RESUMO
Since the delivery of biologic drugs to the brain is greatly hampered by the existence of the blood-brain barrier (BBB), brain shuttles are being developed to enhance therapeutic efficacy. As we have previously shown, efficient and selective brain delivery was achieved with TXB2, a cross-species reactive, anti-TfR1 VNAR antibody. To further explore the limits of brain penetration, we conducted restricted randomization of the CDR3 loop, followed by phage display to identify improved TXB2 variants. The variants were screened for brain penetration in mice using a 25 nmol/kg (1.875 mg/kg) dose and a single 18 h timepoint. A higher kinetic association rate to TfR1 correlated with improved brain penetration in vivo. The most potent variant, TXB4, showed a 3.6-fold improvement over TXB2, which had on average 14-fold higher brain levels when compared to an isotype control. Like TXB2, TXB4 retained brain specificity with parenchymal penetration and no accumulation in other organs. When fused with a neurotensin (NT) payload, it led to a rapid drop in body temperature upon transport across the BBB. We also showed that fusion of TXB4 to four therapeutic antibodies (anti-CD20, anti-EGFRvIII, anti-PD-L1 and anti-BACE1) improved their brain exposure between 14- to 30-fold. In summary, we enhanced the potency of parental TXB2 brain shuttle and gained a critical mechanistic understanding of brain delivery mediated by the VNAR anti-TfR1 antibody.
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Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells have been used to shuttle antibodies and other cargos across the blood brain barrier (BBB) to the brain. For these studies the TXB4 brain shuttle was fused to a TrkB neurotrophin receptor agonist antibody. The TXB4-TrkB fusion retained potent agonist activity at its cognate receptor and after systemic administration showed a 12-fold increase in brain levels over the unmodified antibody. Only the TXB4-TrkB antibody fusion was detected within the brain and localized to TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc), where it was associated with activated ERK1/2 signaling. When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease (PD), TXB4-TrkB, but not the unmodified antibody, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 brain shuttle allows a TrkB agonist antibody to reach neuroprotective concentrations in the brain parenchyma following systemic administration.
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Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin-converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID-19.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , COVID-19/imunologia , COVID-19/prevenção & controle , Chlorocebus aethiops , Humanos , Ligação Proteica , Tubarões/imunologia , Células VeroRESUMO
Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.
Assuntos
Afinidade de Anticorpos , Antígenos CD/imunologia , Transporte Biológico/imunologia , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Bacteriófagos/imunologia , Transporte Biológico/genética , Reações Cruzadas , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos/imunologia , Receptores de Antígenos/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/farmacocinética , TransfecçãoRESUMO
Transferrin receptor 1 (TfR1) mediated transcytosis is an attractive strategy to enhance brain uptake of protein drugs, but translation remains a challenge. Here, a single domain shark antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was used to shuttle protein cargo into the brain. TXB2 was fused to a human IgG1 Fc domain (hFc) or to the amyloid-ß (Aß) antibody bapineuzumab (Bapi). TXB2-hFc displayed 20-fold higher brain concentrations compared with a control VNAR-hFc at 18 hours post-injection in wt mice. At the same time point, brain concentrations of Bapi-TXB2 was threefold higher than Bapi. In transgenic mice overexpressing human Aß, the brain-to-blood concentration ratio increased with time due to interaction with intracerebral Aß deposits. The relatively stable threefold difference between Bapi-TXB2 and Bapi was observed for up to 6 days after injection. PET imaging and ex vivo autoradiography revealed more parenchymal distribution of Bapi-TXB2 compared with Bapi. In conclusion, the TXB2 VNAR shuttle markedly increased brain uptake of protein cargo and increased brain concentrations of the Aß binding antibody Bapi.
Assuntos
Antígenos CD/metabolismo , Produtos Biológicos/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Tromboxano B2/metabolismo , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/genética , Produtos Biológicos/farmacocinética , Barreira Hematoencefálica/diagnóstico por imagem , Sistemas de Liberação de Medicamentos , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Tromboxano B2/genética , TranscitoseRESUMO
B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders.
