RESUMO
Within the neuronal classes of the retina, amacrine cells (ACs) exhibit the greatest neuronal diversity in morphology and function. We show that the selective expression of the transcription factor Gbx2 is required for cell fate specification and dendritic stratification of an individual AC subtype in the mouse retina. We identify Robo1 and Robo2 as downstream effectors that when deleted, phenocopy the dendritic misprojections seen in Gbx2 mutants. Slit1 and Slit2, the ligands of Robo receptors, are localized to the OFF layers of the inner plexiform layer where we observe the dendritic misprojections in both Gbx2 and Robo1/2 mutants. We show that Robo receptors also are required for the proper dendritic stratification of additional AC subtypes, such as Vglut3+ ACs. These results show both that Gbx2 functions as a terminal selector in a single AC subtype and identify Slit-Robo signaling as a developmental mechanism for ON-OFF pathway segregation in the retina.
RESUMO
Our understanding of nervous system function is limited by our ability to identify and manipulate neuronal subtypes within intact circuits. We show that the Gbx2CreERT2-IRES-EGFP mouse line labels two amacrine cell (AC) subtypes in the mouse retina that have distinct morphological, physiological, and molecular properties. Using a combination of RNA-seq, genetic labeling, and patch clamp recordings, we show that one subtype is GABAergic that receives excitatory input from On bipolar cells. The other population is a non-GABAergic, non-glycinergic (nGnG) AC subtype that lacks the expression of standard neurotransmitter markers. Gbx2+ nGnG ACs have smaller, asymmetric dendritic arbors that receive excitatory input from both On and Off bipolar cells. Gbx2+ nGnG ACs also exhibit spatially restricted tracer coupling to bipolar cells (BCs) through gap junctions. This study identifies a genetic tool for investigating the two distinct AC subtypes, and it provides a model for studying synaptic communication and visual circuit function.
Assuntos
Células Amácrinas/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Amácrinas/fisiologia , Animais , Feminino , Junções Comunicantes/metabolismo , Glicina/metabolismo , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/fisiologia , Neurotransmissores/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
It is necessary to develop an understanding of the specific mechanisms involved in alpha-synuclein aggregation and propagation to develop disease modifying therapies for age-related synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. To adequately address this question, we developed a new transgenic mouse model of synucleinopathy that expresses human A53T SynGFP under control of the mouse prion protein promoter. Our characterization of this mouse line demonstrates that it exhibits several distinct advantages over other, currently available, mouse models. This new model allows rigorous study of the initial location of Lewy pathology formation and propagation in the living brain, and strongly suggests that aggregation begins in axonal structures with retrograde propagation to the cell body. This model also shows expeditious development of alpha-synuclein pathology following induction with small, in vitro-generated alpha-synuclein pre-formed fibrils (PFFs), as well as accelerated cell death of inclusion-bearing cells. Using this model, we found that aggregated alpha-synuclein somatic inclusions developed first in neurons, but later showed a second wave of inclusion formation in astrocytes. Interestingly, astrocytes appear to survive much longer after inclusion formation than their neuronal counterparts. This model also allowed careful study of peripheral-to-central spread of Lewy pathology after PFF injection into the hind limb musculature. Our results clearly show evidence of progressive, retrograde trans-synaptic spread of Lewy pathology through known neuroanatomically connected pathways in the motor system. As such, we have developed a promising tool to understand the biology of neurodegeneration associated with alpha-synuclein aggregation and to discover new treatments capable of altering the neurodegenerative disease course of synucleinopathies.
Assuntos
Encéfalo/patologia , Transporte Proteico/fisiologia , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , Animais , Astrócitos/patologia , Axônios/patologia , Modelos Animais de Doenças , Feminino , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/patologiaRESUMO
Immature astrocytes and blood vessels enter the developing mammalian retina at the optic nerve head and migrate peripherally to colonize the entire retinal nerve fiber layer (RNFL). Retinal vascularization is arrested in retinopathy of prematurity (ROP), a major cause of bilateral blindness in children. Despite their importance in normal development and ROP, the factors that control vascularization of the retina remain poorly understood. Because astrocytes form a reticular network that appears to provide a substrate for migrating endothelial cells, they have long been proposed to guide angiogenesis. However, whether astrocytes do in fact impose a spatial pattern on developing vessels remains unclear, and how astrocytes themselves are guided is unknown. Here we explore the cellular mechanisms that ensure complete retinal coverage by astrocytes and blood vessels in mouse. We find that migrating astrocytes associate closely with the axons of retinal ganglion cells (RGCs), their neighbors in the RNFL. Analysis of Robo1; Robo2 mutants, in which RGC axon guidance is disrupted, and Math5 (Atoh7) mutants, which lack RGCs, reveals that RGCs provide directional information to migrating astrocytes that sets them on a centrifugal trajectory. Without this guidance, astrocytes exhibit polarization defects, fail to colonize the peripheral retina, and display abnormal fine-scale spatial patterning. Furthermore, using cell type-specific chemical-genetic tools to selectively ablate astrocytes, we show that the astrocyte template is required for angiogenesis and vessel patterning. Our results are consistent with a model whereby RGC axons guide formation of an astrocytic network that subsequently directs vessel development.
