Week in Review: Number 46

Immune Cell Presence on the Crystalline Lens in Uveitis
Despite being susceptible to a range of potential threats—whether they come from pathogens, chemicals, radiation, or general oxidative damage—it is surprising that ocular tissues such as the cornea, crystalline lens, and retina are thought to be immune privileged, that is, lacking immune cells. So how do these critical tissues protect themselves? Closer investigation by researchers in previous work revealed, for example, that mice engineered to lack a key protein for crystalline lens formation showed immune cells trying to fix the resulting malformed lens. Other researchers observed immune cells in the cornea after damage to the lens, and vice versa immune cells arriving at the lens surface, acting like sentinels to protect this tissue after damage to the cornea (the external ocular surface). In a new study, researchers at Thomas Jefferson University show that immune cells respond to the lens not only after acute eye injury but also in chronic inflammation. Using high-resolution microscopy and a mouse model of uveitis, or inflammation to the middle layer of the eye, the researchers studied the role of immune cells in relation to cataracts associated with uveitis. 

Unlike the sentinels seen after ocular surface injury, the researchers report, "In this case, it was like a battering ram. There were dozens of immune cells, and different types of them, including T-cells and macrophages. It’s clearly a robust immune response and could reflect in part that inflammation in uveitis is so severe." Similarly, it was previously thought that the lens capsule surrounding the lens protects it from the increase in immune cells that populate the aqueous and vitreous chambers during active uveitis. However, high-resolution confocal z-stacks and scanning electron microscopy revealed that immune cells were actually integrated into the lens capsule, increasing not only in number but also in depth (invasion) as the uveitis progressed. Moreover, the immune cells were observed to be able to penetrate the thick lens capsule, infiltrate into and embedding themselves in the lens tissue. Although most of the cells were gone as the uveitis began to resolve, some of these immune cells remained integrated into the lens capsule and lens tissue. Underscoring the complexity of the presence of immune cells in the eye, a topic that has thus far been understudied, the senior author of the study explains, "Till now, the mechanisms for damage that happen in this region of the eye after uveitis have been poorly understood. For the first time, we’ve been able to provide evidence that immune cells could be driving this damage, particularly to the lens.”

SINE RNA Receptor DDX17 Mediates Lupus & AMD
Jayakrishna Ambati, MD, has been a prolific researcher, publishing three papers this year regarding his discoveries connecting atrophic macular degeneration (AMD) with cytoplasmic cDNA and its activation of the inflammasome. His fourth paper this year presents another finding linking triggers of the inflammasome with another disease: lupus. Ambati remarks, "It appears that the new inflammatory pathway we identified could be therapeutically targeted for many chronic diseases." The inflammasome, or pairs of inflammasomes, responsible in this case is the NLRC4-NLRP3 inflammasome. Each are individually large multi-protein complexes that play an important role in protecting the body from pathogens. However, in noninfectious, chronic inflammatory diseases such as lupus and macular degeneration, the researchers found that the NLRC4 inflammasome includes the NLRP3 inflammasome, and is instead independent of NLR family apoptosis inhibitory proteins (NAIPs) that are classically required for NLRC4 inflammasome assembly after bacterial infection. In the "sterile" environment of chronic diseases, that is, in the absence of pathogenic etiologies, researchers wondered what triggers the formation of inflammasomes.

The new research uncovered that the NLRC4-NLRP3 inflammasome is triggered by short interspersed nuclear element RNAs (SINE RNAs), mobile genetic elements known as retrotransposons that make up more than 10% of our genome and is transcribed in response to cellular stresses. SINE RNAs were found to be elevated in both lupus and AMD, among other diseases in previous work. Notably, using human cells transfected with SINE RNA, the research team identified a receptor called DEAD-box helicase 17 (DDX17). When DDX17 was unavailable, there was less interaction between the NLRC4 and NLRP3 proteins in SINE RNA-transfected cells, suggesting that DDX17 is an essential mediator in sterile activation of the NLRC4 inflammasome by SINE RNAs. The finding that DDX17 is colocalized with SINE RNA in the cytoplasm is confirmed in white blood cells isolated from people with active lupus. Additionally, subretinal injection of SINE RNAs in a mouse model of atrophic macular degeneration led to retinal pigmented epithelium (RPE) degeneration in wildtype mice but not in mice engineered with genetic loss of Nlrc4  or Ddx17, demonstrating that the NLRC4 inflammasome and its receptor DDX17 are both necessary to trigger the retinal disease. The finding that there are two inflammasomes involved, both of which are necessary to form an active NLRC4 inflammasome when the complex is triggered by SINE RNAs, also informs therapeutic strategies for both the NLRC4 and NLRP3 inflammasomes. Ambati further comments, “[N]ow that we know what is the sensor—at least a sensor—of SINE retrotransposons, that opens up a whole new intersection between RNA biology and immunology.”

