Monica L. Acosta

Alzheimer's disease in the human eye. Clinical tests that identify ocular and visual information processing deficit as biomarkers

Alzheimers disease (AD) is the most common form of dementia with progressive deterioration of memory and cognition. Complaints related to vision are common among AD patients. Several changes in the retina, lens, and in the vasculature have been noted in the AD eye that may be the cause of visual symptoms experienced by the AD patient. Anatomical changes have been detected within the eye before signs of cognitive impairment and memory loss are apparent. Unlike the brain, the eye is a unique organ that can be visualized noninvasively at the cellular level because of its transparent nature, which allows for inexpensive testing of biomarkers in a clinical setting. In this review, we have searched for candidate biomarkers that could enable diagnosis of AD, covering ocular neurodegeneration associated with functional tests. We explore the evidence that suggests that inexpensive, noninvasive clinical tests could be used to detect AD ocular biomarkers.

Connexin43 in retinal injury and disease.

Gap junctions are specialized cell-to-cell contacts that allow the direct transfer of small molecules between cells. A single gap junction channel consists of two hemichannels, or connexons, each of which is composed of six connexin protein subunits. Connexin43 is the most ubiquitously expressed isoform of the connexin family and in the retina it is prevalent in astrocytes, Müller cells, microglia, retinal pigment epithelium and endothelial cells. Prior to docking with a neighboring cell, Connexin43 hemichannels have a low open probability as open channels constitute a large, relatively non-specific membrane pore. However, with injury and disease Connexin43 upregulation and hemichannel opening has been implicated in all aspects of secondary damage, especially glial cell activation, edema and loss of vascular integrity, leading to neuronal death. We here review gap junctions and their roles in the retina, and then focus in on Connexin43 gap junction channels in injury and disease. In particular, the effect of pathological opening of gap junction hemichannels is described, and hemichannel mediated loss of vascular integrity explained. This latter phenomenon underlies retinal pigment epithelium loss and is a common feature in several retinal diseases. Finally, Connexin43 channel roles in a number of retinal diseases including macular degeneration, glaucoma and diabetic retinopathy are considered, along with results from related animal models. A final section describes gap junction channel modulation and the ocular delivery of potential therapeutic molecules.

Functional activation of glutamate ionotropic receptors in the developing mouse retina

Ionotropic glutamate receptors have been associated with early development of the visual process by regulating cell differentiation, cell motility, and synaptic contacts. We determined the expression of functional ionotropic glutamate receptors during development of the mouse retina by assessing 1‐amino‐4‐guanidobutane (agmatine; AGB) immunolabelling after application of a range of glutamate analogs. Colocalization of AGB with calretinin and islet‐1 allowed the identification of functional receptors in neurochemically defined neurons. Activation with kainate (KA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), and N‐methyl‐D‐aspartate (NMDA) resulted in AGB entry into neurons consistent with that found previous receptor subunit localization studies in the developing retina. Temporal analysis revealed that application of 50 μM KA activated receptors as early as embryonic day 18 in the ventricular zone and in the ganglion cell layer, whereas 30 μM AMPA activated cells predominantly in the ganglion cell layer. Cholinergic amacrine cells showed functional KA and AMPA receptors upon their insertion into the conventional amacrine cell layer from postnatal day 1 (P1). OFF cone bipolar cells showed functional KA receptors from P6, at a developmental age when they are known to make contact with ganglion cells. NMDA activation led to diffuse AGB labeling at birth among cells in the ganglion cell layer, whereas, at P1, regularly spaced cholinergic amacrine cells in the conventional amacrine cell layer started to be responsive to NMDA. Non‐NMDA receptors were first to show functional activation in the developing retina, and cholinergic amacrine cells displayed functional ionotropic glutamate receptors after reaching their final destination. J. Comp. Neurol. 500:923–941, 2007.

Glutamate metabolic pathways and retinal function.

