Browsing by Author "Arshavsky, Vadim"

The vertebrate visual system involves a series of complex steps which cover a number ofdiverse anatomical structures. Light first enters the eye and is projected onto the retina, a thin layer of tissue that lines the posterior of the eye and is comprised of millions of specialized neurons. Rods and cones initiate the process light signaling upon absorption of photons, with stimulation of retinal ganglion cells (RGCs) representing the final step of intraretinal signaling. RGC somas localize to the innermost layers of the retina, while their axons converge at the optic nerve head and bundle together to form the optic nerve carrying visual information to the brain. The RGC structure is unique in that the soma and dendrites are compartmentalized inside of the eye whereas the RGC axons exist primarily outside of the eye as they project via the optic nerve to synaptic targets in the CNS. In the proximal region of the optic nerve is a short, unmyelinated region of axons running between an astrocytic meshwork termed the glial lamina (GL). Seminal studies have shown that mitochondria are particularly abundant in the GL compared to other compartments of RGCs, potentially suggesting a particularly high demand for ATP production in this region. Of relevance, the GL is the first region of axonal degeneration in glaucoma, suggesting an inherent susceptibility of this axonal compartment to cellular stress. The focus of this dissertation centers on elucidating the local landscape and regulation of the GL in healthy mice and disease models. Our earliest work is a comprehensive analysis of the enrichment of mitochondria in the GL in wild type mice, improving upon the low-resolution histological studies on which this concept was based. Using complementary immunofluorescence and electron microscopy techniques, we confirm that mitochondria are more abundant in the GL compared to the retrolaminar (RL) optic nerve, but show that the overall mitochondrial accumulation arises only because of differential mitochondrial abundance in the largest diameter RGC axons. We also show that the mitochondrial accumulation is established by postnatal day 6- v 9, preceding the onset of axonal myelination. Therefore, the enrichment of mitochondria in the GL is not a direct consequence of the unique absence of myelin in this region. The remainder of our work is an exploration of differences between the GL and RL in mitochondrial and axonal morphology as well as in proteomic signatures. In preliminary studies, we have observed distinct mitochondrial morphologies between the two compartments, with small differences in mitochondrial length and cristae structure. We also have found preliminary evidence of unprecedented inter-axonal fusion events between RGC axons of the GL, a phenomenon we term short axonal merging sites (SAMS). SAMS are characterized as two individual axons that run parallel and exhibit focal breakdown of their plasma membranes with apparent fusion between the two. While additional experiments are required to eliminate the possibility that SAMS are non-physiological artifacts of tissue fixation, should we confirm their presence it would open a new direction exploring the functional significance of the fusion events in light signaling to the central nervous system and in the propagation of pathology in optic nerve disorders. Finally, we describe our early efforts proving the feasibility of characterizing the compartment-specific proteomes of optic nerve tissue and of RGC mitochondria specifically. We also describe preliminary studies designed to compare the GL proteomes of DBA/2J mice with early glaucoma to control non-glaucomatous littermates. Completion of this analysis may identify key biological differences between the GL and RL and highlight cellular processes that may subject the GL to early axonal degeneration in optic neuropathies like glaucoma. It is our hope that this work will help to identify pathways that may be targeted pharmacologically to combat RGC neurodegeneration in glaucoma and other blinding diseases.

Rod photoreceptors are specialized neurons responsible for capturing photons and translating visual information into electrical signals. Visual signal transduction in rods is confined to the unique outer segment organelle, a modified primary cilium consisting of a stack of hundreds of flattened disc membranes enveloped by a single plasma membrane. By concentrating important signaling molecules on disc membranes, the outer segment provides an ideal biochemical environment for the production of vision with high sensitivity and temporal resolution.

This dissertation focuses primarily on a molecular dissection of two multifunctional outer segment proteins, R9AP and rhodopsin, and also reassesses the localization of Glut1, a third protein formerly believed to reside in the outer segment. All three experimental lines relied on in vivo expression of novel protein constructs in vertebrate rods using several gene delivery strategies: conventional transgenics, retinal electroporation, and retinal infection with recombinant adeno-associated virus.

