RESUMO
Elevated vascular endothelial growth factor (VEGF) and complement activation are implicated in the pathogenesis of different ocular diseases. The objective of this study was to investigate the hypothesis that dual inhibition of both VEGF and complement activation would confer better protection against ocular inflammation and neovascularization. In this study, we engineered a secreted chimeric VEGF inhibitor domain (VID), a complement inhibitor domain (CID) and a dual inhibitor (ACVP1). Vectors expressing these three inhibitors were constructed and packaged into AAV2 (sextY-F) particles. The expression and secretion of the proteins were validated by Western blot. The effects of these inhibitors expressed from AAV2 vectors were examined in endotoxin-induced uveitis (EIU), experimental autoimmune uveoretinitis (EAU) and choroidal neovascularization (CNV) mouse models. The AAV2 vectors expressing the CID- and ACVP1-attenuated inflammation in EIU and EAU model, whereas the vector expressing VID showed improved retinal structure damaged by EAU, but not affect the infiltration of inflammatory cells in EAU or EIU eyes. Both VID and CID vectors improved laser-induced retinal and choroid/RPE injuries and CNV, whereas ACVP1 vector provided significantly better protection. Our results suggest that gene therapy targeting VEGF and complement components could provide an innovative and long-term strategy for ocular inflammatory and neovascular diseases.
Assuntos
Neovascularização de Coroide/terapia , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Retinite/terapia , Uveíte/terapia , Animais , Doenças Autoimunes , Corioide/imunologia , Corioide/patologia , Neovascularização de Coroide/genética , Neovascularização de Coroide/imunologia , Neovascularização de Coroide/patologia , Dependovirus/metabolismo , Endotoxinas , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Retina/imunologia , Retina/patologia , Retinite/genética , Retinite/imunologia , Retinite/patologia , Uveíte/induzido quimicamente , Uveíte/genética , Uveíte/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid ß 42 (Aß42) to soluble fractions. CTB-MBP oral delivery reduced Aß42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/metabolismo , Cloroplastos/metabolismo , Toxina da Cólera/metabolismo , Proteína Básica da Mielina/metabolismo , Placa Amiloide/tratamento farmacológico , Administração Oral , Doença de Alzheimer/patologia , Animais , Cápsulas , Linhagem Celular Tumoral , Toxina da Cólera/genética , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina/genética , Folhas de Planta/citologia , Placa Amiloide/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
PURPOSE: Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. METHODS: The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. RESULTS: In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor mRNA. CONCLUSIONS: The Mas receptor is expressed in developing and adult mouse retinas, and is more abundant in retinal neurons than in endothelial and Müller glial cells. These observations suggest that Mas receptor-mediated signaling may play important roles that extend beyond mediating the vascular effects of Ang (1-7) in developing and adult retinas. In addition, the relatively high expression of the Mas receptor when compared to AT1R suggests that they may play a more important role in maintaining normal retinal physiology than previously considered.
Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/genética , Receptores Acoplados a Proteínas G/genética , Células Ganglionares da Retina/metabolismo , Angiotensina I/genética , Angiotensina I/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Animais Recém-Nascidos , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Cross-Talk , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina/genética , Células Ganglionares da Retina/ultraestrutura , Pigmentos da Retina/metabolismoRESUMO
AIM: AMPK can be considered as an important target molecule for cancer for its unique ability to directly recognize cellular energy status. The main aim of this study is to explore the role of different AMPK activators in managing cancer cell aggressiveness and to understand the mechanistic details behind the process. MAIN METHODS: First, we explored the AMPK expression pattern and its significance in different subtypes of lung cancer by accessing the TCGA data sets for LUNG, LUAD and LUSC patients and then established the correlation between AMPK expression pattern and overall survival of lung cancer patients using Kaplan-Meire plot. We further carried out several cell-based assays by employing different wet lab techniques including RT-PCR, Western Blot, proliferation, migration and invasion assays to fulfil the aim of the study. KEY FINDINGS: SIGNIFICANCE: This study identifies the importance of AMPK activators as a repurposing agent for combating lung and colon cancer cell aggressiveness. It also suggests SRT-1720 as a potent repurposing agent for cancer treatment especially in NSCLC patients where a point mutation is present in LKB1.
