RESUMO
Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1ß levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis.
Assuntos
Eritropoetina , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritropoetina/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , AVC Isquêmico/tratamento farmacológico , MAP Quinase Quinase Quinases/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
OBJECTIVE: Parkinson's disease (PD) manifests in motor dysfunction, non-motor symptoms, and eventual dementia (PDD). Neuropathological hallmarks include nigrostriatal neurodegeneration, Lewy body (LB) pathology, and neuroinflammation. Alpha-synuclein (α-syn), a primary component of LBs, is implicated in PD pathogenesis, accumulating, and aggregating in both familial and sporadic PD. However, as α-syn pathology is often comorbid with amyloid-beta (Aß) plaques and phosphorylated tau (pTau) tangles in PDD, it is still unclear whether α-syn is the primary cause of neurodegeneration in sporadic PDD. We aimed to determine how the absence of α-syn would affect PDD manifestation. METHODS: IFN-ß knockout (Ifnb-/- ) mice spontaneously develop progressive behavior abnormalities and neuropathology resembling PDD, notably with α-syn+ LBs. We generated Ifnb/Snca double knockout (DKO) mice and evaluated their behavior and neuropathology compared with wild-type (Wt), Ifnb-/- , and Snca-/- mice using immunohistochemistry, electron microscopy, immunoblots, qPCR, and modification of neuronal signaling. RESULTS: Ifnb/Snca DKO mice developed all clinical PDD-like behavioral manifestations induced by IFN-ß loss. Independently of α-syn expression, lack of IFN-ß alone induced Aß plaques, pTau tangles, and LB-like Aß+ /pTau+ inclusion bodies and neuroinflammation. IFN-ß loss caused significant elevated glial and neuronal TNF-α and neuronal TNFR1, associated with neurodegeneration. Restoring neuronal IFN-ß signaling or blocking TNFR1 rescued caspase 3/t-BID-mediated neuronal-death through upregulation of c-FLIPS and lowered intraneuronal Aß and pTau accumulation. INTERPRETATION: These findings increase our understanding of PD pathology and suggest that targeting α-syn alone is not sufficient to mitigate disease. Targeting specific aspects of neuroinflammation, such as aberrant neuronal TNF-α/TNFR1 or IFN-ß/IFNAR signaling, may attenuate disease. ANN NEUROL 2021;90:789-807.
Assuntos
Doenças Neuroinflamatórias/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Progressão da Doença , Corpos de Lewy/patologia , Doença por Corpos de Lewy/metabolismo , Camundongos Knockout , Neuroglia/patologia , Doença de Parkinson/genética , Placa Amiloide/metabolismo , Fator de Necrose Tumoral alfa/deficiênciaRESUMO
Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.
Assuntos
Corioide/irrigação sanguínea , Olho/imunologia , Macrófagos/imunologia , Células Mieloides/imunologia , Neovascularização Fisiológica/fisiologia , Animais , Corioide/embriologia , Biologia Computacional , Simulação por Computador , Olho/citologia , Olho/metabolismo , Feminino , Homeostase/imunologia , Humanos , Imunidade Inata/imunologia , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/fisiologia , Células Mieloides/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Gênica/genéticaRESUMO
Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/- mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-ß-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.
Assuntos
Doenças do Sistema Nervoso Central/imunologia , Doença Enxerto-Hospedeiro/imunologia , Microglia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/imunologia , Doença Aguda , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microglia/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Fator de Necrose Tumoral alfa/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.
Assuntos
Inflamação/imunologia , Monócitos/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Sobrevivência Celular , Encefalomielite Autoimune Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologiaRESUMO
Pseudo-TORCH syndrome (PTS) is characterized by microcephaly, enlarged ventricles, cerebral calcification, and, occasionally, by systemic features at birth resembling the sequelae of congenital infection but in the absence of an infectious agent. Genetic defects resulting in activation of type 1 interferon (IFN) responses have been documented to cause Aicardi-Goutières syndrome, which is a cause of PTS. Ubiquitin-specific peptidase 18 (USP18) is a key negative regulator of type I IFN signaling. In this study, we identified loss-of-function recessive mutations of USP18 in five PTS patients from two unrelated families. Ex vivo brain autopsy material demonstrated innate immune inflammation with calcification and polymicrogyria. In vitro, patient fibroblasts displayed severely enhanced IFN-induced inflammation, which was completely rescued by lentiviral transduction of USP18. These findings add USP18 deficiency to the list of genetic disorders collectively termed type I interferonopathies. Moreover, USP18 deficiency represents the first genetic disorder of PTS caused by dysregulation of the response to type I IFNs. Therapeutically, this places USP18 as a promising target not only for genetic but also acquired IFN-mediated CNS disorders.
Assuntos
Doenças Autoimunes do Sistema Nervoso , Encéfalo/imunologia , Calcinose , Endopeptidases/deficiência , Imunidade Inata , Interferon Tipo I/imunologia , Microglia/imunologia , Malformações do Sistema Nervoso , Transdução de Sinais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/patologia , Encéfalo/patologia , Calcinose/genética , Calcinose/imunologia , Calcinose/patologia , Endopeptidases/imunologia , Feminino , Humanos , Interferon Tipo I/genética , Masculino , Microglia/patologia , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitina TiolesteraseRESUMO
Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.
