Chun-Lan Gao

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.

Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes

During the aging process the retinal pigment epithelium (RPE) accumulates autofluorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis that at least one of the fluorescent components of RPE lipofuscin is formed by reaction of vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All-trans retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two different lipid compositions. One type of liposome consisted of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a primary amine. The other liposome type was identical in composition accept that some of the PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After incubation of the samples, aliquots were examined with fluorescence microscopy to assess whether any lipofuscin-like fluorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chromatography. Photoreceptor outer segments incubated with retinaldehyde developed an intense golden yellow fluorescent emission when illuminated with 395-440 nm light. Similar fluorescence developed in the liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did not develop any detectable fluorescence. The development of fluorescence in the samples in situ correlated with the appearance of an orange colored component in the lipid extracts that displayed a weak red emission upon ultraviolet light illumination. Incorporation of this component into liposomes resulted in the appearance of a golden yellow fluorescent emission. The results of these experiments suggest that retinal, generated during visual pigment bleaching can react with PE in the photoreceptor outer segments to form a fluorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE lipofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably derived from the reaction product of outer segment PE and retinal.

Dietary carnitine supplements slow disease progression in a putative mouse model for hereditary ceroid‐lipofuscinosis

The childhood ceroid‐lipofuscinoses are a group of autosomal recessively inherited disorders characterized by massive accumulation of autofluorescent lysosomal storage bodies in neurons as well as other cell types. The storage body accumulation is accompanied by severe degeneration of the central nervous system that results in blindness, cognitive and psychomotor degeneration, and premature death. On the basis of pathologic and biochemical criteria, a hereditary disease in the mnd mouse strain has been proposed as a model for certain types of human ceroid‐lipofuscinosis. Experimental evidence suggests that the storage body accumulation in humans with juvenile and late‐infantile ceroid‐lipofuscinosis is linked to altered carnitine biosynthesis. On the basis of the latter observation, a study was performed to determine whether dietary carnitine supplements could slow the disease progression in the mnd mouse model. Carnitine supplementation begun at 4 weeks of age did not slow the retinal degeneration that is characteristic of this disease. It did, however, significantly elevate brain carnitine levels, slow the accumulation of autofluorescent storage bodies in brain neurons, and prolong the lifespans of the treated animals. These findings suggest that there is a link between carnitine biosynthesis and the disease pathology and indicate that carnitine supplementation may be beneficial in slowing the disease progression in humans with certain types of hereditary ceroid‐lipofuscinosis. J. Neurosci. Res. 50:123–132, 1997.

Vitamin A incorporation into lipofuscin-like inclusions in the retinal pigment epithelium

Intravitreal injection of the protease inhibitor leupeptin causes a rapid accumulation of lipofuscin-like autofluorescent inclusions in the retinal pigment epithelium (RPE) of the eye. In vitamin A-deprived animals, similar inclusions form in response to leupeptin treatment, but they do not become autofluorescent. Because vitamin A is necessary to the development of fluorescence, it appears likely that retinoids are directly incorporated into the inclusions. Experiments were conducted to determine whether this is the case. Rats were reared on a diet containing retinoic acid as the only retinoid. Retinoic acid cannot be utilized in visual transduction by the retina. When the eyes had been over 90% depleted of visual cycle retinoids, the animals were given a single intramuscular injection of 3H-all-trans retinol. After 7 days, when visual cycle retinoids had returned to an average of almost 70% of normal, the animals were given an intravitreal injection of leupeptin in each eye. At either 1 day or 7 days after the leupeptin treatment, some of the animals were dark-adapted for at least 12 h. The eyes were enucleated and fixed under dim red light. A region of each retina just superior to the optic nerve head was examined with electron microscopic autoradiography. At both one day and 7 days after the leupeptin treatment, the radiolabel in the RPE was primarily associated with the leupeptin induced inclusion bodies. Label was also present in the photoreceptor outer segments. The localization of vitamin A to the leupeptin-induced inclusions in the RPE strongly suggests that vitamin A is covalently bound to outer segment proteins that have been phagocytosed by the RPE but remain undegraded due to protease inhibition. This bound vitamin A is probably responsible for the autofluorescence of the leupeptin-induced inclusions. Vitamin A is not likely to be bound through a Schiff base linkage, since retinal-Schiff base compounds do not exhibit lipofuscin-like fluorescence.

Long-term variations in cyclic light intensity and dietary vitamin A intake modulate lipofuscin content of the retinal pigment epithelium.

Experiments were conducted to determine whether the intensity of cyclic light exposure to the retina over a long period of time affects retinoid‐dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE). Albino rats were maintained from weaning on diets either containing (+A) or lacking (−A) retinyl palmitate, which can be metabolized to the retinoids involved in the visual cycle. Animals in each dietary group were divided between bright (L) and dim (D) cyclic light treatments. Thus, the experiments employed the following four treatment groups: +A/D, +A/L, −A/D, and −A/L. After 6, 12, and 15 months from the start of the treatments, animals in each group were killed for quantitative determination of: 1) retinal photoreceptor densities; 2) RPE lipofuscin content; and 3) RPE lipofuscin fluorescence intensity. Animals in the L groups had a lower volume of RPE lipofuscin than those in the D groups fed the same diet. Among the −A rats, this reduced lipofuscin volume could be attributed to a light‐enhanced depletion of vitamin A from the retina and an accompanying loss of photoreceptor cells. In the +A animals, however, there were no differences in photoreceptor densities between the D and L groups. In the −A rats, the volume of RPE lipofuscin decreased between 6 and 15 months of age, whereas it increased in the +A animals. In contrast, lipofuscin fluorescence intensity increased between 6 and 15 months of age in all four treatment groups. However, in the +A rats, the fluorescence intensity was lower in the L than in the D group at all three ages. In the −A groups, light level had no effect on lipofuscin fluorescence intensity. At all three ages, fluorescence intensity was lower in the −A animals than in +A rats. Thus, at light intensities below those that induce acute retinal degeneration, long‐term exposure to higher intensity light inhibits the age‐related increase in RPE lipofuscin volume. A decrease in the volume of RPE lipofuscin after the retina is depleted of vitamin A suggests that lipofuscin is turned over, and that RPE lipofuscin content is determined by a balance between the rates at which lipofuscin is formed and at which it is eliminated from the RPE. An age‐related increase in lipofuscin‐specific fluorescence intensity after vitamin A depletion from the retina suggests that lipofuscin fluorophores may continue to form slowly from retinoids that have been modified such that they can no longer enter the visual cycle. J. Neurosci. Res. 57:106–116, 1999.