Eye

The structure and transparency of the eye lens are vital for focusing light onto the retina for vision. Lens fiber cells undergo a cellular remodeling program that removes mitochondria (MT), endoplasmic reticulum (ER), and Golgi apparatus (GA) to form mature transparent lens fiber cells. Previous studies established a requirement for the mitochondrial outer membrane protein BNIP3L for the elimination of these non-nuclear organelles in the lens; however, the precise molecular pathways for BNIP3L function remain to be elucidated. BNIP3L contains multiple functional domains whose analysis may illuminate its lens mechanisms including the LIR, BH3, and TM domains. These domains each play an important role in regulation of autophagosome formation and initiation of autophagy. To test each domain’s functionality for BNIP3L-dependent organelle elimination, we designed site-directed mutagenesis studies to delete each domain and test the resulting mutants in initiating the degradation of organelles in ex vivo cultured embryonic chick lenses.

The ocular lens is comprised of an epithelial cell population that undergoes a continuous process of cellular remodeling and differentiation to form elongated transparent fiber cells. This lens differentiation process is hallmarked by the complete elimination of organelles at the center of the lens, elongation of lens fiber cells, and production of lens fiber-cell specific crystallin proteins to form the mature functional structure of the transparent ocular lens. To date, our understanding of the mechanisms that drive the lens differentiation process is incomplete. This dissertation sought to elucidate the potential roles of both hypoxia and epigenetic chromatin remodeling processes as novel regulators of lens differentiation.
The lens lacks a direct blood supply and thus resides in a hypoxic microenvironment. Previous studies revealed the presence of a decreasing oxygen gradient in the region of the lens where cellular remodeling and organelle elimination occur to form mature transparent lens fiber cells. Thus we hypothesized that the hypoxic environment of the lens itself, was required to induce gene expression changes to drive the lens differentiation process. We utilized a multimoics analysis combining CUT&RUN and RNAseq high-throughput sequencing technologies to identify a role for the hypoxia-inducible transcription factor HIF1a as a novel regulator of lens gene expression during lens differentiation.

This study examines gaze patterns of monolinguals and bilinguals encoding speech in familiar and unfamiliar languages. In condition 1 English monolinguals viewed videos in familiar and unfamiliar languages (English and Spanish or Icelandic). They performed a task to ensure encoding: on each trial, two videos of short sentences were presented, followed by an audio-only recording of one of those sentences. Participants choose whether the audio-clip matched the first or second video. Participants gazed significantly longer at speaker's mouths when viewing unfamiliar languages. In condition 2 Spanish-English bilingual's viewed English and Spanish, no difference was found between the languages. In condition 3 the task was removed, English monolinguals viewed 20 English and 20 Icelandic videos, no difference in the gaze patterns was found, suggesting this phenomenon relies on encoding. Results indicate people encoding unfamiliar speech attend to the mouth presumably to extract more accurate audiovisually invariant and highly salient speech information.

The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world for people over 60 years of age. The most severe pathological event of AMD is choroidal neovascularization (CNV), the process of new vessel formation emerging from the choroid. The new vessels extend into the normally avascular photoreceptor cell layer, where they leak fluid and cause photoreceptor cell death. CNV is thought to be initiated by hypoxia and chronic inflammation, which occur due to abnormal, age-related changes within the retinal pigmented epithelium (RPE). These events cause increased expression of the angiogenic protein vascular endothelial growth factor (VEGF) via hypoxiainducible factor-1 (HIF-1), a transcription factor that is vital in regulation of cellular responses to hypoxic and inflammatory conditions. Increased VEGF signaling stimulates proliferation and migration of vascular endothelial cells and facilitates the neovascular process. To target the early pathological events that lead to CNV, we have engineered a novel gene therapy vector that uses HIF-1 regulation to stimulate production of an angiostatic protein, endostatin from the RPE. The purpose of this study was to characterize the activity of our hypoxiaregulated, RPE-specific promoter in vitro, and investigate the effects of regulated endostatin expression, driven by our regulated promoter, on CNV in a mousemodel. We found the regulated promoter construct has robust activity in vitro only in RPE cells, and is conditionally responsive in hypoxic conditions.

Taurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study the neuromodulatory role of taurine in the CNS and to gain insights into its potential sites of action. A combination of techniques was used, including whole-cell patch clamp recording to study taurine's regulation of voltage-gated potassium (K+) and Ca2+ channels and Fluo-4AM Ca2+-imaging to study taurine's regulation of glutamate-induced [Ca2+] I,. Taurine was shown to suppress of glutamate-induced [Ca2+] l, in a dose dependent manner. This suppression was mostly sensitive to the glycine rece ptor antagonist Strychnine but insensitive to any GABA receptor antagonist. The remaining strychnine-insensitive effect was inhibited with the protein kinase A (PKA) inhibitor, PKI, suggesting that there was an additional metabotropic pathway. Moreover, using the protein kinase C (PKC) inhibitor, GF109203X, there was an enhancement in strychnine-insensitive taurine's regulation. Taurine inhibits voltage-gated Ca2+ channels in the retinal neurons and has a dual effect on voltage-gated K+ channels. Taurine causes an increase in K+ current amplitude which is further enhanced with PKI and blocked with GF109203X, suggesting that it is through a PKC-dependent pathway negatively controlled by PKA-dependent pathway.

Although it is well known that the pupil responds dynamically to changes in ambient light levels, the results from this dissertation show for the first time that the pupil also responds dynamically to changes in spatially distributed attention. Using a variety of orientating tasks, subjects alternated between focusing attention on a central stimulus and spreading attention over a larger area. Fourier analysis of the fluctuating pupil diameter indicated that: 1) pupil diameter changed at the rate of attention variation, dilating with broadly spread attention and contracting with narrowly focused attention, and 2) pupillary differences required changes in attentional spread; there were no differences in pupil diameter between sustained broad and sustained spread attention. Given that broadly spread attention increases the relative activation of large receptive fields and narrowly focused attention increases the relative activation of small receptive fields (Balz & Hock, 1997), the results of this study indicate that these attentional effects on receptive field activation can be mediated by changes in pupil diameter. That is, under broad attention, the corresponding pupillary dilation observed would increase spherical aberration, blurring the image thereby reducing high spatial frequency information and decreasing the activation of relatively small cortical receptive fields compared to relatively large receptive fields. This increased perception of low spatial frequencies would be beneficial in cases where attention is spread over a large area. Alternatively, under narrow attention the resulting pupillary constriction reduces spherical aberration sharpening the image and preserving high spatial frequency information resulting in a relatively increased response of small receptive fields.