Metformin Explored as a Treatment for L-ORD
Late-onset retinal degeneration (L-ORD) is a rare genetic disorder of autosomal dominant inheritance. Specifically, L-ORD is caused by a missense substitution in the gene that encodes the protein CTRP5, leading to choroidal neovascularization and deposits of apolipoprotein E (which is involved in lipid metabolism within the retina), and retinal pigmented epithelium (RPE) atrophy (which contain an abundance of fatty acids and lipids). Symptoms begin with nyctalopia, progressing to central vision loss by the sixth decade. Scientists at the National Eye Institute are studying the disease by developing a "disease-in-a-dish" model from induced pluripotent stem cells (iPSCs) to make RPE cells from skin fibroblasts; the fibroblast samples were collected from two siblings with L-ORD (L-ORD-iRPE) and compared with two unaffected siblings who lacked the disease-causing mutation. The L-ORD-iRPE were observed to be dysmorphic (deformed), also showing deposits of apolipoprotein E near the tissue and abnormal secretions of vascular endothelial growth factor (VEGF); these cells also had reduced secretions of both normal and mutant CTRP5 proteins.

Computer modeling of the proteins showed that mutant CTRP5 was less likely to bind with cell receptors that regulate lipid metabolism, in turn leading to chronic activation of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis and lipid metabolism, such as of apolipoprotein E. One of the researchers explains, "AMPK in the RPE itself is a key regulator of the conversion of DHA [docosahexaenoic acid] into protective mediators [against oxidative damage and mutations]." Chemically inhibited AMPK in the L-ORD-iRPE cells led to fewer apolipoprotein E deposits and less abnormal secretion of VEGF. Finally, the researchers tested two therapies on the L-ORD-iRPE cells: a gene augmentation of normal CTRP5, and modulation of AMPK with anti-diabetes drug metformin. Both strategies prevented signs of L-ORD in the RPE model. Senior author of the study states, "Importantly, we now have two potential strategies to disrupt the L-ORD disease process. While gene therapy may be years away, metformin is a drug that’s long been used to treat diabetes." Although L-ORD is a rare genetic disease, it shares some characteristics with the much more common age-related macular degeneration (AMD). The researchers hope that studying this model, and the effect of metformin, will benefit diseases caused by RPE changes.

Association Study Shows Link between AMD Genetic Risk Factors and Thinner Retinal Layers
Age-related macular degeneration (AMD) is a leading cause visual impairment and loss in Western countries, and especially among individuals 55 years of age and older. Researchers in the U.K. looked at medical records from the U.K. Biobank, which includes retinal scans and genetic data from over 30,000 patients. In particular, they compared the latest data of 34 known genetic risk factors of AMD (which together comprise 46% of the disease's genetic variance) with macular thickness, as measured with spectral-domain optical coherence tomography (SD-OCT). The authors report, "Our analysis has interestingly shown that, in the presence of high genetic risk for AMD, there is a significant decrease in the thickness of both the ISOS-RPE and the RPE-BM which may suggest that premature RPE thinning could be a major contributory factor." The inner-segment outer segment-retinal pigment epithelium (ISOS-RPE) thickness measurement, is of particular interest in that it represents the photoreceptor outer segments where light transduction takes place. The researchers conclude, "Our study highlights the premorbid influence of AMD genetic risk variants on macular thickness and may provide mechanistic insight into the pathophysiology of this debilitating disease." Because these structural changes occur prior to onset of disease symptoms or overt clinical signs, they could provide an early assessment of disease risk to guide healthy habits.

Regulators of Adult Visual Cortex Plasticity in Mice
Learning and recovery from injuries depend on the brain's plasticity. This plasticity between neuronal connections relies heavily on the network of macromolecules in between and surrounding the nerve cells, known as the extracellular matrix (ECM). As the brain becomes more mature, the stability of the ECM increases, providing a scaffold for the existing arrangements and synaptic circuits of nerve cells. New experiences require that this extracellular matrix be loosened in order for new connections to form. Similarly, when the brain experiences an injury such as a stroke, it needs to reorganize itself and form new connections. This balance between stability and plasticity is regulated by the proteolytic activity of enzymes such as matrix metalloproteinases (MMPs), which "digest" the ECM in order to "loosen" it. Researchers in Germany studying the visual cortex of mice showed that blocking the matrix metalloproteinases MMP2 and MMP9 can have opposing effects depending on whether the brain is sick, such as after stroke, or healthy. In the primary visual cortex of healthy mice, blocking MMP2 and MMP9 led to decreased ocular dominance plasticity. In mice studied immediately after a stroke, inhibition of MMP2 and MMP9 (which spike for a short time after a stroke) rescued neuronal plasticity that had been compromised by the stroke, that is, the MMPs had a therapeutic effect. The researchers point out that the intentional inhibition of the metaloproteinases immediately after inducing an experimental stroke was in order to simulate treatment. The authors argue that these findings show that levels of MMPs must be precise and optimal in the brain, as both too low or too high an amount can prevent neuronal plasticity.

In Other News
(1) Vision's effect on the development of hearing investigated
(2) Candy-like models to make STEM accessible to students with visual impairment
(3) Visual behavior and autism prediction in infants