Biomedical subjects

Cameron A Ackerley

Publications and source records attributed to Cameron A Ackerley.

10 recordsLinked to original sources

PubMed2005

OCTN3 is a mammalian peroxisomal membrane carnitine transporter.

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.

Animals↗

PubMed2005

Novel glycogen synthase kinase 3 and ubiquitination pathways in progressive myoclonus epilepsy.

Lafora progressive myoclonus epilepsy, caused by defective laforin or malin, insidiously present in normal teenagers with cognitive decline, followed by rapidly intractable epilepsy, dementia and death. Pathology reveals neurodegeneration with neurofibrillary tangle formation and Lafora bodies (LBs). LBs are deposits of starch-like polyglucosans, insufficiently branched and hence insoluble glycogen molecules resulting from glycogen synthase (GS) overactivity relative to glycogen branching enzyme activity. We previously made the unexpected observation that laforin, in the absence of which polyglucosans accumulate, specifically binds polyglucosans. This suggested that laforin's role is to detect polyglucosan appearances during glycogen synthesis and to initiate mechanisms to downregulate GS. Glycogen synthase kinase 3 (GSK3) is the principal inhibitor of GS. Dephosphorylation of GSK3 at Ser 9 activates GSK3 to inhibit GS through phosphorylation at multiple sites. Glucose-6-phosphate is a potent allosteric activator of GS. Glucose-6-phosphate levels are high when the amount of glucose increases and its activation of GS overrides any phospho-inhibition. Here, we show that laforin is a GSK3 Ser 9 phosphatase, and therefore capable of inactivating GS through GSK3. We also show that laforin interacts with malin and that malin is an E3 ubiquitin ligase that binds GS. We propose that laforin, in response to appearance of polyglucosans, directs two negative feedback pathways: polyglucosan-laforin-GSK3-GS to inhibit GS activity and polyglucosan-laforin-malin-GS to remove GS through proteasomal degradation.

Carrier Proteins↗

PubMed2005

Expanded repeat in canine epilepsy.

Epilepsy afflicts 1% of humans and 5% of dogs. We report a canine epilepsy mutation and evidence for the existence of repeat-expansion disease outside humans. A canid-specific unstable dodecamer repeat in the Epm2b (Nhlrc1) gene recurrently expands, causing a fatal epilepsy and contributing to the high incidence of canine epilepsy. Tracing the repeat origins revealed two successive events, starting 50 million years ago, unique to canid evolution. A genetic test, presented here, will allow carrier and presymptomatic diagnosis and disease eradication. Clinicopathologic characterization establishes affected animals as a model for Lafora disease, the most severe teenage-onset human epilepsy.

Alleles↗

PubMed2004

Laforin preferentially binds the neurotoxic starch-like polyglucosans, which form in its absence in progressive myoclonus epilepsy.

Lafora disease (LD) is a fatal and the most common form of adolescent-onset progressive epilepsy. Fulminant endoplasmic reticulum (ER)-associated depositions of starch-like long-stranded, poorly branched glycogen molecules [known as polyglucosans, which accumulate to form Lafora bodies (LBs)] are seen in neuronal perikarya and dendrites, liver, skeletal muscle and heart. The disease is caused by loss of function of the laforin dual-specificity phosphatase or the malin E3 ubiquitin ligase. Towards understanding the pathogenesis of polyglucosans in LD, we generated a transgenic mouse overexpressing inactivated laforin to trap normal laforin's unknown substrate. The trap was successful and LBs formed in liver, muscle, neuronal perikarya and dendrites. Using immunogold electron microscopy, we show that laforin is found in close proximity to the ER surrounding the polyglucosan accumulations. In neurons, it compartmentalizes to perikaryon and dendrites and not to axons. Importantly, it binds polyglucosans, establishing for the first time a direct association between the disease-defining storage product and disease protein. It preferentially binds polyglucosans over glycogen in vivo and starch over glycogen in vitro, suggesting that laforin's role begins after the appearance of polyglucosans and that the laforin pathway is involved in monitoring for and then preventing the formation of polyglucosans. In addition, we show that the laforin interacting protein, EPM2AIP1, also localizes on the polyglucosan masses, and we confirm laforin's intense binding to LBs in human LD biopsy material.

Actins↗

PubMed2004

Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model.