Assuntos
Sistema Nervoso Central/imunologia , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Microglia/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Monócitos/imunologia , Parabiose , Proteínas Proto-Oncogênicas/genética , Transativadores/genéticaRESUMO
Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to ß-hemolytic streptococci.
Assuntos
Macrófagos/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Receptores Toll-Like/metabolismo , Animais , Colo/patologia , Citocinas/metabolismo , Hemólise , Interações Hospedeiro-Patógeno , Imunidade nas Mucosas/genética , Imunidade nas Mucosas/imunologia , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/patologia , Especificidade de Órgãos , Pele/patologia , Receptores Toll-Like/genéticaRESUMO
Microglia are brain macrophages and, as such, key immune-competent cells that can respond to environmental changes. Understanding the mechanisms of microglia-specific responses during pathologies is hence vital for reducing disease burden. The definition of microglial functions has so far been hampered by the lack of genetic in vivo approaches that allow discrimination of microglia from closely related peripheral macrophage populations in the body. Here we introduce a mouse experimental system that specifically targets microglia to examine the role of a mitogen-associated protein kinase kinase kinase (MAP3K), transforming growth factor (TGF)-ß-activated kinase 1 (TAK1), during autoimmune inflammation. Conditional depletion of TAK1 in microglia only, not in neuroectodermal cells, suppressed disease, significantly reduced CNS inflammation and diminished axonal and myelin damage by cell-autonomous inhibition of the NF-κB, JNK and ERK1/2 pathways. Thus, we found TAK1 to be pivotal in CNS autoimmunity, and we present a tool for future investigations of microglial function in the CNS.
Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/imunologia , Marcação de Genes , MAP Quinase Quinase Quinases/metabolismo , Microglia/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Encéfalo/metabolismo , Antígeno CD11c/genética , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/induzido quimicamente , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/genética , Linfonodos/patologia , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , RNA não Traduzido/genética , Receptores de Quimiocinas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: To monitor viability of implanted genetically engineered and microencapsulated human stem cells (MicroBeads) in the mouse eye, and to study the impact of the beads and/or xenogenic cells on retinal integrity. METHODOLOGY/PRINCIPAL FINDINGS: MicroBeads were implanted into the subretinal space of SV126 wild type mice using an ab externo approach. Viability of microencapsulated cells was monitored by noninvasive retinal imaging (Spectralis™ HRA+OCT). Retinal integrity was also assessed with retinal imaging and upon the end of the study by light and electron microscopy. The implanted GFP-marked cells encapsulated in subretinal MicroBeads remained viable over a period of up to 4 months. Retinal integrity and viability appeared unaltered apart from the focal damage due to the surgical implantation, GFAP upregulation, and opsin mistargeting in the immediate surrounding tissue. CONCLUSIONS/SIGNIFICANCE: The accessibility for routine surgery and its immune privileged state make the eye an ideal target for release system implants for therapeutic substances, including neurotrophic and anti-angiogenic compounds or protein based biosimilars. Microencapsulated human stem cells (MicroBeads) promise to overcome limitations inherent with single factor release systems, as they are able to produce physiologic combinations of bioactive compounds.
Assuntos
Olho/citologia , Microesferas , Degeneração Retiniana/terapia , Transplante de Células-Tronco/métodos , Animais , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Oftalmoscopia/métodos , Retina/ultraestrutura , Tomografia de Coerência ÓpticaRESUMO
Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit(+) erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45(+) c-kit(lo) CX(3)CR1(-) immature (A1) cells and matured into CD45(+) c-kit(-) CX(3)CR1(+) (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Fatores Reguladores de Interferon/metabolismo , Microglia/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Fator 4 Semelhante a Kruppel , Camundongos , Microglia/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3-4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.
Assuntos
Terapia Genética/métodos , Retinose Pigmentar/terapia , Animais , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da PolimeraseRESUMO
The rhodopsin gene (RHO) encodes a highly expressed G protein-coupled receptor that is central to visual transduction in rod photoreceptors. A suite of recombinant 2/5 adeno-associated viral (AAV) RHO replacement vectors has been generated in an attempt to recapitulate endogenous rhodopsin levels from exogenously delivered AAV vectors in the retina of mice with a targeted disruption in the rhodopsin gene (Rho(-/-) mice). Approximately 40% of wild-type mouse rhodopsin mRNA levels (RNA taken from whole retinas) was achieved in vivo in AAV-RHO-injected eyes, representing approximately 50-fold increases in expression compared with the initial vector. The main focus of this study was to test whether expression of AAV-RHO replacement in Rho(-/-) mice provided therapeutic benefit, which to date had not been achieved. Rho(-/-) mice neither elaborate rod outer segments nor have rod-derived electroretinograms (ERGs). Our results indicate for the first time in this model that subretinal AAV-RHO delivery leads not only to RHO immunolabeling but the generation of rod outer segments as evaluated by light and transmission electron microscopy. Improved histology was accompanied by rod photoreceptor activity as assessed by ERG for at least 12 weeks postinjection. The most efficient AAV-RHO constructs presented in this study provide sufficient levels of RHO to be of therapeutic benefit in Rho(-/-) mice and therefore represent important steps toward generating potent AAV-RHO replacement genes for gene therapy in RHO-linked human retinopathies.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/fisiologia , Animais , Western Blotting , Eletrorretinografia , Técnicas de Transferência de Genes , Humanos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation. Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation. In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light. Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input.