Assuntos
Anticorpos Biespecíficos/imunologia , Fator Ativador de Células B/antagonistas & inibidores , Mimetismo Molecular/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Linfócitos B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de PeptídeosRESUMO
Amyotrophic Lateral Sclerosis is a devastating neurological disease that is inevitably fatal after 3-5years duration. Treatment options are minimal and as such new therapeutic modalities are required. In this review, we discuss the role of the myostatin pathway as a modulator of skeletal muscle mass and therapeutic approaches using biological based therapies. Both monoclonal antibodies to myostatin and a soluble receptor decoy to its high affinity receptor have been used in clinical trials of neuromuscular diseases and while there have been efficacy signals with the latter approach there have also been safety issues. Our approach is to target the high affinity receptor-binding site on myostatin and to develop a next generation set of therapeutic reagents built on a novel protein scaffold. This is the natural single domain VNAR found in sharks which is extremely versatile and has the ability to develop products with superior properties compared to existing therapeutics.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Anticorpos Monoclonais/imunologia , Miostatina/efeitos dos fármacos , Esclerose Lateral Amiotrófica/metabolismo , Ensaios Clínicos como Assunto , Humanos , Miostatina/imunologiaRESUMO
In amyotrophic lateral sclerosis (ALS), the progressive loss of motor neurons is accompanied by extensive muscle denervation, resulting in paralysis and ultimately death. Upregulation of amyloid beta (A4) precursor protein (APP) in muscle fibres coincides with symptom onset in both sporadic ALS patients and the SOD1(G93A) mouse model of familial ALS. We have further characterized this response in SOD1(G93A) mice and also revealed elevated levels of ß-amyloid (Aß) peptides in the SOD1(G93A) spinal cord, which were predominantly localized within motor neurons and their surrounding glial cells. We therefore examined the effect of genetic ablation of APP on disease progression in SOD1(G93A) mice, which significantly improved multiple disease parameters, including innervation, motor function, muscle contractile characteristics, motor unit and motor neuron survival. These results therefore strongly suggest that APP actively contributes to SOD1(G93A)-mediated pathology. Together with observations from ALS cases, this study indicates that APP may contribute to human ALS pathology.
Assuntos
Substituição de Aminoácidos/genética , Precursor de Proteína beta-Amiloide/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/patologia , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Atrofia , Peso Corporal , Sobrevivência Celular , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Humanos , Longevidade , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Denervação Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Processamento de Proteína Pós-Traducional , Solubilidade , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Regulação para CimaRESUMO
One of the major barriers to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that originate from the myelin sheath and glial scar. So far, only a small number of pharmacological compounds have exhibited functional activity against CNS inhibitors in promoting axon regeneration after injury. To search for novel compounds that enhance neurite outgrowth in vitro, we initiated a screen of a collection of natural products. We identified four compounds with the potential to promote growth over a myelin substrate. Of these, Amphotericin B (AmB) was shown to enhance neurite outgrowth and antagonize activities of major myelin associated inhibitors and glial-scar-derived chondroitin sulfate proteoglycans. AmB was found to activate Akt and thereby suppress the activity of glycogen synthase kinase 3 beta. Also, a cell permeable peptide that inhibits Akt activity was shown to block the effect of AmB in promoting axonal growth, while another peptide that increases Akt activity stimulated axonal growth in the presence of the myelin associated inhibitors. Our results suggest that AmB can promote neurite outgrowth over a wide range of inhibitory substrates via a mechanism that involves activation of Akt.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Axônios/efeitos dos fármacos , Produtos Biológicos/farmacologia , Neurônios/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Glicoproteína Associada a Mielina/antagonistas & inibidores , Glicoproteína Associada a Mielina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Análise de Componente Principal , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.
Assuntos
Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Lipase Lipoproteica/genética , Animais , Ácidos Araquidônicos/metabolismo , Encéfalo/citologia , Glicerídeos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Neurogênese , Plasticidade Neuronal , Transdução de Sinais , Medula Espinal/metabolismo , Sinapses/fisiologiaRESUMO
The N-terminal domain of NogoA, called amino-Nogo, inhibits axonal outgrowth and cell spreading via a largely unknown mechanism. In the present study, we show that amino-Nogo decreases Rac1 activity and inhibits fibroblast spreading. 12-O-Tetradecanoylphorbol-13-acetate-type tumor promoters, such as phorbol 12-myristate 13-acetate (PMA) and teleocidin, increase Rac1 activity and overcome the amino-Nogo-induced inhibition of cell spreading. The stimulating effect of tumor promoters on cell spreading requires activation of protein kinase D and the subsequent activation of Akt1. Furthermore, we identified Akt1 as a new signaling component of the amino-Nogo pathway. Akt1 phosphorylation is decreased by amino-Nogo. Activation of Akt1 with a cell-permeable peptide, TAT-TCL1, blocks the amino-Nogo inhibition. Finally, we provide evidence that these signaling pathways operate in neurons in addition to fibroblasts. Our results suggest that activation of protein kinase D and Akt1 are approaches to promote axonal regeneration after injury.
Assuntos
Proteínas da Mielina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células 3T3 , Animais , Carcinógenos , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Inibidores do Crescimento , Humanos , Camundongos , Regeneração Nervosa , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Neurônios/metabolismo , Proteínas Nogo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Many studies have indicated that the inability of adult mammalian central nervous system (CNS) to regenerate after injury is partly due to the existence of growth-inhibitory molecules associated with CNS myelin. Studies over the years have led to the identification of multiple myelin-associated inhibitors, among which Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (Omgp) represent potentially major contributors to CNS axon regeneration failure. Here we review in vitro and in vivo investigations into these inhibitory ligands and their functional mechanisms, focusing particularly on the neuronal receptors that mediate the inhibitory signals from these myelin molecules. A better understanding of the receptors for myelin-associated inhibitors could provide opportunities to decipher the mechanism of restriction in CNS regeneration, and lead to the development of potential therapeutic targets in neurodegenerative diseases and neurological injury. We will discuss the structures of the receptors and therapeutic opportunities that might arise based on this information.
