Predictors of changing insulin dose requirements and glycaemic control in children, adolescents and young adults with Type 1 diabetes.

AIMS: To investigate trajectories of daily insulin dose requirements and glycaemic control in children, adolescents and young adults with Type 1 diabetes and to identify factors associated with changing insulin needs and deterioration in HbA1c . METHODS: The sample was a dynamic cohort of 635 children, adolescents and young adults with Type 1 diabetes from one centre. Data from clinic visits occurring over 20 years (1993-2013) were extracted from medical records. From age 7-24 years, we evaluated HbA1c and insulin dose according to sex, insulin regimen and weight status. RESULTS: Participants provided a mean ± sd of 10.7±4.3 years of insulin dose data and 12.0±4.6 years of HbA1c data. At first observation, the mean ± sd age was 10.0±2.6 years, diabetes duration was 2.8±2.1 years, insulin dose was 0.8±0.2 units/kg and HbA1c was 74±18 mmol/mol (8.9±1.6%). Insulin dose was higher in girls at ages 8-13 years (P<0.0001 to P<0.01), but higher in boys/young men at ages 16-21 years (P<0.0001 to P=0.04). HbA1c was higher in girls/young women at ages 16-24 years (P<0.0001 to P=0.01). Compared with injection therapy, pump therapy was associated with lower insulin dose at ages 8-24 years (P<0.0001 to P=0.03) and lower HbA1c at ages 8-22 years (P<0.0001 to P=0.005). HbA1c did not differ between overweight/obese and normal weight individuals, but overweight/obese individuals had higher insulin dose at ages 8-13 years (P<0.0001 to P=0.03). CONCLUSIONS: This longitudinal assessment identifies clinically meaningful modifiable (e.g. insulin regimen) and non-modifiable (e.g. sex) factors predictive of insulin requirements and HbA1c levels in young people with Type 1 diabetes; anticipatory insulin adjustments may improve glycaemic control.

Extraneuronal pathology in a canine model of CLN2 neuronal ceroid lipofuscinosis after intracerebroventricular gene therapy that delays neurological disease progression.

CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.

Pharmacokinetics and dynamics of mycophenolate mofetil after single-dose oral administration in juvenile dachshunds.

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young-aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose-bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole-blood mitogen-stimulated T-cell proliferation was determined using a flow cytometric assay. Plasma MPA Cmax (mean ± SD, 9.33 ± 7.04 µg/ml) occurred at <1 hr. The AUC0-∞ (mean ± SD, 12.84±6.62 hr*µg/ml), MRTinf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.

Golden Retriever dogs with neuronal ceroid lipofuscinosis have a two-base-pair deletion and frameshift in CLN5.

We studied a recessive, progressive neurodegenerative disease occurring in Golden Retriever siblings with an onset of signs at 15 months of age. As the disease progressed these signs included ataxia, anxiety, pacing and circling, tremors, aggression, visual impairment and localized and generalized seizures. A whole genome sequence, generated with DNA from one affected dog, contained a plausibly causal homozygous mutation: CLN5:c.934_935delAG. This mutation was predicted to produce a frameshift and premature termination codon and encode a protein variant, CLN5:p.E312Vfs*6, which would lack 39 C-terminal amino acids. Eighteen DNA samples from the Golden Retriever family members were genotyped at CLN5:c.934_935delAG. Three clinically affected dogs were homozygous for the deletion allele; whereas, the clinically normal family members were either heterozygotes (n = 11) or homozygous for the reference allele (n = 4). Among archived Golden Retrievers DNA samples with incomplete clinical records that were also genotyped at the CLN5:c.934_935delAG variant, 1053 of 1062 were homozygous for the reference allele, 8 were heterozygotes and one was a deletion-allele homozygote. When contacted, the owner of this homozygote indicated that their dog had been euthanized because of a neurologic disease that progressed similarly to that of the affected Golden Retriever siblings. We have collected and stored semen from a heterozygous Golden Retriever, thereby preserving an opportunity for us or others to establish a colony of CLN5-deficient dogs.

Validation of the Diabetes Family Impact Scale: a new measure of diabetes-specific family impact.

