Faculty Bibliography

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Ų) compared to that of TLR9-CpG ODN_2395 (2094 Ų), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

Glaucoma is an age-related neurodegenerative disease of the visual system, affecting both the eye and the brain. Yet its underlying metabolic mechanisms and neurobehavioral relevance remain largely unclear. Here, using proton magnetic resonance spectroscopy and functional magnetic resonance imaging, we investigated the GABAergic and glutamatergic systems in the visual cortex of glaucoma patients, as well as neural specificity, which is shaped by GABA and glutamate signals and underlies efficient sensory and cognitive functions. Our study shows that among the older adults, both GABA and glutamate levels decrease with increasing glaucoma severity regardless of age. Further, our study shows that the reduction of GABA but not glutamate predicts the neural specificity. This association is independent of the impairments on the retina structure, age, and the gray matter volume of the visual cortex. Our results suggest that glaucoma-specific decline of GABA undermines neural specificity in the visual cortex and that targeting GABA could improve the neural specificity in glaucoma.

Central insulin resistance, the diminished cellular sensitivity to insulin in the brain, has been implicated in diabetes mellitus, Alzheimer's disease and other neurological disorders. However, whether and how central insulin resistance plays a role in the eye remains unclear. Here, we performed intracerebroventricular injection of S961, a potent and specific blocker of insulin receptor in adult Wistar rats to test if central insulin resistance leads to pathological changes in ocular structures. 80 mg of S961 was stereotaxically injected into the lateral ventricle of the experimental group twice at 7 days apart, whereas buffer solution was injected to the sham control group. Blood samples, intraocular pressure, trabecular meshwork morphology, ciliary body markers, retinal and optic nerve integrity, and whole genome expression patterns were then evaluated. While neither blood glucose nor serum insulin level was significantly altered in the experimental or control group, we found that injection of S961 but not buffer solution significantly increased intraocular pressure at 14 and 24 days after first injection, along with reduced porosity and aquaporin 4 expression in the trabecular meshwork, and increased tumor necrosis factor α and aquaporin 4 expression in the ciliary body. In the retina, cell density and insulin receptor expression decreased in the retinal ganglion cell layer upon S961 injection. Fundus photography revealed peripapillary atrophy with vascular dysregulation in the experimental group. These retinal changes were accompanied by upregulation of pro-inflammatory and pro-apoptotic genes, downregulation of anti-inflammatory, anti-apoptotic, and neurotrophic genes, as well as dysregulation of genes involved in insulin signaling. Optic nerve histology indicated microglial activation and changes in the expression of glial fibrillary acidic protein, tumor necrosis factor α, and aquaporin 4. Molecular pathway architecture of the retina revealed the three most significant pathways involved being inflammation/cell stress, insulin signaling, and extracellular matrix regulation relevant to neurodegeneration. There was also a multimodal crosstalk between insulin signaling derangement and inflammation-related genes. Taken together, our results indicate that blocking insulin receptor signaling in the central nervous system can lead to trabecular meshwork and ciliary body dysfunction, intraocular pressure elevation, as well as inflammation, glial activation, and apoptosis in the retina and optic nerve. Given that central insulin resistance may lead to neurodegenerative phenotype in the visual system, targeting insulin signaling may hold promise for vision disorders involving the retina and optic nerve.

Plasticity in the brain is impacted by an individual's age at the onset of the blindness. However, what drives the varying degrees of plasticity remains largely unclear. One possible explanation attributes the mechanisms for the differing levels of plasticity to the cholinergic signals originating in the nucleus basalis of Meynert. This explanation is based on the fact that the nucleus basalis of Meynert can modulate cortical processes such as plasticity and sensory encoding through its widespread cholinergic projections. Nevertheless, there is no direct evidence indicating that the nucleus basalis of Meynert undergoes plastic changes following blindness. Therefore, using multiparametric magnetic resonance imaging, we examined if the structural and functional properties of the nucleus basalis of Meynert differ between early blind, late blind and sighted individuals. We observed that early and late blind individuals had a preserved volumetric size and cerebrovascular reactivity in the nucleus basalis of Meynert. However, we observed a reduction in the directionality of water diffusion in both early and late blind individuals compared to sighted individuals. Notably, the nucleus basalis of Meynert presented diverging patterns of functional connectivity between early and late blind individuals. This functional connectivity was enhanced at both global and local (visual, language and default-mode networks) levels in the early blind individuals, but there were little-to-no changes in the late blind individuals when compared to sighted controls. Furthermore, the age at onset of blindness predicted both global and local functional connectivity. These results suggest that upon reduced directionality of water diffusion in the nucleus basalis of Meynert, cholinergic influence may be stronger for the early blind compared to the late blind individuals. Our findings are important to unravelling why early blind individuals present stronger and more widespread cross-modal plasticity compared to late blind individuals.

Connecting neuronal activity to perception requires tools that can probe neural codes at cellular and circuit levels, paired with sensitive behavioral measures. In this chapter, we present an overview of current methods for connecting neural codes to perception using precision optogenetics and psychophysical measurements of synthetically induced percepts. We also highlight new methodologies for validating precise control of optical and behavioral manipulations. Finally, we provide a perspective on upcoming developments that are poised to advance the field.
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The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease, and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here, we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma, and endothelium, with subpopulations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a 3D model to study corneal diseases and integrated responses of different cell types.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examine genetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 23 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Modern optical neuroimaging approaches are expanding the ability to elucidate complex brain function. Diverse imaging contrasts enable direct observation of neural activity with functional sensors along with the induced hemodynamic responses. To date, decoupling the complex interplay of neurovascular coupling and dynamical physiological states has remained challenging when employing single-modality functional neuroimaging readings. A hybrid fluorescence optoacoustic tomography platform combined with a custom data processing pipeline based on statistical parametric mapping is devised, attaining the first noninvasive observation of simultaneous calcium and hemodynamic activation patterns using optical contrasts. Correlated changes in the oxy- and deoxygenated hemoglobin, total hemoglobin, oxygen saturation, and rapid GCaMP6f fluorescence signals are observed in response to peripheral sensory stimulation. While the concurrent epifluorescence serves to corroborate and complement the functional optoacoustic observations, the latter further aids in decoupling the rapid calcium responses from the slowly varying background in the fluorescence recordings mediated by hemodynamic changes. The hybrid imaging platform expands the capabilities of conventional neuroimaging methods to provide more comprehensive functional readings for studying neurovascular and neurometabolic coupling mechanisms and related diseases.