Renate Lüllmann-Rauch

Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficientmice

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danons disease is associated with the accumulation of autophagic material in striated myocytes.

Drug-Induced Phospholipidoses

This review deals with drug-induced lipidoses and the possible underlying mechanisms. A variety of drugs, widely differing in their main pharmacological actions, but closely resembling each other with respect to particular physiochemical properties, have been found to cause in animals and man cytological alterations that are reminiscent of the inherited lipid-storage diseases of man. Ultrastructurally, the alterations are characterized by multilamellated or crystalloid cytoplasmic inclusions, i.e., residual bodies resulting from an intralysosomal accumulation of polar lipids. Biochemically, some of the most severely affected tissues have been demonstrated to contain increased amounts of phospholipids and of the drug applied. All drugs hitherto found to induce such alterations are cationic amphiphilic (amphipathic) compounds; they possess a highly apolar ring system and a cationic hydrophilic side chain (e.g., triparanol, chloroquine, chlorzyclizine, 4,4′-diethylaminoethoxyhexestrol, chlorphentermine, ipri...

Array-Based Gene Discovery with Three Unrelated Subjects Shows SCARB2/LIMP-2 Deficiency Causes Myoclonus Epilepsy and Glomerulosclerosis

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.

Normal Lysosomal Morphology and Function in LAMP-1-deficient Mice

Lysosomal membranes contain two highly glycosylated proteins, designated LAMP-1 and LAMP-2, as major components. LAMP-1 and LAMP-2 are structurally related. To investigate the physiological role of LAMP-1, we have generated mice deficient for this protein. LAMP-1-deficient mice are viable and fertile. In LAMP-1-deficient brain, a mild regional astrogliosis and altered immunoreactivity against cathepsin-D was observed. Histological and ultrastructural analyses of all other tissues did not reveal abnormalities. Lysosomal properties, such as enzyme activities, lysosomal pH, osmotic stability, density, shape, and subcellular distribution were not changed in comparison with controls. Western blot analyses of LAMP-1-deficient and heterozygote tissues revealed an up-regulation of the LAMP-2 protein pointing to a compensatory effect of LAMP-2 in response to the LAMP-1 deficiency. The increase of LAMP-2 was neither correlated with an increase in the level oflamp-2 mRNAs nor with increased half-life time of LAMP-2. This findings suggest a translational regulation of LAMP-2 expression.

RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction.

AIMS Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. METHODS AND RESULTS Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3(-/-)) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. CONCLUSION Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.

The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling

The disintegrin and metalloproteinase Adam10 has been implicated in the regulation of key signaling pathways that determine skin morphogenesis and homeostasis. To address the in vivo relevance of Adam10 in the epidermis, we have selectively disrupted Adam10 during skin morphogenesis and in adult skin. K14-Cre driven epidermal Adam10 deletion leads to perinatal lethality, barrier impairment and absence of sebaceous glands. A reduction of spinous layers, not associated with differences in either proliferation or apoptosis, indicates that loss of Adam10 triggers a premature differentiation of spinous keratinocytes. The few surviving K14-Adam10-deleted mice and mice in which Adam10 was deleted postnatally showed loss of hair, malformed vibrissae, epidermal hyperproliferation, cyst formation, thymic atrophy and upregulation of the cytokine thymic stromal lymphopoetin (TSLP), thus indicating non cell-autonomous multi-organ disease resulting from a compromised barrier. Together, these phenotypes closely resemble skin specific Notch pathway loss-of-function phenotypes. Notch processing is indeed strongly reduced resulting in decreased levels of Notch intracellular domain fragment and functional Notch signaling. The data identify Adam10 as the major Site-2 processing enzyme for Notch in the epidermis in vivo, and thus as a central regulator of skin development and maintenance.

Chlorphentermine-induced lipidosislike ultrastructural alterations in lungs and adrenal glands of several species.

Abstract Prolonged administration of the anorectic drug chlorphentermine causes an accumulation of “foam cells” in pulmonary alveoli of guinea pigs, mice, and rabbits. Ultrastructurally, the “foam cells” correspond to enlarged alveolar macrophages, packed with lamellated cytoplasmic inclusions. Lamellar bodies of the same type also occur in increased numbers within nearly all other pulmonary cell types and in the cells of adrenal cortex of all three animal species. These cellular alterations are interpreted to result from an intralysosomal accumulation of phospholipids. In view of the present findings and of the bulk of experimental evidence from the literature, it is concluded that the presence of alveolar “foam cells” in animals treated with an amphiphilic drug is not an isolated occurrence, but rather an indication for a generalized phenomenon, which is believed to be due to a drug-induced phospholipidosis.