The tail-anchored protein R9AP, in conjunction with RGS9-1 and G-beta5, comprises the transducin GTPase activating complex, which catalyzes the rate-limiting step in rod photoresponse recovery. In addition to maximizing the enzymatic activity of the complex, R9AP is responsible for both the post-translational stability and outer segment targeting of RGS9-1-G-beta5. We investigated the mechanism behind R9AP's poorly understood function in protecting RGS9-1-G-beta5 from proteolysis and found that it is performed simply by recruiting the complex to cellular membranes and can be entirely dissociated from R9AP's outer segment targeting function. Furthermore, we demonstrated that replacement of R9AP's transmembrane domain with a lipid anchor preserves the ability of the GTPase activating complex to function in outer segments.

Rhodopsin, the visual pigment of rods, has a second important, yet poorly defined, function as a rod outer segment building block: outer segments disc membranes fail to form in the absence of rhodopsin. Our goal was to identify the molecular features of rhodopsin mechanistically involved in outer segment morphogenesis by designing artificial membrane proteins that could fully substitute for rhodopsin in performing this function. We observed that rhodopsin's C-terminal VXPX outer segment targeting motif is unnecessary for outer segment disc formation since it could be replaced with a targeting motif from an unrelated protein, peripherin. Furthermore, we obtained surprising evidence that rhodopsin's role in this process is limited to providing an abundance of transmembrane protein material to disc membranes.

Finally, while attempting to find a targeting motif to substitute for the VXPX motif of rhodopsin, we made an unexpected observation that the facilitative glucose transporter Glut1, long thought to reside in the outer segment, is actively excluded from this organelle. This revises our understanding of the energy flow in rods by showing that the outer segment is entirely dependent on the inner segment for its energy supply.

The initial steps of vertebrate vision take place in the retina, where light-sensitive rod and cone photoreceptor cells translate light into an electrical signal through a biochemical process called phototransduction. Transducin, a heterotrimeric G protein, is central to this process in rod photoreceptors. In rods, phototransduction begins when transducin is activated by the light-stimulated G protein-coupled receptor rhodopsin, setting off a cascade of cellular events that ultimately generates the visual signal, or photoresponse. Many aspects of transducin’s function were uncovered through studies of knockout mice lacking its individual subunits. Of particular interest is the knockout mouse lacking the transducin γ-subunit, Gγ1 (the Gγ1-/- mouse), which exhibits unique characteristics that have thus far remained incompletely understood. First, Gγ1-/- rods retain the ability to detect light, despite lacking the canonical transducin Gβ1γ1 complex. Second, these cells experience chronic proteostatic stress, consisting of an insufficient capacity for protein degradation by the ubiquitin-proteasome system (UPS), which leads to the progressive dysfunction and eventual death of Gγ1-/- rods.

This dissertation focuses on uncovering the molecular mechanisms underlying the unique functional and pathophysiological consequences of Gγ1 knockout in rod photoreceptors. In Chapter 3, we investigate the mechanism driving light-signaling in Gγ1-/- rods. In Chapter 4, we evaluate whether the chronic proteostatic stress observed in degenerating rods could result from insufficient activity of a specific component of the UPS: the substrate-processing complex formed by the AAA+ ATPase P97 (aka VCP) and associated cofactors.

We determined that the level of photoresponse sensitivity of Gγ1-/- rods was comparable to the expression levels of Gαt and Gβ1, the remaining components of the transducin heterotrimer, in the outer segments of these cells. We found that two additional G protein γ-subunits (Gγ2 and Gγ3) are present in the outer segments of both WT and Gγ1-/- rods. Finally, we demonstrated that Gβ1, which normally forms an inseparable heterodimer with Gγ1, also forms complexes with Gγ2 and Gγ3 in both WT and Gγ1-/- rods. Thus, we conclude that the canonical transducin Gβ1γ1 complex is not the sole Gβγ complex able to facilitate phototransduction and that transducin complexes utilizing alternative γ-subunits support transducin activation in Gγ1-/- rods.