AIMS: To develop and validate the Diabetes Family Impact Scale, a scale to measure the impact of diabetes on families. METHODS: The Diabetes Family Impact Scale was developed by an iterative process, with input from multidisciplinary diabetes providers and parents of children with Type 1 diabetes. The psychometric properties of the Diabetes Family Impact Scale were assessed in parents of children with Type 1 diabetes. This assessment included internal consistency, convergent validity and exploratory factor analysis. RESULTS: Parents (n = 148) of children (mean ± sd age 12.9 ± 3.3 years) with Type 1 diabetes (mean ± sd duration 6.2 ± 3.6 years) completed the 15-item Diabetes Family Impact Scale. After eliminating one item, the 14-item measure demonstrated good internal consistency (Cronbach's α  =  0.84). Correlations between the Diabetes Family Impact Scale and measures of parent diabetes burden (r = 0.48, P < 0.0001), stressful life events (r = 0.28, P = 0.0007), and child's quality of life (r = -0.52 and -0.54, P < 0.0001 for generic and diabetes-specific quality of life, respectively) supported the convergent validity of the instrument. Factor analysis identified four factors corresponding to the four survey domains (school, work, finances and family well-being). CONCLUSIONS: The Diabetes Family Impact Scale measures diabetes-specific family impacts with good internal consistency and convergent validity and may be a useful tool in clinical and research settings.

Characterization of neuronal ceroid-lipofuscinosis in 3 cats.

Three young domestic shorthair cats were presented for necropsy with similar histories of slowly progressive visual dysfunction and neurologic deficits. Macroscopic examination of each cat revealed cerebral and cerebellar atrophy, dilated lateral ventricles, and slight brown discoloration of the gray matter. Histologically, there was bilateral loss of neurons within the limbic, motor, somatosensory, visual, and, to a lesser extent, vestibular systems with extensive astrogliosis in the affected regions of all 3 cases. Many remaining neurons and glial cells throughout the entire central nervous system were distended by pale yellow to eosinophilic, autofluorescent cytoplasmic inclusions with ultrastructural appearances typical of neuronal ceroid-lipofuscinoses (NCLs). Differences in clinical presentation and neurological lesions suggest that the 3 cats may have had different variants of NCL. Molecular genetic characterization in the 1 cat from which DNA was available did not reveal any plausible disease-causing mutations of the CLN1 (PPT1), CLN3, CLN5, CLN8, and CLN10 (CTSD) genes. Further investigations will be required to identify the mutations responsible for NCLs in cats.

Contemporary rates of severe hypoglycaemia in youth with type 1 diabetes: variability by insulin regimen.

AIMS: To determine incidence rates of severe hypoglycaemia and compare incidence rates by insulin regimen in a diverse sample of youth with Type 1 diabetes from two sites. METHODS: In this observational study, 255 youth (51% female) aged 9-15 years receiving varied insulin regimens provided data prospectively for a median of 1.2 years. Reported episodes of severe hypoglycaemia, defined as episodes requiring help from another person for oral treatment or episodes resulting in seizure/coma, and current insulin regimens were collected systematically. Incidence rates were calculated and compared according to insulin regimen in bivariate and multivariate analyses. RESULTS: At first encounter, participants had a median age of 12.2 years (range 9.0-15.0), median diabetes duration of 4.4 years (range 1.0-13.0) and mean HbA(1c) of 67 ± 12 mmol/mol (8.3 ± 1.1%). The incidence rate was 37.6/100 patient-years for all severe hypoglycaemia and 9.6/100 patient-years for seizure/coma. The incidence rate for severe hypoglycaemia was 31.8/100 patient-years on continuous subcutaneous insulin infusion (pump therapy), 34.4/100 patient-years on basal-bolus injections and 46.1/100 patient-years on NPH (NPH vs. pump therapy: P = 0.04). The incidence rate for seizure/coma was 4.5/100 patient-years on pump therapy, 11.1/100 patient-years on basal-bolus injections and 14.4/100 patients-years on NPH (NPH vs. pump therapy: P = 0.004). In the multivariate analysis, the rate of seizure/coma was significantly higher for those on NPH vs. pump therapy (rate ratio 2.9, P = 0.03). CONCLUSIONS: Rates of severe hypoglycaemia in youth with Type 1 diabetes remain high. Pump therapy was associated with lower rates of all severe hypoglycaemia and seizure/coma in comparison with NPH.