Deficiency of the Tetraspanin CD63 Associated with Kidney Pathology but Normal Lysosomal Function

ABSTRACT CD63 is a member of the tetraspanin superfamily that constitutes a main component of the lysosomal membrane. In mice, two CD63 gene loci are present, with only one of these two being functional. We generated and analyzed mice deficient for active CD63. Disruption of CD63 results in a complete loss of CD63 protein expression. Despite its abundance in late endosomes/lysosomes, the lack of CD63 does not cause obvious endosomal/lysosomal abnormalities. CD63 knockout mice are viable and fertile without gross morphological abnormalities in the majority of tissues. No alterations in the populations of immune cells and only minor differences in platelet function were observed. This suggests that the lack of CD63 could be successfully compensated for, most likely by other tetraspanins. However, CD63 deficiency leads to an altered water balance. CD63 knockout mice show an increased urinary flow, water intake, reduced urine osmolality, and a higher fecal water content. In principle cells of the collecting duct of CD63-deficient mice, abnormal intracellular lamellar inclusions were observed. This indicates that the sorting of apical transport proteins might be impaired in these cells. CD63 knockout mice provide an important tool for analyzing the various postulated functions of CD63 in vivo.

Metachromatic leukodystrophy: Molecular genetics and an animal model

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA; EC 3.1.6.8). Deficiency of this enzyme causes intralysosomal storage of the sphingolipid cerebroside sulphate. This lipid is abundant in myelin and it may thus not be surprising that storage mainly affects oligodendrocytes. Patients suffer from a progressive demyelination causing various neurological symptoms. The disease is fatal and treatment is not available. The human ASA gene has been cloned and more than 40 mutations have been analysed that cause metachromatic leukodystrophy. Few of these alleles are frequent among patients, whereas most mutant alleles have only been found in single families. Since MLD has only been described in humans and no naturally occurring animal model has been described, ASA-deficient mice have been generated by homologous recombination. The ASA knockout mice are unable to degrade sulphatide and store the lipid intralysosomally. The pattern of lipid storage in neuronal and nonneuronal tissues resembles that described for patients. In the nervous system, lipid storage is found in oligodendrocytes, astrocytes and some neurons. Animals display an astrogliosis and a decreased average axonal diameter. Purkinje cells and Bergmann glia of the cerebellum are morphologically aberrant. Demyelination is seen in the acoustic ganglion and occurs between the ages of 6 and 12 months. The animals are deaf at this age and display various neuromotor abnormalities. However, compared to humans the mice have a surprisingly mild phenotype, since they have a normal life span and do not develop widespread demyelination. ASA-deficient mice have been transplanted with bone marrow, which was transduced with a retroviral vector expressing arylsulphatase A. The majority of transplanted animals display sustained expression of arylsulphatase A from the retroviral construct up to 5 months after transplantation. However, preliminary data suggest that this therapeutic approach does not reduce storage material.

Bone marrow stem cell-based gene transfer in a mouse model for metachromatic leukodystrophy: effects on visceral and nervous system disease manifestations

Arylsulfatase A (ASA) knockout mice represent an animal model for the lysosomal storage disease metachromatic leukodystrophy (MLD). Stem cell gene therapy with bone marrow overexpressing the human ASA cDNA from a retroviral vector resulted in the expression of high enzyme levels in various tissues. Treatment partially reduces sulfatide storage in livers exceeding 18 ng ASA/mg tissue, while complete reduction was observed in livers exceeding 50 ng ASA/mg tissue. This corresponds to about 80% and 200% of normal enzyme activity. Similar values seem to apply for kidney. A partial correction of the lipid metabolism was detectable in the brain where the galactoerebroside/sulfatide ratio, which is diminished in ASA-deficient mice, increased upon treatment. This partial correction was accompanied by amelioration of neuropathology; axonal cross-sectional areas, which are reduced in deficient mice, were significantly increased in the saphenic and sciatic nerve but not in the optic nerve. Behavioral tests suggest some improvement of neuromotor abilities. The gene transfer did not delay the degeneration occurring in the acoustic ganglion of ASA-deficient animals. The limited success of the therapy appears to be due to the requirement of unexpected high levels of ASA for correction of the metabolic defect.