The lack of an appropriate animal model with multiorgan pathology characteristic of the human form of cystic fibrosis has hampered our understanding of the pathobiology of the disease. We evaluated multiple organs of congenic C57BL/6J cystic fibrosis transmembrane regulator (Cftr)(-/-) and Cftr(+/+) mice maintained from weaning on a liquid diet then sacrificed between 1 and 24 months of age. The lungs of the Cftr(-/-) animals showed patchy alveolar overdistention, interstitial thickening, and fibrosis, with progression up to 6 months of age. The proximal and distal airway surface was encased with mucus-like material but lacked overt evidence of chronic bacterial infections or inflammation. All Cftr(-/-) animals showed progressive liver disease, with hepatosteatosis, focal cholangitis, inspissated secretions, and bile duct proliferation; after 1 year of age there was progression to focal biliary cirrhosis. The intercalated, intralobular and interlobular ducts and acinar lumina of the exocrine pancreas, the parotid and submaxillary glands of the Cftr(-/-) animals were dilated and filled with inspissated material, as well as mild inflammation and acinar cell drop out. Quantitative measurements of the pancreas showed significant acinar atrophy and increased acinar volume in comparison with age-matched Cftr(+/+) littermates. The ileal lumen and crypts were filled with adherent fibrillar material. After 3 months of age the vas deferens of the Cftr(-/-) animals could not be identified. None of the aforementioned pathological changes were observed in the Cftr(+/+) littermates fed the same liquid diet. We show, for the first time, that long-lived C578L/6J Cftr(-/-) mice develop manifestations of cystic fibrosis-like disease in all pathologically affected organs in the human form of cystic fibrosis.

Animals↗

PubMed2003

Abnormalities in villin gene expression and canalicular microvillus structure in progressive cholestatic liver disease of childhood.

BACKGROUND: The molecular basis of clinical cholestasis is a subject of intense investigation. Villin is an actin binding, bundling, and severing protein needed for maintenance of structural integrity of canalicular microvilli, in which membrane transporters required for bile secretion are located. We aimed to investigate the role of canalicular cytoskeletal proteins in three genetically unrelated children with a biliary atresia-like clinical disorder, each of whom developed liver failure requiring liver transplantation. METHODS: Explanted livers from the three patients were examined by standard pathological methods followed by transmission and cryoimmunoelectron microscopy. With archival tissue samples, a panel of cytoskeletal proteins was investigated by immunohistochemistry and western blotting, with purified canalicular membrane preparations. Villin mRNA analyses were undertaken on liver homogenates, with primers from coding regions of the human villin gene. Classic biliary atresia, other types of cholestasis, and normal livers served as controls. FINDINGS: In patients, pronounced ultrastructural deformities of canaliculi and especially of their microvilli were noted, which correlated with absence of villin protein by immunostaining of liver tissue sections and by western blot analysis. Additionally, villin mRNA was strikingly reduced or absent. These results differed greatly from those in controls. INTERPRETATION: These results suggest that the disorder described mimics biliary atresia, but structural and molecular pathological findings differ. We propose that a functional abnormality in villin gene expression is key to the mechanism of cholestasis in patients with progressive cholestasis and hepatic failure.

Biliary Atresia↗

PubMed2003

Mutations in NHLRC1 cause progressive myoclonus epilepsy.

Lafora progressive myoclonus epilepsy is characterized by pathognomonic endoplasmic reticulum (ER)-associated polyglucosan accumulations. We previously discovered that mutations in EPM2A cause Lafora disease. Here, we identify a second gene associated with this disease, NHLRC1 (also called EPM2B), which encodes malin, a putative E3 ubiquitin ligase with a RING finger domain and six NHL motifs. Laforin and malin colocalize to the ER, suggesting they operate in a related pathway protecting against polyglucosan accumulation and epilepsy.

Base Sequence↗

PubMed2003

Presenilin-1, nicastrin, amyloid precursor protein, and gamma-secretase activity are co-localized in the lysosomal membrane.

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.

Alzheimer Disease↗

PubMed2003

Unusual presentation of Lafora's disease.

Lafora's disease is a progressive myoclonus epilepsy with onset in adolescence and a gradual decline in cognitive functions and increase in seizure intractability. We present the case of a 16-year-old with precipitous dementia within 6 months of onset. Peripheral biopsies and EPM2A mutation analysis were negative. The diagnosis could be established only by brain biopsy.

Adolescent↗

PubMed2002

Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion.

Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.

Animals↗