Assuntos
Inibidores do Crescimento/metabolismo , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Regeneração Nervosa/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Axônios/fisiologia , Proteínas Ligadas por GPI , Gangliosídeos/química , Gangliosídeos/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Glicoproteína Mielina-Oligodendrócito , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nogo , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
The diacylglycerol lipases (DAGLalpha/beta) synthesize 2-arachidonylglycerol (2-AG), the major endocannabinoid in the developing and adult brain (eCB). This lipid acts on cannabinoid receptors to regulate axonal growth and guidance, activity-dependent synaptic plasticity and adult neurogenesis, and can also protect neurons from excitotoxicity. 2-AG action is generally terminated by monoacylglycerol lipase (MAGL), however we know very little about the mechanisms that regulate neuronal sensitivity to eCBs. In the present study we show that the brain-derived neurotrophic factor (BDNF) can determine neuronal sensitivity to eCBs. In this context, in cultured cerebellar granule neurons (CGNs) BDNF increases the expression of CB1 receptor transcripts and decreases expression of MAGL transcripts. Using phosphorylation of Akt as the readout, we show that BDNF can promote a stable increase in neuronal sensitivity to eCBs. For example, concentrations of 2-AG and noladin either (NE) that normally do not lead to Akt phosphorylation in control neurons do so in neurons pre-treated with BDNF. In addition, Akt phosphorylation in response to a wide range of concentrations of NE was always greater in neurons pre-treated with BDNF. Our data suggests the existence of a positive feedback loop that might sustain neuronal survival in the normal brain.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Moduladores de Receptores de Canabinoides/biossíntese , Endocanabinoides , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Ácidos Araquidônicos/farmacologia , Ácidos Araquidônicos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Moduladores de Receptores de Canabinoides/genética , Células Cultivadas , Cerebelo/citologia , Regulação para Baixo , Glicerídeos/farmacologia , Glicerídeos/fisiologia , Monoacilglicerol Lipases/biossíntese , Monoacilglicerol Lipases/genética , Neurônios/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor CB1 de Canabinoide/biossíntese , Receptor CB1 de Canabinoide/genética , Regulação para CimaRESUMO
Myostatin is a member of the transformating growth factor-beta (TGF-beta) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a 'double muscling' phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno-associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects.
Assuntos
Dependovirus/metabolismo , Técnicas de Transferência de Genes , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Miostatina/administração & dosagem , Peptídeos/administração & dosagem , Precursores de Proteínas/administração & dosagem , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Hiperplasia , Hipertrofia , Injeções Intravenosas , Camundongos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/patologia , Miostatina/antagonistas & inibidores , Miostatina/metabolismo , Miostatina/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Peptídeos/metabolismo , Peptídeos/farmacologia , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologiaRESUMO
The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.
Assuntos
Envelhecimento/fisiologia , Diferenciação Celular/fisiologia , Ventrículos Cerebrais/crescimento & desenvolvimento , Lipase Lipoproteica/fisiologia , Receptor CB2 de Canabinoide/fisiologia , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Linhagem Celular , Células Cultivadas , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/enzimologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/enzimologia , Neurônios/fisiologia , Células-Tronco/citologia , Células-Tronco/enzimologia , Células-Tronco/fisiologiaRESUMO
While there is now substantial evidence that 5-HT(6) antagonism leads to significantly improved cognitive ability, the mechanism(s) and/or pathway(s) involved are poorly understood. We have evaluated the consequence of chronic administration of the 5-HT(6) receptor antagonists SB-271046 and SB-399885 on neural cell adhesion molecule polysialylation state (NCAM PSA), a neuroplastic mechanism necessary for memory consolidation. Quantitative analysis of NCAM PSA immunopositive neurons in the dentate gyrus of drug-treated animals revealed a dose-dependent increase in polysialylated cell frequency following treatment with both SB-271046 and SB-399885. These effects could not be attributed to increased neurogenesis, as no difference in the rate of bromodeoxyuridine incorporation was apparent between the control and drug-treated groups. A substantial increase in the frequency of polysialylated cells in layer II of the entorhinal and perirhinal cortices was also observed, brain regions not previously associated with neurogenesis. Chronic treatment with SB-271046 or SB-399885 also significantly increased the activation of dentate polysialylation that is specific to learning. This effect does not occur with other cognition-enhancing drugs, such as tacrine, and this action potentially differentiates 5-HT(6) receptor antagonism as an unique neuroplastic mechanism for cognitive processes which may slow or reverse age/neurodegenerative related memory deficits.
Assuntos
Giro Denteado/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/farmacologia , Neurônios/metabolismo , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Ácidos Siálicos/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Antimetabólitos , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Giro Denteado/citologia , Relação Dose-Resposta a Droga , Córtex Entorrinal/citologia , Córtex Entorrinal/efeitos dos fármacos , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH(2)) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists.
Assuntos
Gangliosídeos/metabolismo , Proteínas da Mielina/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS/metabolismo , Chlorocebus aethiops , Análise por Conglomerados , Gangliosídeos/química , Gangliosídeos/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Ácido N-Acetilneuramínico/química , Neuritos/metabolismo , Proteínas NogoRESUMO
Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.