Glutamate is a major neurotransmitter in the CNS but is also a key metabolite intimately coupled to amino acid production/degradation. We consider the effect of inhibition of two key glutamate metabolic enzymes: glutamine synthetase (GS) and aspartate aminotransferase on retinal function assessed using the electroretinogram to consider photoreceptoral (a‐wave) and post‐receptoral (b‐wave) amplitudes. Quantitative immunocytochemistry was used to assess amino acid levels within photoreceptors, ganglion and Müller cells secondary to GS inhibition. Intravitreal injections of methionine sulfoximine reduced GS immunoreactivity in the rat retina. Additionally, glutamate and its precursor aspartate was reduced in photoreceptors and ganglion cells, but elevated in Müller cells. This reduction in neuronal glutamate was consistent with a deficit in neurotransmission (−75% b‐wave reduction). Exogenous glutamine supply completely restored the b‐wave, whereas other amino acid substrates (lactate, pyruvate, α‐ketoglutarate, and succinate) only partially restored the b‐wave (16–20%). Inhibition of the aminotranferases using aminooxyacetic acid had no effect on retinal function. However, aminooxyacetic acid application after methionine sulfoximine further reduced the b‐wave (from −75% to −92%). The above data suggest that de novo glutamate synthesis involving aspartate aminotransferase can partially sustain neurotransmission when glutamate recycling is impaired. We also show that altered glutamate homeostasis results in a greater change in amino acid distribution in ganglion cells compared with photoreceptors.

Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina

Tangential cell dispersion in the retina is a spacing mechanism that establishes a regular mosaic organization among cell types and contributes to their final positioning. The present study has used the X‐inactivation transgenic mouse expressing the lacZ reporter gene on one X chromosome. Due to X chromosome inactivation, 50% of early progenitor cells express β‐galactosidase (β‐Gal); therefore, all cells derived from a particular β‐Gal‐expressing progenitor cell can be identified in labeled columns. The radial segregation of clonally related β‐Gal‐positive and β‐Gal‐negative cells can be used to determine whether single cells transgress a clonal boundary in the retina. We investigated the extent to which particular cell classes tangentially disperse by analyzing the placement of labeled cells expressing particular markers at several ages and quantifying their tangential displacement. Retinal neurons expressing cell markers at postnatal day (P) 1 have a greater degree of tangential dispersion compared with amacrine and bipolar cells at P5–6. We also studied whether there is a functional correlation with these dispersion patterns by investigating the emergence of functional ionotropic glutamate receptors. To determine the degree of functional glutamate receptor activation, agmatine (AGB) was used in combination with cell‐specific labeling. AGB permeates functional glutamate receptor channels following activation with α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), kainate or N‐methyl‐D‐aspartate (NMDA). Within these receptor groups, high concentrations of AMPA, kainate, and NMDA are associated with a high degree of tangential dispersion in the adult. Developmentally, functional kainate and AMPA receptors were detected by P1 and were associated with tangentially dispersed cells. Functional NMDA receptors were not detected as early as kainate and AMPA receptors. These results indicate that cells generated early during development are more likely to disperse tangentially compared with those generated later in development. Therefore, functional AMPA and kainate receptors may play a critical role in tangentially displacing cell types. J. Comp. Neurol. 506:506–523, 2008.

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function.

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick‐end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague–Dawley (SD) rats and a rat model of retinitis pigmentosa [proline‐23‐histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24‐h light exposure period. Short‐term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light‐damaged retinas in both SD and the P23H rat retina.

Retinal amino acid neurochemistry in health and disease.

Advances in basic retinal anatomy, genetics, biochemical pathways and neurochemistry have not only provided a better understanding of retinal function but have also allowed us to link basic science to retinal disease. The link with disease allowed measures to be developed that now provide an opportunity to intervene and slow down or even restore sight in previously ‘untreatable’ retinal diseases. One of the critical advances has been the understanding of the retinal amino acid neurotransmitters, related amino acids, their metabolites and functional receptors. This review provides an overview of amino acid localisation in the retina and examples of how retinal anatomy and amino acid neurochemistry directly links to understanding retinal disease. Also, the implications of retinal remodelling involving amino acid (glutamate) receptors are outlined in this review and insights are presented on how understanding of detrimental and beneficial retinal remodelling will provide better outcomes for patients using strategies for the preservation or restoration of vision. An internet‐based database of retinal images of amino acid labelling patterns and other amino acid‐related images in health and disease is located at http://www.aminoacidimmunoreactivity.com.

