RESUMO
The ß-secretase ß-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Transdução de Sinais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Peixe-Zebra , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid ß lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Descoberta de Drogas , Pirrolidinas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Pirrolidinas/síntese química , Pirrolidinas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer's disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. METHODS AND RESULTS: We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. CONCLUSIONS: This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Biomarcadores/líquido cefalorraquidiano , Western Blotting , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Knockout , Especificidade por SubstratoRESUMO
To investigate further the site where estradiol (E(2)) inhibits food intake, we tested the effects on feeding of subcutaneous and intrahypothalamic implants of 10% E(2) benzoate in cholesterol (CHOL) or CHOL alone. E(2) was implanted subcutaneously in Silastic tubes, and intrahypothalamically via bilateral 29-gauge cannulas into the paraventricular nucleus (PVN) or the medial preoptic area (MPA) of ovariectomized (OVX) Sprague-Dawley and Long-Evans rats. Three-day implant periods followed 3-day baseline periods. Rats were allowed ad libitum access to chow and tap water, and food intake and body weight were measured each day. Subcutaneous 10% E(2) implants in Sprague-Dawley rats reduced food intake 21% on Day 2 and 34% on Day 3 (P's<.01) and decreased 3-day body weight gain 11 g (P<.05). In contrast, 10% E(2) implants in the PVN of Sprague-Dawley rats did not change food intake or body weight. Implants of 10% or 20% E(2) in the MPA also failed to decrease food intake. MPA implants of 10% E(2) decreased body weight gain 8 g (P<.05), but MPA implants of 20% E(2) decreased weight gain only 4 g (P>.05). To determine whether the strain of rat affected our negative results on food intake, we implanted 10% E(2) into the PVN of Long-Evans rats. Again, PVN E(2) did not decrease food intake significantly in comparison to the pretest baseline. PVN E(2) did, however, decrease body weight gain 5 g and decreased food intake 6% compared to rats with implants of CHOL (both P<.05), but these effects appeared to be due to an increase in feeding in the CHOL group in comparison to their baseline. Finally, CHOL and E(2) implants did not impair the responsivity of the PVN because acute implants of norepinephrine (NE) into the PVN of E(2)- or CHOL-treated Long-Evans rats significantly increased food intake. Our results do not support the hypothesis that E(2)'s actions in either the PVN or the MPA are sufficient to account for its inhibitory effects on feeding.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Estradiol/farmacologia , Hipotálamo/fisiologia , Ovariectomia , Animais , Peso Corporal/efeitos dos fármacos , Implantes de Medicamento , Estradiol/administração & dosagem , Feminino , Microinjeções , Núcleo Hipotalâmico Paraventricular/fisiologia , Gravidez , Área Pré-Óptica/fisiologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
We investigated the neural mediation of the feeding suppression through orally administered hydroxycitrate (HCA) in male rats that were fed a high-glucose diet (about 48% glucose). Ten-day ad libitum food intake and body weight regain after previous body weight loss (13% of initial body weight) due to restrictive feeding were measured in rats with sham deafferentation (SHAM; n = 6), subdiaphragmatic vagal deafferentation (SDA; n = 7), and SDA plus celiac-superior mesenteric ganglionectomy (SDA/CGX; n = 9). HCA suppressed the 10-day cumulative food intake in all surgical groups and body weight regain in SDA and SDA/CGX groups. Independent of HCA, SDA and SDA/CGX rats consumed less food and gained less weight compared to SHAM rats. These results demonstrate that all vagal afferents from below the diaphragm and vagal efferents of the dorsal trunk as well as splanchnic nerves (afferents and efferents) are not necessary for the feeding-suppressive effect of HCA in this animal model. Vagal afferents, however, appear to play a role in the control of intake when a high-glucose diet is consumed after a period of restrictive feeding.
