Canine Ceroid Lipofuscinosis Research Articles

Selection response to DNA testing for canine ceroid lipofuscinosis in Tibetan terriers

A late onset form of canine ceroid lipofuscinosis (CCL) is prevalent in Tibetan terriers. The disease is inherited as a monogenic recessive trait caused by aberrant exon skipping in ATP13A2. The aim of the present study was to analyse the frequencies of this mutation in Tibetan terriers registered with the German club for Tibetan dog breeds (Internationaler Klub für Tibetische Hunderassen, KTR) from 1987 to 2012 and to determine responses to selection following the introduction of DNA testing in 2010. The study included DNA extracted from blood samples from 1120/1240 (90.3%) Tibetan terriers registered with the KTR, including 405/420 (96.4%) registered breeding dogs. Mutant allele frequencies before the introduction of DNA testing were 0.20–0.28 in the registered and breeding dog populations, respectively, decreasing to 0.09 and 0.14, respectively, following the introduction of DNA testing.

Increased brain lysosomal pepstatin-insensitive proteinase activity in patients with neurodegenerative diseases

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent k m of 100 μM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

Early detection of canine ceroid-lipofuscinosis (CCL): an ultrastructural study.

English setters from the Koppang line have been used as a model for the juvenile type of Batten disease in human patients. This disorder, canine ceroid-lipofuscinosis (CCL), has been shown by Koppang [1973: Mech Ageing Dev 2:421-445] to be an autosomal recessive disorder. Homozygous animals show the typical storage granules by autofluorescence; however, the autofluorescence material cannot easily be detected in affected dogs before age 3-6 months. At the present time the early stages of CCL can be studied only in litters from homozygous matings. Electron microscopy was used to study the reliability of diagnosing affected animals in heterozygous-heterozygous (HET-HET) and heterozygous-homozygous (HET-HOM) litters at age 2-3 weeks. The electron micrographs of brain biopsies were examined and the number of all dense bodies (d.b.), including linear and fingerprint patterns typical of CCL, were counted per neuron. The results indicated that affected animals have higher numbers of dense bodies than nonaffected carriers. Four-6 months later confirmatory diagnoses were made on a second contralateral biopsy taken and examined as a frozen section by autofluorescence. The diagnoses made by electron microscopy at age 2-3 weeks appeared identical to the results obtained by autofluorescence at 4-6 months of age.

Biosynthesis and metabolism of 4‐hydroxynonenal in canine ceroid‐lipofuscinosis

Canine ceroid-lipofuscinosis (CCL) is a model of the juvenile type of Batten disease in human patients. Abnormalities have been reported previously in 4-hydroxynonenal (HNE) levels in English setters with CCL. The purpose of this study was to examine the sources of HNE in neutrophil membranes and plasma of CCL dogs. The fatty acid composition of neutrophil phospholipids, i.e., phosphatidyl ethanolamine and phosphatidyl serine, was determined by gas-liquid-chromatography (GLC) since some polyunsaturated fatty acids (PUFA) are precursors of HNE. The copper catalyzed peroxidation of low density lipoprotein (LDL) was examined to determine the susceptibility of LDL from CCL dogs to peroxidation. The results indicated that a number of PUFA precursors of HNE decreased in affected an carrier neutrophil phospholipids, indicating that this source of HNE may be disease specific. The Cu++ catalyzed formation of HNE from LDL demonstrated that carrier and normal LDL produced large amounts of HNE, while LDL from affected dogs required much higher concentrations of Cu++ for maximal HNE production. These results provide additional support for the role of HNE in the pathogenetic events in NCL and support the view that lipid peroxidation may be an important contributor to the complex pathogenesis of the NCL.

