantly increased Aß- but not APP/Aß-immunoreactivity in intact 3xTgAD females compared to controls (p<0.001). Moreover, anastrozole significantly increased the number of Aß- compared to APP/Aß-positive hippocampal CA1 neurons in intact and gonadectomized female mice. Concurrently, anastrozole significantly reduced the APP/Aß plaque load in 9 month old female 3xTgAD mice. These data suggest that anastrozole treatment differentially affects select amyloid species which in turn may play a role in the extraneuronal to intraneuronal deposition of this peptide.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Inhibidores de la Aromatasa/farmacología , Hipocampo/patología , Nitrilos/farmacología , Orquiectomía , Ovariectomía , Triazoles/farmacología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Anastrozol , Animales , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismo , Placa Amiloide/patologíaRESUMEN
Amyloid-ß (Aß) plaques occur in close apposition to thickened or swollen cholinergic and galaninergic neurites within the neocortex and hippocampus in Alzheimer's disease (AD). Despite this observation, the effect of Aß deposition upon cholinergic and galaninergic dystrophic neurite formation remains unclear. Therefore, the purpose of this study was to evaluate the interaction between Aß deposition within the neocortex and hippocampus upon cholinergic and galaninergic dystrophic neurite formation. Neocortical and hippocampal tissue harvested from 3- and 12-month-old amyloid-ß protein precursor (AßPP)swe/PS1ΔE9 transgenic (Tg) mice were dual-immunolabeled with antibodies against either choline acetyltransferace and Aß (10D5) or galanin (Gal) and Aß. Stereology was used to quantify amyloid plaques and cholinergic or galaninergic dystrophic neurites. Plaque number was assessed using the optical fractionator; plaque area was calculated with the Cavalieri estimator, and dystrophic neurite numbers and thickness were manually measured. Neither amyloid nor dystrophic neuritic profiles were seen in the brains of 3-month-old Tg mice. In contrast, quantitative analysis revealed significantly more plaques in neocortex than hippocampus, with no difference in regional plaque size in 12-month-old Tg mice. Significantly more cholinergic than galaninergic dystrophic neurites-per-plaque occurred in the neocortex and hippocampus. Additionally, cholinergic dystrophic neurites were thicker than galaninergic dystrophic neurites in both regions. These data suggest that amyloid plaque deposition has a greater impact upon cholinergic than galaninergic dystrophic neurite formation in the neocortex and hippocampus in AßPPswe/PS1ΔE9 Tg mice. These data are also compatible with the hypothesis that galanin is neuroprotective and reduces dystrophic neurite formation in the face of amyloid toxicity.
Asunto(s)
Enfermedad de Alzheimer/patología , Colina O-Acetiltransferasa/metabolismo , Galanina/metabolismo , Hipocampo/patología , Neocórtex/patología , Neuritas/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutación/genética , Neocórtex/metabolismo , Neuritas/patología , Presenilina-1/genéticaRESUMEN
In the field of pharmacological research, the oral consumption of anastrozole, an aromatase inhibitor, when added to an animal's drinking water is hindered by poor drug palatability and environmental loss of drug solution. To overcome these caveats, we developed a novel approach for the oral delivery of anastrozole mixed in a solid hydration gel matrix that functions as a replacement for water. Heated hydration gel was mixed with anastrozole and distributed into a gel delivery device consisting of a 50 mL plastic conical tube containing four stacked 200 µL pipette tips to allow for air pressure induced gel disbursement. Transgenic female 3xTgAD mice were randomized to receive either anastrozole-treated or untreated hydration gel at 3 months of age. Body weights were recorded weekly, and gel consumption was measured every 1-3 days. Six months post treatment mice were killed and serum anastrozole levels were determined using liquid chromatography-mass spectrometry (LC-MS). Anastrozole-treated mice gained significantly more weight despite consuming significantly less hydration gel compared to vehicle treated mice. LC-MS analysis, using a low serum volume (10 µL), revealed average anastrozole serum levels of 2.91 ng/mL. Anastrozole-treated ovarian tissue displayed ovarian cysts, massive edema-like stroma, and also lacked corp lutea compared to control mice. These findings demonstrate that hydration gel delivered using the newly developed oral delivery method is a viable approach for pharmacological research involving compounds with poor palatability, low water solubility, and cost prohibitive compounds where environmental loss needs to be minimized.