Assuntos
Anorexia/fisiopatologia , Regulação do Apetite/fisiologia , Vias Autônomas/fisiologia , Comportamento Alimentar/fisiologia , Ganglionectomia , Vagotomia Troncular , Animais , Anorexia/induzido quimicamente , Depressores do Apetite , Regulação do Apetite/efeitos dos fármacos , Vias Autônomas/cirurgia , Citratos , Ingestão de Alimentos/fisiologia , Inibidores Enzimáticos , Ácidos Graxos/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Gânglios Simpáticos/fisiologia , Gânglios Simpáticos/cirurgia , Masculino , Neurônios Aferentes , Ratos , Ratos Sprague-Dawley , Nervos Esplâncnicos/fisiologia , Nervos Esplâncnicos/cirurgia , Nervo Vago/fisiologia , Nervo Vago/cirurgiaRESUMO
Rats consistently reduce their food intake following injections of bacterial lipopolysaccharides (LPS). Because inhibition of serotonergic (5-HT) activity by 8-OH-DPAT (5-HT(1A) activation) attenuates LPS-induced anorexia, we conducted a series of studies to examine whether other 5-HT-receptors are involved in the mediation of peripheral LPS-induced anorexia. In all experiments, rats were injected with LPS (100 microg/kg body weight [BW] ip) at lights out (hour 0). Antagonists were administered peripherally at hour 4, shortly after the onset of anorexia, which presumably follows the enhanced cytokine production after LPS. Food intake was then recorded during the subsequent 2 h or longer. 5-HT receptor antagonists cyanopindolol and SB 224289 (5-HT(1B)), ketanserin (5-HT(2A)), RS-102221 (5-HT(2C)), and metoclopramide (5-HT(3)) failed to attenuate LPS-induced anorexia. In contrast, both ritanserin (5-HT(2A/C)-receptor antagonist) (0.5 mg/kg BW) and SB 242084 (5-HT(2C)) (0.3 mg/kg BW) attenuated LPS-induced anorexia at doses that did not alter food intake in non-LPS-treated rats (all P<.01). Our results suggest that at least part of the anorexia following peripheral LPS administration is mediated through an enhanced 5-HT-ergic activity and the 5-HT(2C) receptor.
Assuntos
Anorexia/induzido quimicamente , Lipopolissacarídeos/farmacologia , Pindolol/análogos & derivados , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Aminopiridinas/farmacologia , Animais , Anorexia/psicologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Indóis/farmacologia , Masculino , Metoclopramida/farmacologia , Pindolol/farmacologia , Piperidonas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2C de Serotonina , Ritanserina/farmacologia , Compostos de Espiro/farmacologia , Sulfonamidas/farmacologiaRESUMO
We examined the role of serotonin (5-HT) and the 5-HT(1A) and 5-HT(2C) receptors in the anorectic effects of centrally administered lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), and leptin. Food intake was measured in rats after intracerebroventricular (ICV) injections of LPS (20 ng), IL-1 beta (10 ng), or leptin (1 microg) at lights out, followed by intraperitoneal (IP) injections of either the 5-HT(1A) autoreceptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) (125 microg/kg) or the 5-HT(2C) receptor antagonist SB 242084 (0.3 mg/kg) at the onset of anorexia. SB 242084 significantly attenuated the food intake reduction caused by all compounds (all P<.01). IP 8-OH-DPAT attenuated ICV IL-1 beta-induced anorexia (P<.01). We also tested the involvement of the median raphe 5-HT(1A) receptors in peripheral LPS- and IL-1 beta-induced anorexia. Rats were injected intraperitoneally with either LPS (100 microg/kg) or IL-1 beta (2 microg/kg) at lights out, and 8-OH-DPAT (4 nmol) was administered directly into the median raphe nucleus at the onset of anorexia. Median raphe injections of 8-OH-DPAT significantly attenuated both IL-1 beta- and LPS-induced anorexia (both P<.01). These results implicate the 5-HT(2C) receptors in the mediation of central LPS-, IL-1 beta-, and leptin-induced anorexia. Our results also suggest that the midbrain raphe nuclei play a role in mediating the anorectic response to peripheral LPS and IL-1 beta.
Assuntos
Anorexia/induzido quimicamente , Interleucina-1/toxicidade , Leptina/toxicidade , Lipopolissacarídeos/toxicidade , Receptor 5-HT2C de Serotonina/fisiologia , Animais , Anorexia/fisiopatologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Injeções Intraventriculares , Masculino , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/fisiologia , Ratos , Ratos Sprague-Dawley , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
Adenosine A2A antagonists are believed to have therapeutic potential in the treatment of Parkinson's disease (PD). We have characterized the dual adenosine A2A/A1 receptor antagonist JNJ-40255293 (2-amino-8-[2-(4-morpholinyl)ethoxy]-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one). JNJ-40255293 was a high-affinity (7.5 nM) antagonist at the human A2A receptor with 7-fold in vitro selectivity versus the human A1 receptor. A similar A2A:A1 selectivity was seen in vivo (ED50's of 0.21 and 2.1 mg/kg p.o. for occupancy of rat brain A2A and A1 receptors, respectively). The plasma EC50 for occupancy of rat brain A2A receptors was 13 ng/mL. In sleep-wake encephalographic (EEG) studies, JNJ-40255293 dose-dependently enhanced a consolidated waking associated with a subsequent delayed compensatory sleep (minimum effective dose: 0.63 mg/kg p.o.). As measured by microdialysis, JNJ-40255293 did not affect dopamine and noradrenaline release in the prefrontal cortex and the striatum. However, it was able to reverse effects (catalepsy, hypolocomotion, and conditioned avoidance impairment in rats; hypolocomotion in mice) produced by the dopamine D2 antagonist haloperidol. The compound also potentiated the agitation induced by the dopamine agonist apomorphine. JNJ-40255293 also reversed hypolocomotion produced by the dopamine-depleting agent reserpine and potentiated the effects of l-dihydroxyphenylalanine (L-DOPA) in rats with unilateral 6-hydroxydopamine-induced lesions of the nigro-striatal pathway, an animal model of Parkinson's disease. Extrapolating from the rat receptor occupancy dose-response curve, the occupancy required to produce these various effects in rats was generally in the range of 60-90%. The findings support the continued research and development of A2A antagonists as potential treatments for PD.
