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1.
Sci Transl Med ; 10(459)2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232227

RESUMO

ß-Site APP (amyloid precursor protein) cleaving enzyme 1 (BACE1) is the ß-secretase enzyme that initiates production of the toxic amyloid-ß peptide that accumulates in the brains of patients with Alzheimer's disease (AD). Hence, BACE1 is a prime therapeutic target, and several BACE1 inhibitor drugs are currently being tested in clinical trials for AD. However, the safety of BACE1 inhibition is unclear. Germline BACE1 knockout mice have multiple neurological phenotypes, although these could arise from BACE1 deficiency during development. To address this question, we report that tamoxifen-inducible conditional BACE1 knockout mice in which the Bace1 gene was ablated in the adult largely lacked the phenotypes observed in germline BACE1 knockout mice. However, one BACE1-null phenotype was induced after Bace1 gene deletion in the adult mouse brain. This phenotype showed reduced length and disorganization of the hippocampal mossy fiber infrapyramidal bundle, the axonal pathway of dentate gyrus granule cells that is maintained by neurogenesis in the mouse brain. This defect in axonal organization correlated with reduced BACE1-mediated cleavage of the neural cell adhesion protein close homolog of L1 (CHL1), which has previously been associated with axon guidance. Although our results indicate that BACE1 inhibition in the adult mouse brain may avoid phenotypes associated with BACE1 deficiency during embryonic and postnatal development, they also suggest that BACE1 inhibitor drugs developed for treating AD may disrupt the organization of an axonal pathway in the hippocampus, an important structure for learning and memory.


Assuntos
Envelhecimento/metabolismo , Secretases da Proteína Precursora do Amiloide/deficiência , Ácido Aspártico Endopeptidases/deficiência , Axônios/metabolismo , Hipocampo/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Ácido Aspártico Endopeptidases/metabolismo , Cognição , Epilepsia/patologia , Epilepsia/fisiopatologia , Deleção de Genes , Hipocampo/patologia , Hipocampo/fisiopatologia , Potenciação de Longa Duração , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/metabolismo , Neurogênese , Fenótipo , Especificidade por Substrato
2.
Science ; 340(6135): 924-f, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23704555

RESUMO

Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) reported that bexarotene rapidly reduces ß-amyloid (Aß) levels and plaque burden in two mouse models of Aß deposition in Alzheimer's disease (AD). We now report that, although bexarotene reduces soluble Aß40 levels in one of the mouse models, the drug has no impact on plaque burden in three strains that exhibit Aß amyloidosis.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Tetra-Hidronaftalenos/farmacologia , Tetra-Hidronaftalenos/uso terapêutico , Animais , Masculino
3.
J Biol Chem ; 287(46): 38408-25, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22988240

RESUMO

BACE1 is the ß-secretase enzyme that initiates production of the ß-amyloid peptide involved in Alzheimer disease. However, little is known about the functions of BACE1. BACE1-deficient mice exhibit mild but complex neurological phenotypes suggesting therapeutic BACE1 inhibition may not be completely free of mechanism-based side effects. Recently, we have reported that BACE1 null mice have axon guidance defects in olfactory sensory neuron projections to glomeruli in the olfactory bulb. Here, we show that BACE1 deficiency also causes an axon guidance defect in the hippocampus, a shortened and disorganized infrapyramidal bundle of the mossy fiber projection from the dentate gyrus to CA3. Although we observed that a classical axon guidance molecule, EphA4, was cleaved by BACE1 when co-expressed with BACE1 in HEK293 cells, we could find no evidence of BACE1 processing of EphA4 in the brain. Remarkably, we discovered that the axon guidance defects of BACE1(-/-) mice were strikingly similar to those of mice deficient in a recently identified BACE1 substrate, the neural cell adhesion molecule close homolog of L1 (CHL1) that is involved in neurite outgrowth. CHL1 undergoes BACE1-dependent processing in BACE1(+/+), but not BACE1(-/-), hippocampus, and olfactory bulb, indicating that CHL1 is a BACE1 substrate in vivo. Finally, BACE1 and CHL1 co-localize in the terminals of hippocampal mossy fibers, olfactory sensory neuron axons, and growth cones of primary hippocampal neurons. We conclude that BACE1(-/-) axon guidance defects are likely the result of abrogated BACE1 processing of CHL1 and that BACE1 deficiency produces a CHL1 loss-of-function phenotype. Our results imply the possibility that axon mis-targeting may occur in adult neurogenic and/or regenerating neurons as a result of chronic BACE1 inhibition and add a note of caution to BACE1 inhibitor development.


Assuntos
Secretases da Proteína Precursora do Amiloide/fisiologia , Ácido Aspártico Endopeptidases/fisiologia , Moléculas de Adesão Celular/fisiologia , Doença de Alzheimer/genética , Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Modelos Neurológicos , Neurônios/metabolismo , Fenótipo
4.
Mol Neurodegener ; 6: 88, 2011 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-22204380

