Martin L. Katz

Fluorophores of the human retinal pigment epithelium: Separation and spectral characterization

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.

Embryonic stem cell-derived neural progenitors incorporate into degenerating retina and enhance survival of host photoreceptors

Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type–specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.

Effects of antioxidant nutrient deficiency on the retina and retinal pigment epithelium of albino rats: a light and electron microscopic study

Abstract Male Sprague-Dawley rats were maintained, from weaning, on diets deficient either in vitamin E, selenium, chromium and sulfur amino acids (−E−Se−S−Cr), or only in vitamin E and selenium (−E−Se+S+Cr). Control animals (+E+Se+S+Cr) received all four nutrients. After 24–26 weeks on their respective diets, both deficient groups showed a dramatic accumulation of an autofluorescent pigment, similar to lipofuscin, in the retinal pigment epithelium. This increased autofluorescence was correlated with a large increase in the number of electron-dense inclusion bodies observed in the pigment epithelium by transmission electron microscopy. Accompanying the build-up of autofluorescent pigment was the development of an irregularity and an overall increase in retinal pigment epithelium cell height. There was also an increase in the number of lipid droplets in the retinal pigment epithelium, particularly in the periphery of the eye. In the eyes of deficient animals, cells were occasionally seen which appeared to have detached from Bruchs membrane and migrated into the region of the photoreceptor outer segments. Photoreceptor outer segment phagocytosis by the retinal pigment epithelium appeared to be decreased as a consequence of dietary antioxidant deficiency, since a reduction of over 75% in the number of phagosomes per unit retinal pigment epithelium cell length was seen in some areas of the eye in both deficient groups. These changes in the retinal pigment epithelium were accompanied by a pronounced loss of photoreceptor cells, particularly from the central retina (20–34% fewer cells than supplemented controls). The disk membranes of the photoreceptor outer segments of deficient animals were often swollen, disoriented and vesiculated, and areas were frequently seen where outer segment debris had accumulated at the interface between the photoreceptors and retinal pigment epithelium. Many of the changes in the retina and retinal pigment epithelium were more severe in the fully deficient (−E−Se−S−Cr) group, than in the group deficient only in vitamin E and selenium.

Evidence of cell loss from the rat retina during senescence

In order to determine whether neuronal cell death in the retina accompanies senescence, retinas from pigmented ACI rats of various ages were analysed for changes in dimensions, cell densities, DNA content, and rhodopsin levels. Between the ages of 4 and 30 months, there were significant declines in the densities of nuclei in all three nuclear layers of the retina. Over this age span, nuclear density in the outer nuclear layer (ONL) declined by 32-41%, depending on the region of the retina examined, while inner nuclear layer (INL) nuclear density decreased by 25-34%. Over the same age range, the nuclear density in the ganglion cell layer (GCL) declined by 25-28%. The eye increased in size over the 4-30 month age span, particularly between 4 and 12 months. The retinal stretching that resulted from this growth could only partially account for the age-related decline in retinal cell densities. Depending on the region of the retina examined, decreases in nuclear density of 17-26% for the ONL, 9-17% for the INL, and 16-20% for the GCL were apparently due to cell death. Measurements of retinal DNA content support the conclusion that significant cell loss from the retina occurs during senescence; between 4 and 30 months of age retinal DNA content decreased by about 13%. Age-related changes in retinal rhodopsin content did not parallel apparent changes in photoreceptor number. Between 4 and 11 months of age, rhodopsin levels rose by about 10%, and then remained constant up to 26 months. Over this same age span, there was an apparent decrease in photoreceptor number of about 15%.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in the retinal pigment epithelium of pigmented rats

A number of significant age-related changes were found to occur in the retinal pigment epithelium (RPE) of normal pigmented ACI rats. Age-related morphological changes in the RPE included: changes in RPE cell height, an increase in lipofuscin content, alterations in the apical microvilli and basal infoldings, and an apparent thickening of the RPE basal lamina. Unusual banded structures were often seen within the basal infoldings of the RPE in senescent but not in young adult rats. Lipid droplets were relatively common in the RPE of senescent rats, but were quite rare in 4- and 11-month-old animals. In addition, contact between the RPE apical microvilli and the photoreceptor outer segments appeared to become less intimate with age. Phagocytosis of rod outer segment discs appeared to be reduced between 4 and 32 months of age. This reduction was greater than could be accounted for solely by a concomitant age-related decrease in the number of photoreceptors per unit retinal length. RPE-choroid acid phosphatase activity (a lysosomal marker), on the other hand, did not appear to change with age. The molar ratio of vitamin A-palmitate to vitamin A-stearate in the PRE-choroid rose significantly with age, from about 1.85 at both 4 and 11 months, to over 2.5 at 28 months. At each age, the palmitate- to stearate-ester ratio was the same in the dark-adapted and light-adapted eyes.

A mouse gene knockout model for juvenile ceroid-lipofuscinosis (batten disease)

The human hereditary ceroid‐lipofuscinoses are a group of autosomal recessively inherited diseases characterized by massive accumulations of autofluorescent lysosomal storage bodies in the cells of many tissues and by neuronal degeneration throughout the central nervous system. There are a number of clinically and genetically distinct forms of ceroid‐lipofuscinosis, the most common of which is the juvenile type, also known as Batten disease and CLN3. To study the mechanisms that lead to pathology in CLN3 and to evaluate potential therapies, a mouse model has been generated by targeted disruption of the mouse ortholog of the CLN3 gene (Cln3). As in affected humans, mice homozygous for the disrupted Cln3 allele show accumulation of autofluorescent storage material in neurons and other cell types. The storage material consists of membrane‐bounded intracellular inclusions with ultrastructural features typical of the ceroid‐lipofuscinoses. The accumulation of this storage material validates the Cln3 knockout mice as a model for the human disorder. J. Neurosci. Res. 57:551–556, 1999.

Lipofuscin accumulation resulting from senescence and vitamin E deficiency: Spectral properties and tissue distribution

The corrected fluorescence emission spectra and tissue distributions of the autofluorescent pigments which accumulate during normal aging and as a consequence of vitamin E deficiency were studied in albino rats. In the retinal pigment epithelium, both the age-related pigment (lipofuscin) and the pigment related to vitamin E deficiency had essentially identical emission spectra. Peak emission occurred from 590 to 650 nm. Young animals which had been kept on a vitamin E deficient diet for 17 weeks after weaning showed significant accumulations of autofluorescent pigment in uterus, duodenum, and retinal pigment epithelium, but not in the spinal cord or inferior olivary nucleus of the brain. Old animals (96 weeks) fed a commercial diet with adequate vitamin E had accumulated lipofuscin in the retinal pigment epithelium, spinal cord gray matter, and inferior olivary nucleus, but not in the duodenum or uterus. Thus, while the auto-fluorescent pigments related to aging and vitamin E deficiency have similar properties, their tissue distributions are quite different.

A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan terriers

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.