RESUMO
Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuits in vivo by simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia cells (MGs). We conducted in vivo time-lapse multiphoton imaging of the rapidly developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MGs are absent. We found that removal of MGs had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.
Assuntos
Neuroglia/fisiologia , Neurônios/fisiologia , Retina/citologia , Sinapses/fisiologia , Aminoácidos , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Imageamento Tridimensional/métodos , Proteínas Luminescentes/genética , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Neuroglia/ultraestrutura , Neurônios/classificação , Neurônios/ultraestrutura , Fotodegradação , Receptores de Glutamato/metabolismo , Sinapses/ultraestrutura , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Age-related hearing loss (ARHL), also known as presbycusis, is a widespread and debilitating condition impacting many older adults. Conventionally, researchers utilize mammalian model systems or human cadaveric tissue to study ARHL pathology. Recently, the zebrafish has become an effective and tractable model system for a wide variety of genetic and environmental auditory insults, but little is known about the incidence or extent of ARHL in zebrafish and other non-mammalian models. Here, we evaluated whether zebrafish exhibit age-related loss in auditory sensitivity. The auditory sensitivity of adult wild-type zebrafish (AB/WIK strain) from three adult age subgroups (13-month, 20-month, and 37-month) was characterized using the auditory evoked potential (AEP) recording technique. AEPs were elicited using pure tone stimuli (115-4500 Hz) presented via an underwater loudspeaker and recorded using shielded subdermal metal electrodes. Based on measures of sound pressure and particle acceleration, the mean AEP thresholds of 37-month-old fish [mean sound pressure level (SPL) = 122.2 dB ± 2.2 dB SE re: 1 µPa; mean particle acceleration level (PAL) = -27.5 ± 2.3 dB SE re: 1 ms-2] were approximately 9 dB higher than that of 20-month-old fish [(mean SPL = 113.1 ± 2.7 dB SE re: 1 µPa; mean PAL = -37.2 ± 2.8 dB re: 1 ms-2; p = 0.007)] and 6 dB higher than that of 13-month-old fish [(mean SPL = 116.3 ± 2.5 dB SE re: 1 µPa; mean PAL = -34.1 ± 2.6 dB SE re: 1 ms-2; p = 0.052)]. Lowest AEP thresholds for all three age groups were generally between 800 Hz and 1850 Hz, with no evidence for frequency-specific age-related loss. Our results suggest that zebrafish undergo age-related loss in auditory sensitivity, but the form and magnitude of loss is markedly different than in mammals, including humans. Future work is needed to further describe the incidence and extent of ARHL across vertebrate groups and to determine which, if any, ARHL mechanisms may be conserved across vertebrates to support meaningful comparative/translational studies.
Assuntos
Presbiacusia , Peixe-Zebra , Estimulação Acústica , Animais , Limiar Auditivo , Potenciais Evocados Auditivos , SomRESUMO
The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein-protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.
Assuntos
Proteínas 14-3-3/metabolismo , Anexina A2/metabolismo , Biomarcadores Tumorais/metabolismo , Epiderme/metabolismo , Exorribonucleases/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Células Cultivadas , Cristalização , Células Epidérmicas , Exorribonucleases/genética , Proteínas Filagrinas , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Ligação Proteica , Transporte Proteico/fisiologia , Proteínas S100/metabolismo , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.
Assuntos
Epiderme/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células Epidérmicas , Proteínas Filagrinas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Filamentos Intermediários/genética , Queratina-10/metabolismo , Queratinócitos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Domínios e Motivos de Interação entre Proteínas/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Caspase-14 is a cysteine endoproteinase that is expressed in the epidermis and a limited number of other tissues. It is activated during keratinocyte differentiation by zymogen processing, but its precise function is unknown. To obtain caspase-14 for functional studies, we engineered and expressed a constitutively active form of human caspase-14 (Rev-hC14) in Escherichia coli and cultured mammalian cells. Rev-hC14 required no proteolytic processing for activity, showed strong activity against the caspase substrate WEHD, and was inhibited by the pan-caspase inhibitor zVAD-fmk. Mammalian cells that expressed active caspase-14 showed normal cell adherence and morphology. Using positional scanning of synthetic tetrapeptide libraries, we determined the substrate preference of human caspase-14 to be W (or Y)-X-X-D. These studies affirm that caspase-14 has a substrate specificity similar to the group I caspases, and demonstrate that it functions in a distinct manner from executioner caspases to carry out specific proteolytic events during keratinocyte differentiation.