Our examination of proteostatic stress in degenerating rods focused on two mouse models of retinal degeneration: the Gγ1-/- mouse and the knockin mouse expressing a single copy of the rhodopsin P23H mutation (the P23H mouse). Rods of both strains exhibit proteostatic stress, consisting of an insufficient capacity for protein degradation by the UPS, linked to the requirement to degrade misfolded photoreceptor proteins. We investigated whether insufficient UPS function in these cells results from an insufficient cellular capacity for substrate processing by P97 complexes, a critical step in the proteasomal degradation of a large subset of UPS targets. Gγ1-/- and P23H retinas displayed strikingly different patterns of accumulation of two complementary in vivo proteasomal activity reporters whose degradation is either P97-dependent or P97-independent. Based on these patterns, we conclude that the proteostatic stress observed in Gγ1-/- and P23H rods likely originates from distinct pathophysiological mechanisms in which protein degradation by the UPS may or may not be limited by the cellular capacity for substrate processing by P97 complexes. We show that UPS function in Gγ1-/- rods is likely limited by insufficient P97-dependent substrate processing, whereas proteasomal degradation itself limits UPS function in P23H rods. Finally, we found that, despite being aphenotypic in several other tissues, P97 overexpression is toxic to rod photoreceptors and increases proteostatic stress in Gγ1-/- rods.

Together, these studies broaden our understanding of photoreceptor cell biology. In addition to illuminating the mechanisms underlying light-signaling in rods, the work described in this dissertation highlights phototransduction in Gγ1-/- rods as a compelling example of the functional interchangeability of G protein γ-subunits. To our knowledge, this represents the first direct demonstration of multiple Gβγ complexes performing the same function in a living animal. Further, this work highlights the complexity of pathophysiological mechanisms related to degrading misfolded proteins in mutant photoreceptors, which must be accounted for in the development of effective strategies to ameliorate these blinding conditions.

The complex process of visually interpreting our environment begins with the task of detecting the light that enters our eyes. This task is performed by the rod and cone photoreceptors, which both contain a highly evolved sensory cilium called the outer segment. The outer segment is a specialized cellular compartment that contains all of the protein machinery involved in converting the initial light signal into an electrical signal that can be ultimately transmitted to the brain. Outer segments are cylindrical structures that envelop an array of individual, densely packed membrane discs. Discs are renewed throughout the lifetime of a photoreceptor, with older material being shed at the tip and new material added at the base of the outer segment. Many studies conducted over the past 35 years conclude that disc formation starts with evagination of the plasma membrane at the outer segment base, followed by membrane expansion and, in the case of rods, subsequent disc enclosure. Despite the intense interest in the topic, the molecular mechanisms governing how outer segment discs are formed and renewed are not well understood.

The focus of this dissertation centers on elucidating the molecular role of peripherin/retinal degeneration slow (rds) in outer segment disc morphogenesis, including study of peripherin/rds trafficking from its site of synthesis in the endoplasmic reticulum to its site of function in the outer segment. Peripherin/rds is expressed specifically in photoreceptor outer segments, where it fulfills a critical role in assembling and/or maintaining the structure of this organelle. Mutation or loss of peripherin/rds in humans is often associated with visual impairments, and its knockout in mice results in rudimentary ciliary stumps completely lacking disc structures.

We found that early outer segment morphogenesis steps in mice lacking peripherin/RDS proceed normally for the first week. However, in the second week of postnatal development at the onset of disc formation, mice lacking peripherin/RDS produce extracellular vesicles next to their connecting cilia rather than discs. We characterized these vesicles and determined that they are enriched in outer segment proteins, are ~230 nm in size, and are formed as outward buds of the plasma membrane. These characteristics allowed us to classify these extracellular vesicles as ciliary ectosomes. Furthermore, we determined that ectosome shedding is arrested upon expression of the peripherin/rds C-terminal cytoplasmic sequence, which allows for the accumulation of excessive membranous material. Thus, we conclude that peripherin/rds transforms the functional dynamics of photoreceptor primary cilium from shedding massive amounts of ectosomes to retaining these membranes in the outer segment to eventually become photoreceptor discs. This novel function of peripherin is performed by its C-terminal cytoplasmic sequence and represents the first step in disc morphogenesis.