Asunto(s)
Experimentación Animal , Administración Oral , Enfermedad de Alzheimer/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/genética , Anastrozol , Animales , Antipsicóticos/administración & dosificación , Antipsicóticos/sangre , Peso Corporal/efectos de los fármacos , Cromatografía Liquida/métodos , Modelos Animales de Enfermedad , Femenino , Geles , Humanos , Espectrometría de Masas , Ratones , Ratones Transgénicos , Nitrilos/administración & dosificación , Nitrilos/sangre , Presenilina-1/genética , Reproducibilidad de los Resultados , Triazoles/administración & dosificación , Triazoles/sangre , Proteínas tau/genéticaRESUMEN
Although the M(1) muscarinic receptor is a potential therapeutic target for Alzheimer's disease (AD) based on its wide spread distribution in brain and its association with learning and memory processes, whether its receptor response is altered during the onset of AD remains unclear. A novel [(35)S]GTPgammaS binding/immunocapture assay was employed to evaluated changes in M(1) receptor function in cortical tissue samples harvested from people who had no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD. M(1) function was stable across clinical groups. However, [(3)H]-oxotremorine-M radioligand binding studies revealed that the concentration of M(1) cortical receptors increased significantly between the NCI and AD groups. Although M(1) receptor function did not correlate with cognitive function based upon mini-mental status examination (MMSE) or global cognitive score (GCS), functional activity was negatively correlated with the severity of neuropathology determined by Braak staging and NIA-Reagan criteria for AD. Since M(1) agonists have the potential to modify the pathologic hallmarks of AD, as well as deficits in cognitive function in animal models of this disease, the present findings provide additional support for targeting the M(1) receptor as a potential therapeutic for AD.
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Acetilcolina/metabolismo , Enfermedad de Alzheimer/metabolismo , Corteza Cerebral/metabolismo , Receptor Muscarínico M1/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Unión Competitiva/fisiología , Biomarcadores/análisis , Biomarcadores/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Inmunoensayo/métodos , Masculino , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Pruebas Neuropsicológicas , Oxotremorina/metabolismo , Valor Predictivo de las Pruebas , Ensayo de Unión Radioligante/métodos , Receptor Muscarínico M1/análisis , Radioisótopos de Azufre/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The triple transgenic mouse (3xTgAD), harboring human APP(Swe), PS1(M146V) and Tau(P301L) genes, develops age-dependent forebrain intraneuronal Abeta and tau as well as extraneuronal plaques. We evaluated brainstem AD-like pathology using 6E10, AT8, and Alz50 antibodies and unbiased stereology in young and old 3xTgAD mice. Intraneuronal Abeta occurred in the tectum, periaqueductal gray, substantia nigra, red nucleus, tegmentum and mesencephalic V nucleus at all ages. Abeta-positive neuron numbers significantly decreased in the superior colliculus and substantia nigra while AT8-positive superior colliculus, red nucleus, principal sensory V, vestibular nuclei, and tegmental neurons significantly increased between 2 and 12 months. Alz50-positive neuron numbers increased only in the inferior colliculus between these ages. Dual labeling revealed a few Abeta- and tau-positive neurons. Plaques occurred only in the pons of female 3xTgAD mice starting at 9 months. 3xTgAD mice provide a platform to define in vivo mechanisms of Abeta and tau brainstem pathology.
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Envejecimiento , Enfermedad de Alzheimer/patología , Tronco Encefálico/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Antígenos/metabolismo , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas/patología , Presenilina-1/genética , Nexinas de Proteasas , Receptores de Superficie Celular/genética , Caracteres Sexuales , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Raloxifene and arzoxifene are benzothiophene selective estrogen receptor modulators (SERMs) of clinical use in postmenopausal osteoporosis and treatment of breast cancer and potentially in hormone replacement therapy. The benefits of arzoxifene are attributed to improved bioavailability over raloxifene, whereas the arzoxifene metabolite, desmethylarzoxifene (DMA) is a more potent antiestrogen. As polyaromatic phenolics, benzothiophene SERMs undergo oxidative metabolism to electrophilic quinoids. The long-term clinical use of SERMs demands increased understanding of correlations between structure and toxicity, with metabolism being a key component. A homologous series of 4'-substituted 4'-desmethoxyarzoxifene derivatives was developed, and metabolism was studied in liver and intestinal microsomes. Formation of glutathione conjugates was assayed in rat liver microsomes and novel adducts were characterized by liquid chromatography-tandem mass spectrometry. Formation of glucuronide conjugates was assayed in human intestine and liver microsomes, demonstrating formation of glucuronides ranging from 5 to 100% for the benzothiophene SERMs: this trend was inversely correlated with the loss of parent SERM in rat liver microsomal incubations. Molecular orbital calculations generated thermodynamic parameters for oxidation that correlated with Hammett substituent constants; however, metabolism in liver microsomes correlated with a combination of both Hammett and Hansch lipophilicity parameters. The results demonstrate a rich oxidative chemistry for the benzothiophene SERMs, the amplitude of which can be powerfully modulated, in a predictable manner, by structural tuning of the 4'-substituent. The predicted extensive metabolism of DMA was confirmed in vivo and compared with the relatively stable arzoxifene and F-DMA.