Batten's disease: failure of allogeneic bone marrow transplantation to arrest disease progression in a canine model

We investigated whether the course of canine ceroid lipofuscinosis (CCL), a model of Batten's disease in man, was affected by allogeneic bone marrow transplantation. Four English setters with CCL, 4 1/2 months of age, were given 9.2 Gy of total body irradiation, followed by the infusion of bone marrow cells from healthy DLA identical sibling donors. All transplanted dogs had complete hematologic reconstitution. However, at 12-13 months posttransplant, all dogs developed characteristic and progressive signs of CCL. Autopsies revealed cerebral atrophy and findings of ceroid storage not different from those in non-transplanted controls. These findings suggest that bone marrow cells do not contain or release the gene product(s) necessary to correct the disease. It appears unlikely that with our current knowledge, allogeneic marrow transplantation would be beneficial in the treatment of Batten's disease.

The English setter with ceroid-lipofuscinosis: a suitable model for the juvenile type of ceroid-lipofuscinosis in humans.

Genetic and histological examinations (light and EM) of tissues of an inbred line of English setters have proved that these dogs suffer a general metabolic autosomal recessive disease, canine ceroid-lipofuscinosis (CCL) almost identical to the human Stengel-Batten-Spielmeyer-Vogt disease, or neuronal ceroid-lipofuscinosis (NCL). A controlled longitudinal morphologic study showed that the formation and accumulation of an autofluorescent lipopigment, identified as "ceroid" in the isolated state, appears in the neurons already in 2-day-old puppies and increases linearly with time. Clinical signs and symptoms develop after a distinct loss of neurocytoplasm and its functional organelles is demonstrable. At that time, loss of functional neuronal cytoplasm appears to result from pigment formation. Nerve cells which have suffered this fate will eventually die and disappear. The process leads to severe global neurologic disturbance and cerebral atrophy. By the time of death from the disease at age 20 to 27 months, brain weight is reduced to 60-70% of normal control animals. The English setter with CCL differs somewhat from humans in the degree of morphological damage to various layers of the retina. In human with NCL, there is a pronounced loss of photoreceptors in the end-stage of the disease, but in CCL only minimal structural damage is observed in the retina. For experimental treatment protocols for NCL, the CCL setter is a useful model.

Enhanced synthesis of prostaglandins and hydroxyeicosatetraenoic acids in retina from a canine model of Batten's disease.

The metabolism of [1-14C]arachidonic acid (20:4, n-6) was studied in intact retina and retinal pigment epithelial cells from normal English setters and English setters affected with hereditary canine ceroid lipofuscinosis. Acylation of arachidonic acid into membrane glycerolipids and oxygenation by lipoxygenase and cyclooxygenase to eicosanoids were measured by radiochromatographic techniques. In addition, the histopathology of accumulated ceroid particles in retinal ganglion cells and pigment epithelial cells was studied by electron microscopy. Synthesis of prostaglandins and hydroxyeicosatetraenoic acids was increased in canine ceroid lipofuscinosis retina, but not in retinal pigment epithelium. Prostaglandin D2, the putative neuronal eicosanoid, was increased nearly eightfold, whereas other eicosanoids increased two- to threefold. Ultrastructural studies revealed accumulation of ceroid and deterioration of neuronal and pigment epithelial cell architecture. These experiments demonstrate that, although lipopigment accumulates in both tissues, alterations of eicosanoid synthesis are specific for the retina, a neuronal tissue. The specific increase in prostaglandin D2 and the specificity of changes for the retina indicate that enhanced eicosanoid synthesis may be a result of an impairment of the control of oxygenation of arachidonic acid in neurons.

Electrocardiographic and histologic abnormalities in canine ceroid-lipofuscinosis (CCL)

Electrocardiograms were recorded in a longitudinal study on six adult dogs with canine ceroid-lipofuscinosis (CCL) and compared with those of four unaffected animals. Two dogs exhibited minor conduction abnormalities, QRS-notching and LVH at 12 months of age. By 24 to 25 months of age, the underlying cardiac rhythm was that of a pronounced sinus arrhythmia. Some dogs developed premature atrial beats, A-V dissociation or tachycardia. No gross cardiac abnormalities were noted at necropsy, but histologically, autonomic ganglion cells in the atrium, near the conducting system and in ventricular myocardial cells were filled with autofluorescent lipopigments. Quantification by micro-spectrophotometry showed peak emission at 538 nm. At the ultrastructural level, these fluorescent particles consisted of collections of curvilinear and lamellar profiles, similar to those present in other viscera. Ceroid-lipofuscinosis has been utilized for many years as a model for human ceroid-lipofuscinosis and the results described here are similar to those previously reported in hearts of patients with this disease. Because related lipopigments accumulate in normal aging of the heart and contraction/conduction abnormalities develop with time, the canine model may also be useful in aging studies as well. The studies described here are the first to demonstrate possible structural-functional relationships in the heart of dogs with CCL.