Accumulation of glial fibrillary acidic protein and histone H4 in brain storage bodies of Tibetan terriers with hereditary neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases characterized by massive accumulation of autofluorescent storage bodies in neurons and other cells. A late-onset form of NCL occurs in Tibetan terrier dogs. Gel electrophoretic analyses of isolated storage body proteins from brains of affected dogs indicated that a protein of approximately 50 kDa was consistently prominent and a 16 kDa component was present in some brain storage body preparations. Mass spectral analysis identified the 50 kDa protein as glial fibrillary acidic protein (GFAP), isoform 2. GFAP identification was supported by immunoblot and immunohistochemical analyses. Histone H4 was the major protein in the 16 kDa component. Specific accumulation of GFAP and histone H4 in storage bodies has not been previously reported for any of the NCLs. Tibetan terrier NCL may be the canine correlate of one of the human adult-onset NCLs for which the genetic bases and storage body compositions have not yet been determined.

Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BACKGROUND: No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. HYPOTHESIS/OBJECTIVE: Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. ANIMALS: Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. METHODS: Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. RESULTS: Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9-51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2-61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6-59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0-12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

GM2 Gangliosidosis in Shiba Inu Dogs with an In-Frame Deletion in HEXB.

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.

Homozygous PPT1 Splice Donor Mutation in a Cane Corso Dog With Neuronal Ceroid Lipofuscinosis.

A 10-month-old spayed female Cane Corso dog was evaluated after a 2-month history of progressive blindness, ataxia, and lethargy. Neurologic examination abnormalities indicated a multifocal lesion with primarily cerebral and cerebellar signs. Clinical worsening resulted in humane euthanasia. On necropsy, there was marked astrogliosis throughout white matter tracts of the cerebrum, most prominently in the corpus callosum. In the cerebral cortex and midbrain, most neurons contained large amounts of autofluorescent storage material in the perinuclear area of the cells. Cerebellar storage material was present in the Purkinje cells, granular cell layer, and perinuclear regions of neurons in the deep nuclei. Neuronal ceroid lipofuscinosis (NCL) was diagnosed. Whole genome sequencing identified a PPT1c.124 + 1G>A splice donor mutation. This nonreference assembly allele was homozygous in the affected dog, has not previously been reported in dbSNP, and was absent from the whole genome sequences of 45 control dogs and 31 unaffected Cane Corsos. Our findings indicate a novel mutation causing the CLN1 form of NCL in a previously unreported dog breed. A canine model for CLN1 disease could provide an opportunity for therapeutic advancement, benefiting both humans and dogs with this disorder.

Australian Cattle Dogs with Neuronal Ceroid Lipofuscinosis are Homozygous for a CLN5 Nonsense Mutation Previously Identified in Border Collies.

BACKGROUND: Neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease, has been diagnosed in young adult Australian Cattle Dogs. OBJECTIVE: Characterize the Australian Cattle Dog form of NCL and determine its molecular genetic cause. ANIMALS: Tissues from 4 Australian Cattle Dogs with NCL-like signs and buccal swabs from both parents of a fifth affected breed member. Archived DNA samples from 712 individual dogs were genotyped. METHODS: Tissues were examined by fluorescence, electron, and immunohistochemical microscopy. A whole-genome sequence was generated for 1 affected dog. A TaqMan allelic discrimination assay was used for genotyping. RESULTS: The accumulation of autofluorescent cytoplasmic storage material with characteristic ultrastructure in tissues from the 4 affected dogs supported a diagnosis of NCL. The whole-genome sequence contained a homozygous nonsense mutation: CLN5:c.619C>T. All 4 DNA samples from clinically affected dogs tested homozygous for the variant allele. Both parents of the fifth affected dog were heterozygotes. Archived DNA samples from 346 Australian Cattle Dogs, 188 Border Collies, and 177 dogs of other breeds were homozygous for the reference allele. One archived Australian Cattle Dog sample was from a heterozygote. CONCLUSIONS AND CLINICAL IMPORTANCE: The homozygous CLN5 nonsense is almost certainly causal because the same mutation previously had been reported to cause a similar form of NCL in Border Collies. Identification of the molecular genetic cause of Australian Cattle Dog NCL will allow the use of DNA tests to confirm the diagnosis of NCL in this breed.

Decreased plasma carnitine and trimethyl-L-lysine levels associated with lysosomal accumulation of a trimethyl-L-lysine containing protein in Batten disease.

