RESUMO
The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.
Assuntos
Apoptose/fisiologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Neurônios/citologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Caspase 3/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Fosforilação , Potássio/metabolismo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , Proteína Reelina , Serina Endopeptidases/genética , Transdução de SinaisRESUMO
Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.
Assuntos
Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Animais , Proteína Morfogenética Óssea 15 , Linhagem Celular , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Fator 9 de Diferenciação de Crescimento , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Camundongos , Ratos , Proteínas Smad/metabolismo , Timidina , TrítioRESUMO
Disruption of the circadian rhythms is a frequent preclinical and clinical manifestation of Alzheimer's disease. Furthermore, it has been suggested that shift work is a risk factor for Alzheimer's disease. Previously, we have reported association of intolerance to shift work (job-related exhaustion in shift workers) with a variant rs12506228A, which is situated close to melatonin receptor type 1A gene (MTNR1A) and linked to MTNR1A brain expression levels. Here, we studied association of that variant with clinical and neuropathological Alzheimer's disease in a Finnish whole-population cohort Vantaa 85+ (n = 512, participants over 85 years) and two follow-up cohorts. Rs12506228A was associated with clinical Alzheimer's disease (p = 0.000073). Analysis of post-mortem brain tissues showed association with higher amount of neurofibrillary tangles (p = 0.0039) and amyloid beta plaques (p = 0.0041). We then followed up the associations in two independent replication samples. Replication for the association with clinical Alzheimer's disease was detected in Kuopio 75+ (p = 0.012, n = 574), but not in the younger case-control sample (n = 651 + 669). While melatonin has been established in regulation of circadian rhythms, an independent role has been also shown for neuroprotection and specifically for anti-amyloidogenic effects. Indeed, in vitro, RNAi mediated silencing of MTNR1A increased the amyloidogenic processing of amyloid precursor protein (APP) in neurons, whereas overexpression decreased it. Our findings suggest variation close to MTNR1A as a shared genetic risk factor for intolerance to shift work and Alzheimer's disease in old age. The genetic associations are likely to be mediated by differences in MTNR1A expression, which, in turn, modulate APP metabolism.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Emaranhados Neurofibrilares/genética , Placa Amiloide/genética , Receptor MT1 de Melatonina/genética , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Ritmo Circadiano , Estudos de Coortes , Feminino , Finlândia , Predisposição Genética para Doença , Humanos , Técnicas In Vitro , Masculino , Melatonina/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/metabolismo , Placa Amiloide/patologia , Receptor MT1 de Melatonina/metabolismo , Receptores de MelatoninaRESUMO
Amyloid-ß precursor protein (APP) plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Fragmentos de Peptídeos/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Exossomos/metabolismo , Meia-Vida , Camundongos , Ligação ProteicaRESUMO
Neuronal LRRTM3 (leucine-rich repeat transmembrane 3) protein has been reported to promote amyloid-ß protein precursor (AßPP) processing and LRRTM3 is a candidate gene in late-onset Alzheimer's disease. To address the role of LRRTM3 in AßPP processing and amyloid-ß (Aß) production in vivo, we analyzed amyloidogenic processing of AßPP in the brains of LRRTM3-deficient mice and transgenic AßPP/PS1 mice with or without LRRTM3. We did not find differences between the genotypes in the levels of Aß or AßPP C-terminal fragments indicating that LRRTM3 is not an essential regulator of Aß production in adult mice. Moreover, Aß levels in primary cortical neurons were similar between the genotypes, indicating that LRRTM3 is not required for Aß generation in developing mice.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Moléculas de Adesão Celular Neuronais/deficiência , Proteínas de Membrana/deficiência , Proteínas do Tecido Nervoso/deficiência , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/fisiologia , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Genótipo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/deficiência , Fragmentos de Peptídeos/genéticaRESUMO
Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.