Arachidonic acid-induced platelet aggregation and malondialdehyde production in dogs of various ages and in dogs affected with canine ceroid lipofuscinosis

The arachidonate pathway of canine platelets was assessed in dogs of varying ages by examination of platelet aggregation to arachidonic acid and by measurement of arachidonic acid-induced platelet malondialdehyde production. As age increased, arachidonic acid-induced platelet aggregation showed a progressive prolongation in the lag phase before aggregation. The production of malondialdehyde by canine platelets significantly decreased with increasing age. Dogs affected with ceroid lipofuscinosis showed similar declines in platelet aggregation and malondialdehyde production, but at an earlier age. This suggests that changes occur in canine platelet reactivity during natural aging and in a disease of “premature” aging. The lack of change in platelet numbers and volume suggests that the alterations reported were related to the aging process rather than to advancing vascular disease. The findings support the free radical theory of aging.

Change in content, incorporation and lipoxygenation of docosahexaenoic acid in retina and retinal pigment epithelium in canine ceroid lipofuscinosis

We have examined the metabolism of docosahexaenoic acid (22:6, n − 3) in retina and retinal pigment epithelium of normal dogs and those affected with canine ceroid lipofuscinosis (CCL), a hereditary degenerative neurological disorder. In the CCL retina, there was a decrease in 22:6 content in phospholipids, particularly phosphatidylethanolamine. This decrease in 22:6 was compensated by an increase in arachidonic acid (20:4, n − 6). In contrast, CCL retinal pigment epithelium had higher levels of 22:6 and lower levels of 20:4 in phosphatidylethanolamine. The in vitro incorporation of radiolabeled 22:6 into glycerolipids of CCL retina and retinal pigment epithelium was increased as compared to control. The major lipoxygenase reaction product of 22:6, (11-hydroxy-4,7,9( trans)13,16,19)-22:6, increased 31% in CCL retina, but not in the retinal pigment epithelium. This is the first report describing alterations in content, incorporation and lipoxygenation of 22:6 in an animal model of a human disease (Batten's disease).

Computed tomographic and nuclear magnetic resonance correlation of canine ceroid-lipofuscinosis with aging

Canine ceroid lipofuscinosis is a degenerative neuronal disorder which has as a distinct pathologic counterpart, neuronal ceroid lipofuscinosis or Batten's disease in humans. The disease occurs in English setters and is associated with abnormal accumulation of autofluorescent lipopigments. As such, a study of this animal model may allow insight into the aging process as well. This investigation explores the computed tomographic features of canine ceroid-lipofuscinosis and correlates these findings (progressive atrophy, inherent changes in CT/NMR tissue characteristics) with clinical and pathologic features.

Free radicals and aging. Electron spin resonance studies on neuronal lipopigments and cells grown in vitro