Functional and anatomical remodeling in human retinal detachment.

Rhegmatogenous retinal detachment is by far the most common indication for retinal surgery and a major cause of severe vision loss. Increased levels of glutamate found in the vitreous of human patients and persistent remodeling, even after reattachment, suggest substantial neurochemical, functional and anatomical changes have occurred in the detached retina. Therefore, this study was designed to characterize the morphological changes and glutamate receptor functionality in human rhegmatogenous retinal detachment. A cation channel permeating probe, agmatine (1-amino-4-guanidobutane; AGB), was employed to track endogenous and kainate (KA) driven channel functionality combined with immunocytochemical characterization of cellular remodeling. In the detached retina increased AGB permeability was identified in the outer retina while there was a decrease in the inner retina in basal conditions. KA receptors exhibited increased AGB permeability in ON bipolar cells and decreased permeability in calbindin labeled inner retinal cells. All retinal detachment samples demonstrated ectopic synaptic protein expression, photoreceptor processes extending toward the inner retina, and other remodeling features of retinal degeneration. These anatomical changes have been demonstrated in animal studies and are novel features unreported in primary cases of human retinal detachment. We conclude that deafferentation in retinal detachment leads to alteration of the glutamatergic pathway.

Retinal metabolic state of the proline-23-histidine rat model of retinitis pigmentosa

We determined the metabolic changes that precede cell death in the dystrophic proline-23-histidine (P23H) line 3 (P23H-3) rat retina compared with the normal Sprague-Dawley (SD) rat retina. Metabolite levels and metabolic enzymes were analyzed early in development and during the early stages of degeneration in the P23H-3 retina. Control and degenerating retinas showed an age-dependent change in metabolite levels and enzymatic activity, particularly around the time when phototransduction was activated. However, lactate dehydrogenase (LDH) activity was significantly higher in P23H-3 than SD retina before the onset of photoreceptor death. The creatine/phosphocreatine system did not contribute to the increase in ATP, because phosphocreatine levels, creatine kinase, and expression of the creatine transporter remained constant. However, Na(+)-K(+)-ATPase and Mg(2+)-Ca(2+)-ATPase activities were increased in the developing P23H-3 retina. Therefore, photoreceptor apoptosis in the P23H-3 retina occurs in an environment of increased LDH, ATPase activity, and higher-than-normal ATP levels. We tested the effect of metabolic challenge to the retina by inhibiting monocarboxylate transport with alpha-cyano-4-hydroxycinnamic acid or systemically administering the phosphodiesterase inhibitor sildenafil. Secondary to monocarboxylate transport inhibition, the P23H-3 retina did not demonstrate alterations in metabolic activity. However, administration of sildenafil significantly reduced LDH activity in the P23H-3 retina and increased the number of terminal deoxynucleotidyl transferase biotin-dUPT nick end-labeled photoreceptor cells. Photoreceptor cells with a rhodopsin mutation display an increase in apoptotic markers secondary to inhibition of a phototransduction enzyme (phosphodiesterase), suggesting increased susceptibility to altered cation entry.

Octodon degus (Molina 1782): A Model in Comparative Biology and Biomedicine

One major goal of integrative and comparative biology is to understand and explain the interaction between the performance and behavior of animals in their natural environment. The Caviomorph, Octodon degu, is a native rodent species from Chile, and represents a unique model to study physiological and behavioral traits, including cognitive and sensory abilities. Degus live in colonies and have a well-structured social organization, with a mostly diurnal-crepuscular circadian activity pattern. More notable is the fact that in captivity, they reproduce and live between 5 and 7 yr and show hallmarks of neurodegenerative diseases (including Alzheimers disease), diabetes, and cancer.