ABSTRACT
Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.
Subject(s)
Interleukin-1 Receptor-Associated Kinases , Mice, Knockout , Oxidative Stress , Retinal Degeneration , Retinal Pigment Epithelium , Animals , Humans , Male , Mice , Cellular Senescence , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Macular Degeneration/metabolism , Macular Degeneration/pathology , Macular Degeneration/genetics , Mice, Inbred C57BL , Mitochondria/metabolism , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Degeneration/genetics , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathologyABSTRACT
Unchecked, chronic inflammation is a constitutive component of age-related diseases, including age-related macular degeneration (AMD). Here we identified interleukin-1 receptor-associated kinase (IRAK)-M as a key immunoregulator in retinal pigment epithelium (RPE) that declines with age. Rare genetic variants of IRAK-M increased the likelihood of AMD. IRAK-M expression in RPE declined with age or oxidative stress and was further reduced in AMD. IRAK-M-deficient mice exhibited increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M disrupted RPE cell homeostasis, including compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of AAV-expressing IRAK-M rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in IRAK-M-deficient mice. Our data support that replenishment of IRAK-M expression may redress dysregulated pro-inflammatory processes in AMD, thereby treating degeneration.
ABSTRACT
Age-related macular degeneration (AMD), a degenerative disease affecting the retinal pigment epithelium (RPE) and photoreceptors in the macula, is the leading cause of central blindness in the elderly. AMD progresses to advanced stages of the disease, atrophic AMD (aAMD), or in 15% of cases "wet" or neovascular AMD (nAMD), associated with substantial vision loss. Whilst there has been advancement in therapies treating nAMD, to date, there are no licenced effective treatments for the 85% affected by aAMD, with disease managed by changes to diet, vitamin supplements, and regular monitoring. AMD has a complex pathogenesis, involving highly integrated and common age-related disease pathways, including dysregulated complement/inflammation, impaired autophagy, and oxidative stress. The intricacy of AMD pathogenesis makes therapeutic development challenging and identifying a target that combats the converging disease pathways is essential to provide a globally effective treatment. Interleukin-33 is a cytokine, classically known for the proinflammatory role it plays in allergic disease. Recent evidence across degenerative and inflammatory disease conditions reveals a diverse immune-modulatory role for IL-33, with promising therapeutic potential. Here, we will review IL-33 function in disease and discuss the future potential for this homeostatic cytokine in treating AMD.
Subject(s)
Geographic Atrophy , Wet Macular Degeneration , Aged , Angiogenesis Inhibitors , Cytokines/metabolism , Geographic Atrophy/pathology , Humans , Interleukin-33/metabolism , Retinal Pigment Epithelium/pathology , Vascular Endothelial Growth Factor A/metabolism , Visual Acuity , Wet Macular Degeneration/drug therapy , Wet Macular Degeneration/metabolismABSTRACT
Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited childhood neurodegenerative disorders. In addition to the accumulation of auto-fluorescent storage material in lysosomes, NCLs are largely characterised by region-specific neuroinflammation that can predict neuron loss. These phenotypes suggest alterations in the extracellular environment-making the secretome an area of significant interest. This study investigated the secretome in the CLN6 (ceroid-lipofuscinosis neuronal protein 6) variant of NCL. To investigate the CLN6 secretome, we co-cultured neurons and glia isolated from Cln6nclf or Cln6± mice, and utilised mass spectrometry to compare protein constituents of conditioned media. The significant changes noted in cathepsin enzymes, were investigated further via western blotting and enzyme activity assays. Viral-mediated gene therapy was used to try and rescue the wild-type phenotype and restore the secretome-both in vitro in co-cultures and in vivo in mouse plasma. In Cln6nclf cells, proteomics revealed a marked increase in catabolic and cytoskeletal-associated proteins-revealing new similarities between the pathogenic signatures of NCLs with other neurodegenerative disorders. These changes were, in part, corrected by gene therapy intervention, suggesting these proteins as candidate in vitro biomarkers. Importantly, these in vitro changes show promise for in vivo translation, with Cathepsin L (CTSL) activity reduced in both co-cultures and Cln6nclf plasma samples post gene-therapy. This work suggests the secretome plays a role in CLN6 pathogenesis and highlights its potential use as an in vitro model. Proteomic changes present a list of candidate biomarkers for monitoring disease and assessing potential therapeutics in future studies.