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Moduladores Selectivos de los Receptores de Estrógeno/metabolismo , Tiofenos/metabolismo , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Humanos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Oxidación-Reducción , Ratas , Moduladores Selectivos de los Receptores de Estrógeno/química , Espectrometría de Masas en Tándem , Tiofenos/químicaRESUMEN
UNLABELLED: The lack of a safe and reliable alternative to hormone therapy (HT) for treating menopausal symptoms underscores the need for alternative therapies. OBJECTIVE: The purpose of this study was to assess the in vivo estrogenic effects of the botanical dietary supplements Trifolium pratense (red clover) and Humulus lupulus (hops), and two compounds obtained from H. lupulus, isoxanthohumol and 8-prenylnaringenin (8-PN) using the ovariectomized uterotrophic adult rat model. A H. lupulus extract and a 30% isoflavone extract of T. pratense were tested at three escalating doses as was one dose of isoxanthohumol for 21d. 8-Prenylnaringenin, the major estrogen in H. lupulus, was also tested at three relevant escalating doses. In order to determine the in vivo metabolism of 8-PN, the major phases I and II metabolites were also identified. The primary outcome measure, uterus weight gain, indicated that H. lupulus and T. pratense did not have an estrogenic effect on the uterus, and none of the secondary outcome measures were positive. In contrast, there was a clear dose response when 8-PN was evaluated where the middle and high doses of 8-PN were active. 8-Prenylnaringenin in rat plasma, liver, and mammary gland was measured and the major phases I and II 8-PN metabolites were detected. Our findings suggest that while both the H. lupulus and T. pratense extracts do not have an effect on the rat uterus, 8-PN at equivalent doses to those previously used in humans did have an effect, and may therefore have a deleterious effect in women.
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Estrógenos/metabolismo , Flavanonas/farmacología , Humulus/metabolismo , Extractos Vegetales/farmacología , Trifolium/metabolismo , Xantonas/farmacología , Animales , Peso Corporal/efectos de los fármacos , Cromatografía Liquida , Eosina Amarillenta-(YS)/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Flavanonas/análisis , Flavanonas/química , Hematoxilina/metabolismo , Espectrometría de Masas , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Útero/citología , Útero/efectos de los fármacos , Útero/crecimiento & desarrollo , Xantonas/químicaRESUMEN
Oxidative metabolism of estrogens has been associated with genotoxicity. O-methylation of catechol estrogens is considered as a protective mechanism. 4-Methoxyequilenin (4-MeOEN) is the O-methylated product of 4-hydroxyequilenin (4-OHEN). 4-OHEN, the major catechol metabolite of the equine estrogens present in the most widely prescribed hormone replacement therapeutics, causes DNA damage via quinone formation. In this study, estrogen receptor (ERa) binding of 4-MeOEN was compared with estradiol (E2) and equilenin derivatives including 4-BrEN using computer modeling, estrogen response element (ERE)-luciferase induction in MCF-7 cells, and alkaline phosphatase (AP) induction in Ishikawa cells. 4-MeOEN induced AP and luciferase with nanomolar potency and displayed a similar profile of activity to E2. Molecular modeling indicated that MeOEN could be a ligand for ERa despite no binding being observed in the ERa competitive binding assay. Methylation of 4-OHEN may not represent a detoxification pathway, since 4-MeOEN is a full estrogen agonist with nanomolar potency.