Assuntos
Proteínas Quinases Ativadas por AMP , Movimento Celular , Proliferação de Células , Neoplasias Pulmonares , Monoaminoxidase , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Monoaminoxidase/metabolismo , Monoaminoxidase/genética , Linhagem Celular Tumoral , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidores da Monoaminoxidase/farmacologia , Regulação Neoplásica da Expressão Gênica , Invasividade NeoplásicaRESUMO
The genesis and functioning of the central nervous system are one of the most intricate and intriguing aspects of embryogenesis. The big lacuna in the field of human CNS development is the lack of accessibility of the human brain for direct observation during embryonic and fetal development. Thus, it is imperative to establish alternative animal models to gain deep mechanistic insights into neurodevelopment, establishment of neural circuitry, and its function. Neurodevelopmental events such as neural specification, differentiation, and generation of neuronal and non-neuronal cell types have been comprehensively studied using a variety of animal models and in vitro model systems derived from human cells. The experimentations on animal models have revealed novel, mechanistic insights into neurogenesis, formation of neural networks, and function. The models, thus serve as indispensable tools to understand the molecular basis of neurodevelopmental disorders (NDDs) arising from aberrations during embryonic development. Here, we review the spectrum of in vivo models such as fruitfly, zebrafish, frog, mice, and nonhuman primates to study neurogenesis and NDDs like microcephaly and Autism Spectrum Disorder. We also discuss nonconventional models such as ascidians and the recent technological advances in the field to study neurogenesis, disease mechanisms, and pathophysiology of human NDDs. This article is categorized under: Cancer > Stem Cells and Development Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development Congenital Diseases > Genetics/Genomics/Epigenetics.
Assuntos
Transtorno do Espectro Autista , Microcefalia , Transtornos do Neurodesenvolvimento , Humanos , Animais , Camundongos , Peixe-Zebra , Neurogênese/fisiologiaRESUMO
A local renin-angiotensin system (RAS) exists in the retina and plays a critical role in retinal neurovascular function. The protective axis of RAS comprising of angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor attenuate the deleterious actions of increased levels of angiotensin II (Ang II), the main effector peptide of RAS. A new peptide, alamandine, and its receptor Mas-related G protein-coupled receptor D (MrgD) have been recently identified that share structural and functional similarity to Ang-(1-7) and its receptor, Mas, establishing another new protective axis of RAS. Here, we examined the expression and cellular localization of MrgD in the retina, the effect of MrgD deficiency on mouse retinal structure and function, as well as the biological function of alamandine in cultured retinal cells. We showed that MrgD is expressed in the retinal neurons, retinal vasculature, Müller glial and RPE cells, similar to Mas receptor expression. MrgD-deficient mice did not exhibit gross change in retinal morphology and thickness; however, these mice did show a progressive decrease in both scotopic and photopic a-wave and b-wave amplitudes, and increase in retinal capillary loss with age compared to age-matched wild-type mice. In vitro studies in human retinal cells showed that alamandine attenuated the Ang II and LPS-induced increases in inflammatory cytokine gene expression, NF-κB activation, Ang II and hydrogen peroxide-induced production of reactive oxygen species, comparable to that mediated by Ang-(1-7). These results support the notion that alamandine/MrgD may represent another new protective axis of RAS in the retina exerting anti-oxidative and anti-inflammatory effects.
Assuntos
Oligopeptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Envelhecimento/patologia , Angiotensina II , Animais , Células Cultivadas , Eletrorretinografia , Humanos , Ligantes , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genéticaRESUMO
Age-related macular degeneration (AMD) is a progressive retinal neurodegenerative disorder characterized by extracellular deposits known as drusen. A major constituent of drusen deposits are Alzheimer disease-associated amyloid ß (Aß) peptides. To understand the etiology of Aß proteostasis in AMD, we delivered recombinant adeno-associated virus (AAV) encoding Aß42 and Aß40 peptides fused to BRI2 protein by intraocular injection in C57BL/6J mice. Endogenous protease cleavage of such constructs leads to production of secreted Aß42 and Aß40 respectively. We demonstrate that overexpression of secreted Aß40 or Aß42 resulted in dramatic induction of drusen-like deposits by 2 months' post-injection. These drusen-like deposits were immunopositive for Aß and complement proteins but did not stain for conventional amyloid dyes, such as Thioflavin S. Both injected cohorts showed gliosis and degenerative changes, though ERG responses were minimally affected. Intriguingly, simultaneous overexpression of BRI-Aß40 or BRI-Aß42 together resulted in dose-dependent and cumulative changes reminiscent of AMD type pathology - drusen-like deposits, severe reduction in ERG responses, photoreceptor cell loss and gliosis. Here, we have established a physiological model of Aß containing deposits in wild-type mice that recapitulates major retinal pathophysiological features of AMD and will be instrumental in mechanistic understanding and development of therapeutic strategies against AMD.
