Investigations of the molecular mechanisms of aging and age-related macular degeneration
Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the developed world. Current treatment options for AMD are available only to a limited number of patients with advanced “wet AMD”. However, these treatments are initiated after vision loss has started and are designed to treat the lesion, i.e., sealing off leaking blood vessels, but do not address disease pathogenesis. Ideally, the most efficacious therapy for AMD should be started prior to vision loss and target the fundamental molecular defect, which requires defining disease mechanism.
Help define the molecular defects in retinal cells that are responsible for the pathology associated with AMD.
Perform biochemical analyses of human donor retinas that have been evaluated for their stage of AMD using the Minnesota Grading System (Olsen and Feng, 2004). Our collaborators on this project include Dr. Timothy Olsen and the Minnesota Lions Eye Bank.
Converging evidence from several laboratories, including our own, have implicated mitochondrial damage in the AMD disease process. In our first proteomic analysis of the retinal pigment epithelium (RPE), we identified altered mitochondrial proteins with AMD progression (Nordgaard et al., 2006), thus prompting an in-depth investigation of the mt proteome. The second proteomic analysis suggested potential damage to mtDNA with AMD (Nordgaard et al., 2008). To test if mtDNA damage was associated with AMD progression, we employed an analysis of mtDNA lesions in the macula of human donor RPE using a polymerase chain reaction strategy. Since aging is a strong risk factor for AMD, we evaluated the extent of mtDNA damage in human RPE with both aging and AMD progression to distinguish damage associated with normal from pathologic aging (Karunadharma et al., 2010). Our data revealed low mtDNA damage with normal aging compared to AMD and elevated damage preceding significant macular degeneration and vision loss in AMD donors. Collectively, these results support a role for mtDNA damage in AMD pathology.
Our studies are consistent with the idea that increased mtDNA damage could be one factor leading to RPE dysfunction (see Figure 1). Reactive oxygen species generated under normal conditions, such as light and phagocytosis, place the RPE under considerable oxidative stress. With normal aging, mtDNA damage is maintained at low levels with an increase only in the “common deletion”. This common deletion is associated with the repair process, and thus the accumulation of this defect reflects ongoing repair over the individual’s lifetime. With AMD, we see mitochondrial genome-wide damage that could potentially exceed a critical threshold that reduces proper energy generation. Impairment of the RPE can lead to an imbalance in signaling factors (such as VEGF) and apoptosis, resulting in inappropriate blood vessel growth and RPE atrophy that are manifested as “wet” and “dry” AMD, respectively.
Investigations of Retinal Proteasome
The proteasome proteolytic complex plays a fundamental role in processes essential for cell viability, such as cell cycle regulation, control of signal transduction and gene expression, and the degradation of oxidized and misfolded proteins. The 20S catalytic core of the proteasome is a barrel-shaped structure, consisting of four heptameric rings. The two outer rings contain the constitutively expressed α subunits that interact with a number of regulatory complexes, i.e., PA28, PA700. The two inner rings contain the β subunits. Three of the β subunits ( b1, b 2, b5) contain the catalytic sites that perform distinct proteolytic activities referred to as caspase-like (b1), trypsin-like (b2) and chymotrypsin-like (b5). These catalytic subunits form the core of the standard proteasome. In nascent proteasomes, the standard subunits can be replaced by LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i) to form the core of the immunoproteasome. A third type of catalytic core, referred to as the intermediate-type proteasome, contains a mixture of both standard and immunoproteasome catalytic subunits.
While the standard proteasome is the predominant core particle in most tissues, the immunoproteasome is the major proteasome species found in tissues and cells of the immune system. However, immunoproteasome is also found in limited abundance in cells outside the immune system, including neurons (photoreceptors and Purkinje cells) and glia (Mueller cells and astrocytes) of the retina and brain (Ferrington et al., 2008). A recent focus of our lab has been to define conditions that provoke the upregulation of immunoproteasome in the central nervous system. Data derived from this experimental approach provide some indication that immunoproteasome’s function goes beyond its well-defined role in immune surveillance. For example, immunoproteasome is upregulated in the central nervous system in response to acute injury (Ferrington et al., 2008), disease (Ethen et al., 2006), and age (Hussong et al., 2010), suggesting a role in responding to stress and injury. Additionally, immunoproteasome expression in the non-injured retina and brain, and its recent localization to synapse in the brain and outer plexiform layer in the retina implies a role in normal, neuronal function.
Immunoproteasome possesses activities that are essential for maintaining retinal integrity and for coping with stress.
Compare WT with KO mice lacking one (lmp2-/- and lmp7-/-) or two catalytic subunits of the immunoproteasome (lmp2-/- / lmp7-/-) to determine the in vivo consequences of inhibiting immunoproteasome expression. Comparing KO mice will help distinguish conditions when specific subunits are preferentially required. Multiple functional and biochemical analyses are underway to help define the site of action for immunoproteasome in the retina. These results will add significantly to our understanding of how the immunoproteasome regulates specific aspects of retinal function and the cellular stress response. Results from these studies will provide novel information of fundamental significance to retina cell biology.