Assuntos
Astrócitos/fisiologia , Axônios/fisiologia , Neovascularização Fisiológica/fisiologia , Retina/citologia , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Toxina Diftérica/farmacologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Células Ganglionares da Retina/citologia , Proteína Homeobox SIX3RESUMO
Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca2+) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca2+ arise from the precise localization of Ca2+ signals into microdomains containing specific Ca2+ effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca2+ signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in Xenopus laevis spinal cord neurons both in vivo and in vitro Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca2+ within filopodia. In addition, blocking calpain cleavage of talin and FAK in vivo promotes Rohon-Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca2+ signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK.SIGNIFICANCE STATEMENT The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may identify areas of therapeutic intervention in disease or injury. One important signal that controls growth cones is that of local Ca2+ transients, which control the rate and direction of axon outgrowth. We demonstrate here that Ca2+-dependent inhibition axon outgrowth and guidance is mediated by calpain proteolysis of the adhesion proteins talin and focal adhesion kinase. Our findings provide mechanistic insight into Ca2+/calpain regulation of growth cone motility and axon guidance during neuronal development.
Assuntos
Orientação de Axônios/fisiologia , Calpaína/fisiologia , Adesão Celular/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteólise , Talina/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Cones de Crescimento/metabolismo , Humanos , Medula Espinal/embriologia , Medula Espinal/metabolismo , Xenopus laevisRESUMO
Neuronal growth cones are exquisite sensory-motor machines capable of transducing features contacted in their local extracellular environment into guided process extension during development. Extensive research has shown that chemical ligands activate cell surface receptors on growth cones leading to intracellular signals that direct cytoskeletal changes. However, the environment also provides mechanical support for growth cone adhesion and traction forces that stabilize leading edge protrusions. Interestingly, recent work suggests that both the mechanical properties of the environment and mechanical forces generated within growth cones influence axon guidance. In this review we discuss novel molecular mechanisms involved in growth cone force production and detection, and speculate how these processes may be necessary for the development of proper neuronal morphogenesis.
RESUMO
TRPM8 has been implicated in pain and migraine based on dorsal root- and trigeminal ganglion-enriched expression, upregulation in preclinical models of pain, knockout mouse studies, and human genetics. Here, we evaluated the therapeutic potential in pain of AMG2850 ((R)-8-(4-(trifluoromethyl)phenyl)-N-((S)-1,1,1-trifluoropropan-2-yl)-5,6-dihydro-1,7-naphthyridine-7(8H)-carboxamide), a small molecule antagonist of TRPM8 by in vitro and in vivo characterization. AMG2850 is potent in vitro at rat TRPM8 (IC90 against icilin activation of 204 ± 28 nM), highly selective (>100-fold IC90 over TRPV1 and TRPA1 channels), and orally bioavailable (F po > 40 %). When tested in a skin-nerve preparation, AMG2850 blocked menthol-induced action potentials but not mechanical activation in C fibers. AMG2850 exhibited significant target coverage in vivo in a TRPM8-mediated icilin-induced wet-dog shake (WDS) model in rats (at 10 mg/kg p.o.). However, AMG2850 did not produce a significant therapeutic effect in rat models of inflammatory mechanical hypersensitivity or neuropathic tactile allodynia at doses up to 100 mg/kg. The lack of efficacy suggests that either TRPM8 does not play a role in mediating pain in these models or that a higher level of target coverage is required. The potential of TRPM8 antagonists as migraine therapeutics is yet to be determined.