Batten disease, or juvenile neuronal ceroid-lipofuscinosis, is an autosomal-recessive hereditary disorder that leads to blindness, severe neurological degeneration, and premature death. The disease is characterized by massive accumulation of lysosomal storage bodies in most tissues. A significant constituent of the storage material is a protein that appears to be almost identical to a small hydrophobic inner mitochondrial membrane protein, subunit c of ATP synthase. The protein isolated from the storage bodies contains an epsilon-N-trimethyl-L-lysine (TML) residue at amino acid position 43. The presence of TML in the stored protein suggests that one of the lysine residues in subunit c is normally trimethylated, and this trimethylation may act as a signal to initiate degradation of the protein. Free TML produced by the degradation of TML-containing proteins is the first intermediate in the carnitine biosynthetic pathway. It is possible that trimethylated subunit c is a major source of the free TML used in carnitine biosynthesis. If this is the case, one would predict that the genetic defect resulting in the accumulation of TML containing subunit c would also reduce systemic levels of free TML and carnitine. To evaluate this possibility, plasma TML and carnitine levels were measured in affected human subjects, heterozygous carriers, and normal controls. Both TML and carnitine levels were significantly depressed in the affected individuals. This suggests that subunit c is normally a major source of TML for carnitine biosynthesis. In Batten disease, failure to degrade the TML-containing form of subunit c is probably responsible for the reduction in plasma TML and carnitine levels.

Methylated lysine in storage body protein of sheep with hereditary ceroid-lipofuscinosis.

Juvenile ceroid-lipofuscinosis (Batten disease) is a hereditary storage disease with an autosomal-recessive mode of transmission. This disorder has been identified in humans, dogs and sheep. It is characterized by massive accumulations of autofluorescent storage bodies in many tissues. This storage body accumulation is accompanied by functional decline and degeneration of the affected tissues, and ultimately results in premature death. The primary defect responsible for juvenile ceroid-lipofuscinosis has not been identified. Previous studies have indicated that the storage material is primarily protein. Why this protein accumulates in storage bodies remains to he determined. In affected humans, the storage body protein appears to be abnormally rich in a methylated derivative of lysine (epsilon-N-trimethyllysine). Studies were undertaken to determine whether the storage bodies from sheep with hereditary ceroid-lipofuscinosis were also characterized by the presence of this modified amino acid. Chromatographic and mass spectral analyses of hydrolysates of the storage body protein indicated a significant fraction of the lysine residues in this protein were present as the epsilon-N-trimethyl derivative. This modified amino acid was not detected in hydrolysates of protein from normal sheep tissues or from tissues of sheep with GM1 gangliosidosis, nor did it appear to be present in the storage body protein from a human subject with the late infantile form of ceroid-lipofuscinosis. Thus, it is apparently specific to the storage body protein that accumulates in the juvenile type of this disease. The abnormal presence of epsilon-N-trimethyllysine in proteins could interfere with their sorting or degradation within cells and thus cause them to accumulate in the storage bodies characteristic of the human juvenile and ovine ceroid-lipofuscinoses.

Dietary vitamins A and E influence retinyl ester composition and content of the retinal pigment epithelium.

Experiments were conducted to determine the influence of dietary levels of vitamin A and alpha-tocopherol on the amounts and composition of retinyl esters in the retinal pigment epithelium of light-adapted albino rats. Groups of rats were fed diets containing alpha-tocopherol and either no retinyl palmitate, adequate retinyl palmitate, or excessive retinyl palmitate. Other groups of rats received diets lacking alpha-tocopherol and containing the same three levels of retinyl palmitate. Retinoic acid was added to diets lacking retinyl palmitate. After 27 weeks, the animals were light-adapted to achieve essentially total visual pigment bleaches, and the neural retinas and retinal pigment epithelium-eyecups were then dissected from each eye for vitamin A ester determinations. Almost all of the retinyl esters were found in the retinal pigment epithelium-eyecup portions of the eyes, mainly as retinyl palmitate and retinyl stearate. Maintaining rats on a vitamin A-deficient, retinoic acid-containing diet led to significant reductions in retinal pigment epithelial retinyl ester levels in rats fed both the vitamin E-supplemented and vitamin E-deficient diets; contrary to expectations, the effect of dietary vitamin A deficiency was more pronounced in the vitamin E-supplemented rats. Vitamin A deficiency in retinoic acid-maintained animals also led to significant reductions in retinyl palmitate-to-stearate ester ratios in the retinal pigment epithelia of both vitamin E-supplemented and vitamin E-deficient rats. Excessive dietary intake of vitamin A had little, if any, effect on retinal pigment epithelial retinyl ester content or composition. Vitamin E deficiency resulted in significant increases in retinal pigment epithelial retinyl palmitate content and in palmitate-to-stearate ester ratios in rats fed all three levels of vitamin A, but had little effect on retinal pigment epithelial retinyl stearate content. In other tissues, vitamin E deficiency has been shown to lower vitamin A levels, and it is widely accepted that this effect is due to autoxidative destruction of vitamin A. The increase in retinal pigment epithelial vitamin A ester levels in response to vitamin E deficiency indicates that vitamin E does not regulate vitamin A levels in this tissue primarily by acting as an antioxidant, but rather may act as an inhibitor of vitamin A uptake and/or storage. The effect of vitamin E on pigment epithelial vitamin A levels may be mediated by the vitamin E-induced change in retinyl palmitate-to-stearate ratios.

