measurement-energy-metabolism-explanted-retinal-tissue-using

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Washington University School of Medicine

SYNE1 (synaptic nuclear envelope 1) encodes multiple isoforms of Nesprin1 (nuclear envelope spectrin 1) that associate with the nuclear envelope (NE) through a C-terminal KASH (Klarsicht/Anc1/Syne homology) domain (Figure 1A) [1-4]. This domain interacts directly with the SUN (Sad1/Unc84) domain of Sun proteins [5-7], a family of transmembrane proteins of the inner nuclear membrane (INM) [8, 9], to form the so-called LINC complexes (linkers of the nucleoskeleton and cytoskeleton) that span the entire NE and mediate nuclear positioning [10-12]. In a stark departure from this classical depiction of Nesprin1 in the context of the NE, we report here that rootletin recruits Nesprin1α at the ciliary rootlets of photoreceptors and identify asymmetric NE aggregates of Nesprin1α and Sun2 that dock filaments of rootletin at the nuclear surface. In NIH 3T3 cells, we show that recombinant rootletin filaments also dock to the NE through the specific recruitment of an ∼600-kDa endogenous isoform of Nesprin1 (Nes1) and of Sun2. In agreement with the association of Nesprin1α with photoreceptor ciliary rootlets and the functional interaction between rootletin and Nesprin1 in fibroblasts, we demonstrate that multiple isoforms of Nesprin1 are integral components of ciliary rootlets of multiciliated ependymal and tracheal cells. Together, these data provide a novel functional paradigm for Nesprin1 at ciliary rootlets and suggest that the wide spectrum of human pathologies linked to truncating mutations of SYNE1 [13-15] may originate in part from ciliary defects.

Multiple Isoforms of Nesprin1 Are Integral Components of Ciliary Rootlets.

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye.

Membrane lipid composition is central to the highly specialized functions of neurological tissues. In the retina, abnormal lipid metabolism causes severe forms of blindness, often through poorly understood neuronal cell death. Here, we demonstrate that deleting the de novo lipogenic enzyme fatty acid synthase (FAS) from the neural retina, but not the vascular retina, results in progressive neurodegeneration and blindness with a temporal pattern resembling rodent models of retinitis pigmentosa. Blindness was not rescued by protection from light-evoked activity; by eating a diet enriched in palmitate, the product of the FAS reaction; or by treatment with the PPARα agonist fenofibrate. Vision loss was due to aberrant synaptic structure, blunted responsiveness to glial-derived neurotrophic factor and ciliary neurotrophic factor, and eventual apoptotic cell loss. This progressive neurodegeneration was associated with decreased membrane cholesterol content, as well as loss of discrete n-3 polyunsaturated fatty acid- and saturated fatty acid-containing phospholipid species within specialized membrane microdomains. Neurotrophic signaling was restored by exogenous cholesterol delivery. These findings implicate de novo lipogenesis in neurotrophin-dependent cell survival by maintaining retinal membrane configuration and lipid composition, and they suggest that ongoing lipogenesis may be required to prevent cell death in many forms of retinopathy.

Teresa Doggett

Department of Ophthalmology and Visual Science

Washington University School of Medicine

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Teresa Doggett

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology and Visual Science

Washington University School of Medicine

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Department of Ophthalmology and Visual Science