Assuntos
Hiperalgesia/tratamento farmacológico , Naftiridinas/farmacologia , Naftiridinas/uso terapêutico , Canais de Cátion TRPM/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Cálcio/metabolismo , Temperatura Baixa , Cricetinae , Cricetulus , Adjuvante de Freund , Humanos , Masculino , Mentol/farmacologia , Camundongos Endogâmicos C57BL , Dor/tratamento farmacológico , Pirimidinonas , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesõesRESUMO
Intracellular Ca(2+) signals control the development and regeneration of spinal axons downstream of chemical guidance cues, but little is known about the roles of mechanical cues in axon guidance. Here we show that transient receptor potential canonical 1 (TRPC1) subunits assemble mechanosensitive (MS) channels on Xenopus neuronal growth cones that regulate the extension and direction of axon outgrowth on rigid, but not compliant, substrata. Reducing expression of TRPC1 by antisense morpholinos inhibits the effects of MS channel blockers on axon outgrowth and local Ca(2+) transients. Ca(2+) influx through MS TRPC1 activates the protease calpain, which cleaves the integrin adaptor protein talin to reduce Src-dependent axon outgrowth, likely through altered adhesion turnover. We found that talin accumulates at the tips of dynamic filopodia, which is lost upon cleavage of talin by active calpain. This pathway may also be important in axon guidance decisions since asymmetric inhibition of MS TRPC1 is sufficient to induce growth cone turning. Together our results suggest that Ca(2+) influx through MS TRPC1 on filopodia activates calpain to control growth cone turning during development.
Assuntos
Axônios/metabolismo , Calpaína/metabolismo , Cones de Crescimento/metabolismo , Canais de Cátion TRPC/metabolismo , Talina/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Feminino , Masculino , Neurônios/citologia , Neurônios/metabolismo , Proteólise , Pseudópodes/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Canais de Cátion TRPC/genética , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Pain is the leading cause of emergency department visits, hospitalizations, and daily suffering in individuals with sickle cell disease (SCD). The pathologic mechanisms leading to the perception of pain during acute RBC sickling episodes and development of chronic pain remain poorly understood and ineffectively treated. We provide the first study that explores nociceptor sensitization mechanisms that contribute to pain behavior in mice with severe SCD. Sickle mice exhibit robust behavioral hypersensitivity to mechanical, cold, and heat stimuli. Mechanical hypersensitivity is further exacerbated when hypoxia is used to induce acute sickling. Behavioral mechanical hypersensitivity is mediated in part by enhanced excitability to mechanical stimuli at both primary afferent peripheral terminal and sensory membrane levels. In the present study, inhibition of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) with the selective antagonist A-425619 reversed the mechanical sensitization at both primary afferent terminals and isolated somata, and markedly attenuated mechanical behavioral hypersensitivity. In contrast, inhibition of TRPA1 with HC-030031 had no effect on mechanical sensitivity. These results suggest that the TRPV1 receptor contributes to primary afferent mechanical sensitization and a substantial portion of behavioral mechanical hypersensitivity in SCD mice. Therefore, TRPV1-targeted compounds that lack thermoregulatory side effects may provide relief from pain in patients with SCD.
Assuntos
Anemia Falciforme/metabolismo , Hiperalgesia/metabolismo , Isoquinolinas/farmacologia , Nociceptores/metabolismo , Dor/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Potenciais de Ação , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/patologia , Animais , Capsaicina/efeitos adversos , Capsaicina/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Hipóxia , Masculino , Camundongos , Camundongos Endogâmicos , Microeletrodos , Nociceptores/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/patologia , Medição da Dor/métodos , Técnicas de Patch-Clamp , Canais de Cátion TRPV/metabolismo , Ureia/farmacologiaRESUMO
BACKGROUND: TRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal. RESULTS: Acute application of HC-030031, a selective TRPA1 antagonist, inhibited all formalin responses in rat C fibers but had no effect on TRPV1 function, assessed by capsaicin responsiveness. Genetic ablation experiments corroborated the pharmacological findings as C fibers from wild type mice responded to both formalin and capsaicin, but fibers from their TRPA1-deficient littermates responded only to capsaicin. HC-030031 markedly reduced the mechanically-evoked action potential firing in rat and wild type mouse C fibers, particularly at high-intensity forces, but had no effect on the mechanical responsiveness of Adelta fiber nociceptors. Furthermore, HC-030031 had no effect on mechanically-evoked firing in C fibers from TRPA1-deficient mice, indicating that HC-030031 inhibits mechanically-evoked firing via a TRPA1-dependent mechanism. CONCLUSION: Our data show that acute pharmacological blockade of TRPA1 at the cutaneous receptive field inhibits formalin-evoked activation and markedly reduces mechanically-evoked action potential firing in C fibers. Thus, functional TRPA1 at sensory afferent terminals in skin is required for their responsiveness to both noxious chemical and mechanical stimuli.