Finally, the morphogenesis study of peripherin/rds is complemented by a study of its trafficking. Understanding how peripherin is delivered from the site of its synthesis is critical for its function at the outer segment. We show that the peripherin/rds targeting sequence is confined within ten amino acid residues, which do not overlap with the putative fusogenic domain, and that only a single amino acid within this region is irreplaceable, a highly conserved valine at position 332.

Collectively, these studies shed considerable light on the molecular role played by peripherin. Peripherin is a photoreceptor specific protein that transforms the primary sensory cilium into a specialized sensory cilium capable of building the discs required for efficient photon capture. While work in this direction provides a significant advance in our understanding of peripherin’s role in disc morphogenesis, questions such as whether peripherin participates in disc enclosure, remain to be solved.

Human vision begins in the retina, where ~100 million photoreceptor neurons absorb light and respond to it, transferring the information to the brain where ultimately an image is created. Just like a camera’s sensor, an essential quality of photoreceptors for functional vision is their incredible sensitivity—our rod photoreceptors can detect the smallest unit of light possible, a single photon. To achieve this level of sensitivity, the photoreceptor evolved a primary cilium-derived light sensor organelle called the outer segment, which is a massive 30µm-long cylinder filled with a stack of ~1000 perfectly flattened disc membranes. The disc membrane houses the protein machinery necessary for generating light responses, including rhodopsin, the transmembrane photopigment protein responsible for absorbing light. By stacking 1000 discs, each with two rhodopsin containing membrane bilayers, the light absorbing membrane surface area of the retina is increased ~2000 fold, enabling the incredible sensitivity of the rod photoreceptor.

We completed a mass spectrometry project identifying the proteins which specifically reside in the photoreceptor disc. Except for one 6 kDa protein called PRCD, the proteins identified were previously known to reside in photoreceptor discs and their functions were well studied. PRCD is a recently discovered protein whose mutations are linked to retinal degeneration in canine and human patients, and had previously unknown localization in any cell type. Virtually nothing was known about this protein, so this dissertation sought to biochemically characterize the protein, understand how its mutation leads to blindness in dogs and humans, and elucidate its function in photoreceptor discs.

To biochemically characterize the protein, we generated an antibody to its C terminus, and used it to confirm its localization specific to discs by immunohistochemistry. By analyzing the multiple bands PRCD protein produces on Western blot, we discovered that the protein is post translationally modified by lipid acylation and phosphorylation. Furthermore, mutagenesis experiments determined that the lipid is attached to the single cysteine residue of PRCD, which is mutated in blind canine and human patients. This disease-causing mutation results in complete mislocalization of PRCD from the outer segment, and its degradation—effectively resulting in a null PRCD mutant allele.

Pull down experiments revealed PRCD specifically binding to rhodopsin, which was confirmed by reciprocal immunoprecipitation and co-chromatography experiments. Bolstering this result, we found that PRCD was nearly absent from rhodopsin knockout mouse retinas and without outer segment localization. This result contrasted a large cohort of other outer segment proteins; all of them except guanylate cyclase 1 were trafficked to the outer segment and expressed in relative abundance. Through reciprocal co-immunoprecipitations, we discovered that guanylate cyclase 1 is also a rhodopsin binding protein, and that this interaction is dependent on gentle detergent conditions, likely hindering its identification in the past. These results reveal that the bulk of disc specific proteins have their own, uncharacterized trafficking pathway(s), independent of rhodopsin.

To elucidate the function of PRCD in photoreceptor discs, we generated and characterized a PRCD knockout mouse, which develops a normally layered retina. The abundance and localization of disc proteins is normal in young animals, and so are their rod photoresponses. PRCD knockout mouse photoreceptors degenerate slowly, and by electron microscopy, outer segments from PRCD knockout mice are disorganized and display a phenotype similar to dogs containing C2Y mutation in PRCD. The studies presented in this dissertation are the first to lay the biochemical ground work for characterizing PRCD, and elucidate its function in photoreceptor disc membranes.