Electron spin resonance (ESR) spectroscopy has been used in an attempt to observe free radicals which may be connected to the aging processes. Isolated lipofuscin particles from the human brain, which may act as traps for reactive ESR centers, have been studied. The lipopigments exhibit ESR signals which were attributed to metal ions. Both high-spin ferric iron and copper (II) complexes were observed. The copper resonance showed axial symmetry and the observed ESR parameters ( A ‖ = 185 gauss, g ‖ = 2.25 and g ⊥ = 2.066) indicate that the copper ions are of the “non-blue” (type (so-called “type 2”). The data suggest that the copper ions might be ligated to two nitrogen and two oxygen atoms. Ceroid particles from the brains of dogs with end-stage “canine ceroid lipofuscinosis” yield similar resonances involving metal complexes. The concentration of the copper ions in the lipopigments seems to increase slightly towards the end stage of life. Retinal pigment epithelial cells from the eyes of dogs with canine ceroid lipofuscinosis were grown in vitro and examined with ESR when the cells reached confluency and then after being maintained confluent for 2 weeks. The spectra indicate an accumulation of metal ions due to this “aging”. Phase-contrast microscopy reveals an increase in perinuclear dense bodies during the time in confluency. A weak ESR signal, compatible with an “oxygen-centered” radical, was also found to increase slightly during the “aging” period.

Morphological and biochemical abnormalities in a model of retinal degeneration: Canine ceroid-lipofuscinosis (CCL)

Human ceroid-lipofuscinosis is marked by blindness, dementia, ataxia, and premature death. A canine model for this disease exists in English setters whose clinical, pathological and biochemical changes resemble the human disorder. In both syndromes, autofluorescent lipopigments, i.e.; lipofuscin and ceroid (“granular”, “fingerprint” and/or “curvilinear bodies”) are found in the nervous system, viscera, retina, and pigment epithelium (RPE). Retinal neurons of affected animals between 6 and 22 months of age, contain a variety of abnormal intracellular pigment inclusions. Pigment epithelial cells also contain distinctive cytosomes. Electroretinograms from affected animals showed a reduction in b-wave amplitude. Leukocyte, retinal, and RPE peroxidases, were decreased in affected animals, and also showed age-related changes. In the normal canine eye, peroxidase was associated with fractions containing plasma membranes and melanolysosomes. Improved fractionation techniques localized normal peroxidase to “heavy” fractions (1.24–1.28 g/ml), and peroxidase was decreased in these fractions in CCL animals. A new particle containing hexosaminidase, galactosidase, and acid lipase was observed in affected animals. When retinal homogenates from CCL dogs were injected into the vitreous of rabbit eyes they completely abolished the ERG recording. No such change was observed with homogenates from unaffected animals. The accumulation of large numbers of dense bodies in the retina and RPE in dogs with CCL, along with a decrease in peroxidase, suggests an impairment of degradative mechanisms. Furthermore, ceroid appears to be cytotoxic to the retina and RPE. The relationship of these cytotoxic properties to the accumulation of ceroid in the eye, is the subject of our future research.

Ultrastructure of the Retina in Canine Neuronal Ceroid Lipofuscinosis

Electron microscopic studies on the retinae of 3 English setters, homozygous for the trait of canine neuronal ceroid lipofuscinosis (NCL), revealed preservation of photoreceptors early and late in the

Canine ceroid-lipofuscinosis — A model for human neuronal ceroid-lipofuscinosis and aging

A controlled longitudinal morphologic study revealed that the formation and accumulation of an autofluorescent lipopigment, identified as “ceroid” in the isolated state, commences in utero and progresses linearly with time. Only after a distinct loss of neurocytoplasm and its functional organelles is demonstrable, do clinical signs and symptoms develop. At that time, loss of functional neuronal cytoplasm appears to result from pigment formation. Nerve cells which have suffered this fate will eventually die and disappear. The process leads to a severe global neurologic disturbance and a cerebral atrophy. By the time of death from the disease at age 25 months, the brain weight is reduced to 70% of normal controls. Extraneural accumulations of ceroid pigments are massive and lead to an increase in the size of many lymph nodes but damage of viscera, demonstrable either by functional tests or by unequivocal morphologic criteria, was not observed. The morphogenesis of pigment accumulation has not been conclusively revealed. However, it appears reasonably safe to assume that a “prepigment” forms randomly in the cytoplasm which in the process of repetitive focal cell degradation becomes incorporated in autophagic vacuoles and by lysosomal digestion, which removes the catabolizable moieties, the residual pigment is being formed. The internal structures of the pigment bodies span a wide range and their architecture does not appear to be of pathognostic or pathogenetic significance.