Assuntos
Antiparkinsonianos/farmacologia , Indenos/farmacologia , Pirimidinas/farmacologia , Antagonistas do Receptor A1 de Adenosina/química , Antagonistas do Receptor A1 de Adenosina/farmacocinética , Antagonistas do Receptor A1 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/farmacocinética , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Antiparkinsonianos/química , Antiparkinsonianos/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Células CHO , Cricetulus , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Indenos/química , Indenos/farmacocinética , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Norepinefrina/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/fisiopatologia , Pirimidinas/química , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Ratos Wistar , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Non-competitive antagonists of the N-methyl-d-aspartate receptor (NMDA) such as phencyclidine (PCP) elicit schizophrenia-like symptoms in healthy individuals. Similarly, PCP dosing in rats produces typical behavioral phenotypes that mimic human schizophrenia symptoms. Although schizophrenic behavioral phenotypes of the PCP model have been extensively studied, the underlying alterations of intrinsic neuronal properties and synaptic transmission in relevant limbic brain microcircuits remain elusive. Acute brain slice electrophysiology and immunostaining of inhibitory neurons were used to identify neuronal circuit alterations of the amygdala and hippocampus associated with changes in extinction of fear learning in rats following PCP treatment. Subchronic PCP application led to impaired long-term potentiation (LTP) and marked increases in the ratio of NMDA to 2-amino-3(5-methyl-3-oxo-1,2-oxazol-4-yl)propionic acid (AMPA) receptor-mediated currents at lateral amygdala (LA) principal neurons without alterations in parvalbumin (PV) as well as non-PV, glutamic acid decarboxylase 67 (GAD 67) immunopositive neurons. In addition, LTP was impaired at the Schaffer collateral to CA1 hippocampal pathway coincident with a reduction in colocalized PV and GAD67 immunopositive neurons in the CA3 hippocampal area. These effects occurred without changes in spontaneous events or intrinsic membrane properties of principal cells in the LA. The impairment of LTP at both amygdalar and hippocampal microcircuits, which play a key role in processing relevant survival information such as fear and extinction memory concurred with a disruption of extinction learning of fear conditioned responses. Our results show that subchronic PCP administration in rats impairs synaptic functioning in the amygdala and hippocampus as well as processing of fear-related memories.
Assuntos
Medo , Transtornos da Memória/etiologia , Neurônios/fisiologia , Esquizofrenia/complicações , Esquizofrenia/patologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Tonsila do Cerebelo/patologia , Animais , Área Sob a Curva , Biofísica , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/toxicidade , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Técnicas In Vitro , Masculino , Neurônios/efeitos dos fármacos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Fenciclidina/toxicidade , Quinoxalinas/farmacologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Esquizofrenia/induzido quimicamente , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
Anorexia is an element of the acute-phase immune response. Its mechanisms remain poorly understood. Activation of inducible cyclooxygenase-2 (COX-2) in blood-brain-barrier endothelial cells and subsequent release of prostaglandins (e.g., prostaglandin E2, PGE2) may be involved. Therefore, we sought to relate the effects of prostaglandins on the anorexia following gram-negative bacterial lipopolysaccharide treatment (LPS) to neural activity in the dorsal and median raphe nuclei (DRN and MnR) in rats. COX-2 antagonist (NS-398, 10mg/kg; IP) administration prior to LPS (100µg/kg; IP) prevented anorexia and reduced c-Fos expression the DRN, MnR, nucleus tractus solitarii and several related forebrain areas. These data indicate that COX-2-mediated prostaglandin synthesis is necessary for LPS anorexia and much of the initial LPS-induced neural activation. Injection of NS-398 into the DRN and MnR (1ng/site) attenuated LPS-induced anorexia to nearly the same extent as IP NS-398, suggesting that prostaglandin signaling in these areas is necessary for LPS anorexia. Because the DRN and MnR are sources of major serotonergic projections to the forebrain, these data suggest that serotonergic neurons originating in the midbrain raphe play an important role in acute-phase response anorexia.