Assuntos
Peptídeos beta-Amiloides/genética , Dependovirus/genética , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Degeneração Macular/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise , Epitélio Pigmentado da Retina/citologia , Transfecção/métodosRESUMO
Calsyntenin-2 has an evolutionarily conserved role in cognition. In a human genome-wide screen, the CLSTN2 locus was associated with verbal episodic memory, and expression of human calsyntenin-2 rescues the associative learning defect in orthologous Caenorhabditis elegans mutants. Other calsyntenins promote synapse development, calsyntenin-1 selectively of excitatory synapses and calsyntenin-3 of excitatory and inhibitory synapses. We found that targeted deletion of calsyntenin-2 in mice results in a selective reduction in functional inhibitory synapses. Reduced inhibitory transmission was associated with a selective reduction of parvalbumin interneurons in hippocampus and cortex. Clstn2(-/-) mice showed normal behavior in elevated plus maze, forced swim test, and novel object recognition assays. However, Clstn2(-/-) mice were hyperactive in the open field and showed deficits in spatial learning and memory in the Morris water maze and Barnes maze. These results confirm a function for calsyntenin-2 in cognitive performance and indicate an underlying mechanism that involves parvalbumin interneurons and aberrant inhibitory transmission.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Transtornos Cognitivos/metabolismo , Interneurônios/metabolismo , Proteínas de Membrana/deficiência , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Comportamento Exploratório/fisiologia , Feminino , Imunofluorescência , Masculino , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Inibição Neural/fisiologia , Parvalbuminas/metabolismo , Reconhecimento Psicológico/fisiologia , Memória Espacial/fisiologia , Técnicas de Cultura de TecidosRESUMO
Purpose: Dysfunction of the renin-angiotensin system (RAS) contributes to pathogenesis of diabetic retinopathy (DR). Yet RAS blockers have only limited beneficial effects on progression of DR in clinical trials. The natriuretic peptide system offsets RAS, so that enhancing the activity of this system on top of RAS blockade might be beneficial. Neprilysin has an important role in the degradation of natriuretic peptides. Therefore, we hypothesize that dual angiotensin receptor-neprilysin inhibition (ARNI) may outperform angiotensin receptor blocker (ARB) in protection against DR. We tested this hypothesis in streptozotocin-induced diabetic transgenic (mRen2)27 rats. Methods: Adult male diabetic (mRen2)27 rats were followed for 5 or 12 weeks. Treatment with vehicle, irbesartan (ARB), or ARB combined with the neprilysin inhibitor thiorphan (irbesartan+thiorphan [ARNI]) occurred during the final 3 weeks. Retinal cell death, gliosis, and capillary loss were evaluated. Real-time polymerase chain reaction (RT-PCR) analyses were performed to quantify the retinal level of inflammatory cell markers. Results: Both ARB- and ARNI-treated groups showed similarly reduced retinal apoptotic cell death, gliosis, and capillary loss compared to the vehicle-treated group in the 5-week study. Treatment with ARNI reduced the expression of inflammatory markers more than ARB treatment in the 5-week study. In the 12-week study, ARNI treatment showed significantly more reduction in apoptotic cell death (51% vs. 25% reduction), and capillary loss (68% vs. 43% reduction) than ARB treatment. Conclusions: Treatment with ARNI provides better protection against DR in diabetic (mRen2)27 transgenic rats, compared to ARB alone. This approach may be a promising treatment option for patients with DR.
Assuntos
Compostos de Bifenilo/farmacologia , Diabetes Mellitus Experimental , Retinopatia Diabética/tratamento farmacológico , Sistema Renina-Angiotensina/fisiologia , Tetrazóis/farmacologia , Tiorfano/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacocinética , Animais , Western Blotting , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/metabolismo , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Irbesartana , Masculino , Neprilisina , RNA/genética , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Perturbations of cell surface synapse-organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-ß) as a synapse-organizing protein unique in binding and recruiting α-neurexins, but not ß-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3-shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3â»/â» mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Hipocampo/citologia , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/citologia , Ratos , Receptores de Superfície Celular/metabolismo , Sinapses/genéticaRESUMO
Balanced development of excitatory and inhibitory synapses is required for normal brain function, and an imbalance in this development may underlie the pathogenesis of many neuropsychiatric disorders. Compared with the many identified trans-synaptic adhesion complexes that organize excitatory synapses, little is known about the organizers that are specific for inhibitory synapses. We found that Slit and NTRK-like family member 3 (Slitrk3) actS as a postsynaptic adhesion molecule that selectively regulates inhibitory synapse development via trans-interaction with axonal tyrosine phosphatase receptor PTPδ. When expressed in fibroblasts, Slitrk3 triggered only inhibitory presynaptic differentiation in contacting axons of co-cultured rat hippocampal neurons. Recombinant Slitrk3 preferentially localized to inhibitory postsynaptic sites. Slitrk3-deficient mice exhibited decreases in inhibitory, but not excitatory, synapse number and function in hippocampal CA1 neurons and exhibited increased seizure susceptibility and spontaneous epileptiform activity. Slitrk3 required trans-interaction with axonal PTPδ to induce inhibitory presynaptic differentiation. These results identify Slitrk3-PTPδ as an inhibitory-specific trans-synaptic organizing complex that is required for normal functional GABAergic synapse development.