Late-infantile ceroid-lipofuscinosis: lysine methylation of mitochondrial ATP synthase subunit c from lysosomal storage bodies.

Late-infantile ceroid-lipofuscinosis is a fatal autosomal recessively inherited disease characterized by massive accumulations of lysosomal storage bodies in many tissues. A major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase. Juvenile ceroid-lipofuscinosis, a disease that is similar to but genetically distinct from the late-infantile disorder, also involves lysosomal accumulation of the subunit c protein. In the juvenile disease, the stored form of the protein contains an epsilon-N-trimethyllysine (TML) residue at position 43. Analyses were performed to determine whether subunit c protein stored in the late-infantile disease is also trimethylated at lysine residue 43. Amino acid composition analysis of the subunit c protein stored in brains from subjects with the late-infantile disease indicated that one of the two lysine residues in the protein is trimethylated. Data from molecular mass analysis of the protein was consistent with the presence of three methyl groups not present in the unmodified protein. The TML in the storage body subunit c protein was found by amino acid sequence analysis to occur exclusively at residue 43. The lysine at this position in the stored protein was completely methylated. Recent studies suggest that the subunit c protein from normal mitochondria may also have the same amino acid modification. Thus, it appears that specific methylation of lysine residue 43 of mitochondrial ATP synthase subunit c is probably a normal post-translational modification, and that the lysosomal storage of this protein in late-infantile, as well as in juvenile ceroid-lipofuscinosis, does not result from a defect in its methylation.

The autofluorescent products of lipid peroxidation may not be lipofuscin-like.

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of lipofuscin-like fluorescence in the retinal pigment epithelium in response to protease inhibitor treatment.

Experiments were conducted to determine whether photoreceptor outer segment components contain precursors for lipofuscin fluorophores that accumulate in the retinal pigment epithelium (RPE) during senescence. Intravitreal injection of the lysosomal protease inhibitor leupeptin caused a rapid accumulation of inclusions with lipofuscin-like autofluorescence in the RPE of albino rats. These inclusions appeared to be derived from photoreceptor outer segments, which are normally phagocytosed and degraded by the RPE. The tripeptide leu-gly-gly, which is similar to leupeptin except that it does not inhibit proteolysis, had no effect on RPE autofluorescent pigment content. Likewise, netilmicin, a purported inhibitor of lysosomal phospholipases, did not enhance autofluorescent pigment deposition in the RPE. These findings are consistent with other experiments suggesting that RPE age-pigment fluorophores are derived from molecular components of the photoreceptor outer segments. The effect of leupeptin suggests that outer segment proteins may be among the precursors for these fluorophores. The RPE appears to differ from other cell types that accumulate lipofuscin in that the majority of the precursors for this pigment in the RPE are taken up by phagocytosis rather than being generated within the cells themselves.

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.

Storage protein in hereditary ceroid-lipofuscinosis contains S-methylated methionine.

Hereditary ceroid-lipofuscinoses are neurodegenerative disorders characterized by the accumulation in numerous tissues of a storage material with lipofuscin-like fluorescence properties. Investigations were undertaken to determine the chemical nature of this storage material isolated from the cerebral cortex of human subjects with the late infantile form of the disease. The storage material was mainly protein that consisted of a mixture of polypeptides ranging in apparent molecular weight from 13 to 67 kDa. Protein-bound fluorophores apparently were largely responsible for the autofluorescence of the storage bodies. The disease-related storage body protein was rich in S-methylmethionine [(3-amino-3-carboxypropyl) dimethyl sulfonium ion], an amino acid that does not normally occur in animal proteins. Methylation of proteins to form this unusual charged amino acid may impair proteolytic degradation or other aspects of protein metabolism, and account for the accumulation of protein-filled inclusions in cells of individuals with ceroid-lipofuscinoses. Similar amino acid modifications that block proteolysis could be involved in age pigment accumulation.