RESUMO

BACKGROUND: The ß-secretase, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), is a prime therapeutic target for lowering cerebral ß-amyloid (Aß) levels in Alzheimer's disease (AD). Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1-/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs), a well-studied model of axon targeting in vivo. RESULTS: We bred BACE1-/- mice with gene-targeted mice in which GFP is expressed from the loci of two odorant-receptors (ORs), MOR23 and M72, and olfactory marker protein (OMP) to produce offspring that were heterozygous for MOR23-GFP, M72-GFP, or OMP-GFP and were either BACE1+/+ or BACE1-/-. BACE1-/- mice had olfactory bulbs (OBs) that were smaller and weighed less than OBs of BACE1+/+ mice. In wild-type mice, BACE1 was present in OSN axon terminals in OB glomeruli. In whole-mount preparations and tissue sections, many OB glomeruli from OMP-GFP; BACE1-/- mice were malformed compared to wild-type glomeruli. MOR23-GFP; BACE1-/- mice had an irregular MOR23 glomerulus that was innervated by randomly oriented, poorly fasciculated OSN axons compared to BACE1+/+ mice. Most importantly, M72-GFP; BACE1-/- mice exhibited M72 OSN axons that were mis-targeted to ectopic glomeruli, indicating impaired axon guidance in BACE1-/- mice. CONCLUSIONS: Our results demonstrate that BACE1 is required for the accurate targeting of OSN axons and the proper formation of glomeruli in the OB, suggesting a role for BACE1 in axon guidance. OSNs continually undergo regeneration and hence require ongoing axon guidance. Neurogenesis and the regeneration of neurons and axons occur in other adult populations of peripheral and central neurons that also require axon guidance throughout life. Therefore, BACE1 inhibitors under development for the treatment of AD may potentially cause axon targeting defects in these neuronal populations as well.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Axônios/fisiologia , Bulbo Olfatório/anatomia & histologia , Bulbo Olfatório/crescimento & desenvolvimento , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/crescimento & desenvolvimento , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Axônios/ultraestrutura , Camundongos , Camundongos Knockout , Bulbo Olfatório/anormalidades , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Hum Mol Genet ; 19(16): 3105-13, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20511334

RESUMO

Empirical evidence supporting a genetic basis for the etiology of congenital heart disease (CHD) is limited and few disease-causing mutations have been identified. To identify novel CHD genes, we performed a forward genetic screen to identify mutant mouse lines with heritable CHD. Lines with recessive N-ethyl-N-nitrsourea-induced CHD-causing mutations were identified using a three-generation backcross. A hierarchical screening protocol was used to test the hypothesis that the fetal-to-neonatal circulatory transition unmasks the specific structural heart defects observed in CHD. Mice with heart defects were efficiently ascertained by selecting for pups exhibiting perinatal lethality and characterizing their cardiac pathology. A marked increase of perinatal lethality was observed in the mutagen-treated cohort compared with an untreated backcross population. Cardiac pathology on perinatal lethals revealed cardiovascular defects in 79 pups from 47 of 321 mutagenized lines. All identified structural abnormalities were analogous to previously described forms of human CHD. Furthermore, the phenotypic recurrence and variance patterns across all lines were similar to human CHD prevalence and recurrence patterns. We mapped the locus responsible for heritable atrioventricular septal defects in six lines (avc1-6). Our screen demonstrated that 'sporadic' CHD may have major genetic component and established a practical, efficient approach for identifying CHD candidate genes.


Assuntos
Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Cardiopatias Congênitas/genética , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Mapeamento Cromossômico , Cruzamentos Genéticos , Modelos Animais de Doenças , Etilnitrosoureia , Feminino , Testes Genéticos/métodos , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/diagnóstico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Miocárdio/patologia
6.
Apoptosis ; 13(1): 41-51, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17955374

RESUMO

Patients with mutations in the death receptor CD95 (Fas/APO-1) frequently develop B-cell lymphoma. However, solid tumors have not been found in the context of defective CD95. This could be due to the fatal autoimmune proliferative disease that develops in the absence of functional CD95 or to a difference in CD95 signaling in lymphoid versus nonlymphoid tissues. To test this we reconstituted mice that harbor a point mutation in the death domain of CD95 (lpr(cg) mice), either in one or in both alleles, with bone marrow from wild-type (wt) mice. After a year one third of the lpr(cg)/lpr(cg) mice developed spontaneous hepatic neoplasms. In contrast only one of the wt/lpr(cg) mice and none of the wt mice developed liver cancer. The agonistic anti-CD95 antibody Jo2 induced massive apoptosis in the liver of wt mice but not in the livers of either wt/lpr(cg) or lpr(cg)/lpr(cg) mice. The susceptibility of lpr(cg)/lpr(cg) mice to liver cancer cannot solely be due to impaired CD95 mediated apoptosis because there was no clear correlation between apoptosis resistance and tumor formation. A gene chip analysis identified genes selectively upregulated in the liver of wt and wt/lpr(cg) mice which may protect these mice from developing liver cancer. Our data represent the first case of CD95 protecting from developing a solid cancer.


Assuntos
Sistema Hematopoético/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos
7.
Anal Chem ; 78(14): 4945-51, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16841915

RESUMO

This paper describes a label-free assay for measuring endogenous caspase protease activities in cell lysates. The assay format, termed SAMDI-MS (self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry), is based on the enzymatic modification of peptides immobilized to monolayer substrates, followed by direct detection of the products with mass spectrometry. Monolayers presenting peptide substrates for either caspase-3 or -8 were treated with lysates from Jurkat cells that were stimulated with staurosporine and SKW6.4 cells that were stimulated with LzCD95L. In both cases, the SAMDI assays reported on the activation of endogenous caspase enzymes with levels of detection that are similar to those observed using the commonly employed fluorogenic assays. The use of longer peptide substrates, which are not compatible with the fluorogenic assays, provided for a better resolution of the two caspase activities. This work is significant because it demonstrates that the SAMDI assay can be used to measure endogenous enzyme activities and because it avoids the loss of activity and specificity that often accompany label-dependent assay formats.


Assuntos
Caspase 3/análise , Caspase 3/metabolismo , Caspase 8/análise , Caspase 8/metabolismo , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Espectrometria de Fluorescência/métodos , Sequência de Aminoácidos , Apoptose , Caspase 3/química , Caspase 8/química , Linhagem Celular , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Especificidade por Substrato
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