Assuntos
Potenciais Pós-Sinápticos Inibidores/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural/fisiologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia/genética , Feminino , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/metabolismo , Humanos , Potenciais Pós-Sinápticos Inibidores/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Inibição Neural/genética , Terminações Pré-Sinápticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Sinapses/genética , Transmissão Sináptica/genética , Transfecção , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γ(del/del) null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs.
RESUMO
Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express noncatalytic isoforms, suggesting additional functions. Here we discovered noncatalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans binding to axonal protein tyrosine phosphatase receptor PTPσ. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC-neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.
Assuntos
Densidade Pós-Sináptica/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptor trkC/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Sinapses/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/fisiologia , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Densidade Pós-Sináptica/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Receptor trkC/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Transecting the corpus callosum of postnatal day (P)1-6 rats disinhibits the production of spindle bursts (SBs) within primary somatosensory cortex (S1), most notably during periods of sleep-related myoclonic twitching. Here we investigated developmental changes in this callosally mediated disinhibition and its association with cortical plasticity. Recordings in P2-15 subjects revealed that callosotomy-induced disinhibition is a transient feature of early development that disappears abruptly after P6. This abrupt switch was accompanied by sharp decreases in myoclonic twitching and equally sharp increases in spontaneous SBs and in the number of GABAergic and glutamatergic presynaptic terminals in S1. Expression of the K+Cl- cotransporter 2 (KCC2) also increased across these ages. To determine whether these developmental changes are associated with alterations in cortical plasticity, pups were callosotomized at P1, P6, or P8, and tested over the subsequent week. Regardless of age, callosotomy immediately disrupted SBs evoked by forepaw stimulation. Over the next week, the P1 and P6 callosotomy groups exhibited full recovery of function; in contrast, the P8 group did not exhibit recovery of function, thus indicating an abrupt decrease in cortical plasticity between P6 and P8. Together, our data demonstrate that callosotomy-induced disinhibition is a transient phenomenon whose disappearance coincides with the onset of increased intrinsic connectivity, establishment of excitatory-inhibitory balance, and diminished plasticity in S1. Accordingly, our findings indicate that callosotomy-induced disinhibition of twitch-related SBs is a bioassay of somatosensory cortical plasticity and, in addition, support the hypothesis that myoclonic twitches, like retinal waves, actively contribute to cortical development and plasticity.
Assuntos
Corpo Caloso/crescimento & desenvolvimento , Potenciais Somatossensoriais Evocados/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Sono/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Bicuculina/análogos & derivados , Bicuculina/farmacologia , Corpo Caloso/fisiologia , Eletroencefalografia , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Feminino , Ácido Glutâmico/metabolismo , Masculino , Muscimol/farmacologia , Inibição Neural/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Recuperação de Função Fisiológica/fisiologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/crescimento & desenvolvimento , Córtex Somatossensorial/metabolismo , Simportadores/metabolismo , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
Although the role of developmental apoptosis in shaping the complement and connectivity of sensory and motoneurons is well documented, the extent to which cell death affects the 13 cardinal classes of spinal interneurons is unclear. Using a series of genetic manipulations in vivo, we demonstrate for the first time a differential pattern of developmental apoptosis in molecularly identified spinal interneuron populations, and implicate the adhesion molecule family encoded by the 22-member protocadherin-gamma (Pcdh-gamma) gene cluster in its control. In constitutive Pcdh-gamma null mouse embryos, many interneuron populations undergo increased apoptosis, but to differing extents: for example, over 80% of En1-positive V1 neurons are lost, whereas only 30% of Chx10-positive V2a neurons are lost and there is no reduction in the number of V1-derived Renshaw cells. We show that this represents an exacerbation of a normal, underlying developmental pattern: the extent of each population's decrease in Pcdh-gamma mutants is precisely commensurate both with the extent of its loss during normal embryogenesis and with the extent of its increase in Bax(-/-) mice, in which apoptosis is genetically blocked. Interneuron apoptosis begins during the first wave of synaptogenesisis in the spinal cord, occurring first among ventral populations (primarily between E14 and E17), and only later among dorsal populations (primarily after P0). Utilizing a new, conditional Pcdh-gamma mutant allele, we show that the gamma-Pcdhs can promote survival non-cell-autonomously: mutant neurons can survive if they are surrounded by normal neurons, and normal neurons can undergo apoptosis if they are surrounded by mutant neurons.