Subject(s)
Membrane Proteins/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Animals , Biomarkers , Cathepsin L/biosynthesis , Coculture Techniques , Computational Biology , Disease Models, Animal , Female , Gene Expression Regulation , Genetic Therapy , Male , Mice , Mice, Knockout , Neuroglia/metabolism , Neuronal Ceroid-Lipofuscinoses/diagnosis , Neuronal Ceroid-Lipofuscinoses/drug therapy , Neurons/metabolism , Primary Cell Culture , ProteomicsABSTRACT
The leading cause of central vision loss, age-related macular degeneration (AMD), is a degenerative disorder characterized by atrophy of retinal pigment epithelium (RPE) and photoreceptors. For 15% of cases, neovascularization occurs, leading to acute vision loss if left untreated. For the remaining patients, there are currently no treatment options and preventing progressive RPE atrophy remains the main therapeutic goal. Previously, we have shown treatment with interleukin-33 can reduce choroidal neovascularization and attenuate tissue remodelling. Here, we investigate IL-33 delivery in aged, high-fat diet (HFD) fed mice on a wildtype and complement factor H heterozygous knockout background. We characterize the non-toxic effect following intravitreal injection of IL-33 and further demonstrate protective effects against RPE cell death with evidence of maintaining metabolic retinal homeostasis of Cfh+/-~HFD mice. Our results further support the potential utility of IL-33 to prevent AMD progression.
Subject(s)
Aging , Interleukin-33/pharmacology , Retinal Degeneration/etiology , Retinal Degeneration/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Aging/genetics , Aging/metabolism , Animals , Disease Models, Animal , Humans , Immunohistochemistry , Macular Degeneration/etiology , Macular Degeneration/metabolism , Macular Degeneration/pathology , Mice , Mice, Knockout , Retinal Degeneration/drug therapy , Retinal Degeneration/pathology , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/ultrastructure , Treatment OutcomeABSTRACT
The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2-ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2-ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2-ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2-ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2-ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain.
ABSTRACT
Forebrain embryonic zinc finger (Fezf2) encodes a transcription factor essential for the specification of layer 5 projection neurons (PNs) in the developing cerebral cortex. As with many developmental transcription factors, Fezf2 continues to be expressed into adulthood, suggesting it remains crucial to the maintenance of neuronal phenotypes. Despite the continued expression, a function has yet to be explored for Fezf2 in the PNs of the developed cortex. Here, we investigated the role of Fezf2 in mature neurons, using lentiviral-mediated delivery of a shRNA to conditionally knockdown the expression of Fezf2 in the mouse primary motor cortex (M1). RNA-sequencing analysis of Fezf2-reduced M1 revealed significant changes to the transcriptome, identifying a regulatory role for Fezf2 in the mature M1. Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analyses of Fezf2-regulated genes indicated a role in neuronal signaling and plasticity, with significant enrichment of neuroactive ligand-receptor interaction, cell adhesion molecules and calcium signaling pathways. Gene Ontology analysis supported a functional role for Fezf2-regulated genes in neuronal transmission and additionally indicated an importance in the regulation of behavior. Using the mammalian phenotype ontology database, we identified a significant overrepresentation of Fezf2-regulated genes associated with specific behavior phenotypes, including associative learning, social interaction, locomotor activation and hyperactivity. These roles were distinct from that of Fezf2-regulated genes identified in development, indicating a dynamic transition in Fezf2 function. Together our findings demonstrate a regulatory role for Fezf2 in the mature brain, with Fezf2-regulated genes having functional roles in sustaining normal neuronal and behavioral phenotypes. These results support the hypothesis that developmental transcription factors are important for maintaining neuron transcriptomes and that disruption of their expression could contribute to the progression of disease phenotypes.
ABSTRACT
The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.
Subject(s)
DNA-Binding Proteins/metabolism , Motor Cortex/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Animals , DNA-Binding Proteins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Matrix Attachment Region Binding Proteins/metabolism , Mice, Transgenic , Motor Cortex/cytology , Nerve Tissue Proteins/genetics , Neuroanatomical Tract-Tracing Techniques , Neurons/cytology , Polymerase Chain Reaction , Pyramidal Tracts/cytology , Pyramidal Tracts/metabolism , Repressor Proteins/metabolism , Tissue Culture Techniques , Transcription Factors/metabolism , Transfection , Tumor Suppressor Proteins/metabolismABSTRACT
The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.