Sonja Forss-Petter

TARGETED INACTIVATION OF THE X-LINKED ADRENOLEUKODYSTROPHY GENE IN MICE

In its severe form, X‐linked adrenoleukodystrophy (ALD) is a lethal neurologic disease of children, characterized by progressive cerebral demyelination and adrenal insufficiency. Associated with a biochemical defect of peroxisomal β‐oxidation, very long‐chain fatty acids (VLCFA) build up in tissues that have a high turnover of lipids, such as central nervous system (CNS) white matter, adrenal cortex, and testis. Whether the abnormal accumulation of VLCFA is the underlying cause of demyelination or merely an associated biochemical marker is unknown. ALD is caused by mutations in the gene for a peroxisomal membrane protein (ALDP) that shares structural features with ATP‐binding‐cassette (ABC) transporters. To analyze the cellular function of ALDP and to obtain an animal model of this debilitating disease, we have generated transgenic mice with a targeted inactivation of the ald gene. Motor functions in ALDP‐deficient mice developed at schedule, and unexpectedly, adult animals appeared unaffected by neurologic symptoms up to at least 6 months of age. Biochemical analyses demonstrated impaired β‐oxidation in mutant fibroblasts and abnormal accumulation of VLCFAs in the CNS and kidney. In 6‐month‐old mutants, adrenal cortex cells displayed a ballooned morphology and needle‐like lipid inclusions, also found in testis and ovaries. However, lipid inclusions and demyelinating lesions in the CNS were not a feature. Thus, complete absence of ALDP expression results in a VLCFA storage disease but does not impair CNS function of young adult mice by pathologic and clinical criteria. This suggests that additional genetic or environmental conditions must be fulfilled to model the early‐onset and lethality of cerebral ALD in transgenic mice. J. Neurosci. Res. 50:829–843, 1997. © 1997 Wiley‐Liss, Inc.

T-Cell Apoptosis in Inflammatory Brain Lesions : Destruction of T Cells Does Not Depend on Antigen Recognition

Elimination of inflammatory T cells by apoptosis appears to play an important role in the down-regulation of inflammation in the central nervous system. Here we report that apoptosis of T lymphocytes occurs to a similar extent in different models of autoimmune encephalomyelitis. Apoptosis is restricted to cells located in the neuroectodermal parenchyma, thereby leaving T cells present in the brains connective tissue compartments unharmed. Death of T cells in the parenchyma does not depend on antigen presentation by resident microglial cells or astrocytes. Adoptive transfer experiments with T lymphocytes carrying a specific genetic marker revealed that in the central nervous system these cells are destroyed regardless of their antigen specificity or state of activation. Although many of both antigen-dependent and -independent mechanisms in the induction of T-cell apoptosis may act simultaneously, our results suggest that the nervous system harbors a specific, currently undefined, mechanism that effectively eliminates infiltrating T lymphocytes.

Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus

The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire.

Peroxisomal alterations in Alzheimer’s disease

In Alzheimer’s disease (AD), lipid alterations are present early during disease progression. As some of these alterations point towards a peroxisomal dysfunction, we investigated peroxisomes in human postmortem brains obtained from the cohort-based, longitudinal Vienna-Transdanube Aging (VITA) study. Based on the neuropathological Braak staging for AD on one hemisphere, the patients were grouped into three cohorts of increasing severity (stages I–II, III–IV, and V–VI, respectively). Lipid analyses of cortical regions from the other hemisphere revealed accumulation of C22:0 and very long-chain fatty acids (VLCFA, C24:0 and C26:0), all substrates for peroxisomal β-oxidation, in cases with stages V–VI pathology compared with those modestly affected (stages I–II). Conversely, the level of plasmalogens, which need intact peroxisomes for their biosynthesis, was decreased in severely affected tissues, in agreement with a peroxisomal dysfunction. In addition, the peroxisomal volume density was increased in the soma of neurons in gyrus frontalis at advanced AD stages. Confocal laser microscopy demonstrated a loss of peroxisomes in neuronal processes with abnormally phosphorylated tau protein, implicating impaired trafficking as the cause of altered peroxisomal distribution. Besides the original Braak staging, the study design allowed a direct correlation between the biochemical findings and the amount of neurofibrillary tangles (NFT) and neuritic plaques, quantified in adjacent tissue sections. Interestingly, the decrease in plasmalogens and the increase in VLCFA and peroxisomal volume density in neuronal somata all showed a stronger association with NFT than with neuritic plaques. These results indicate substantial peroxisome-related alterations in AD, which may contribute to the progression of AD pathology.

Current and Future Pharmacological Treatment Strategies in X-Linked Adrenoleukodystrophy

Mutations in the ABCD1 gene cause the clinical spectrum of the neurometabolic disorder X‐linked adrenoleukodystrophy/adrenomyeloneuropathy (X‐ALD/AMN). Currently, the most efficient therapeutic opportunity for patients with the cerebral form of X‐ALD is hematopoietic stem cell transplantation and possibly gene therapy of autologous hematopoietic stem cells. Both treatments, however, are only accessible to a subset of X‐ALD patients, mainly because of the lack of markers that can predict the onset of cerebral demyelination. Moreover, for female or male X‐ALD patients with AMN, currently only unsatisfying therapeutic opportunities are available. Thus, this review focuses on current and urgently needed future pharmacological therapies. The treatment of adrenal and gonadal insufficiency is well established, whereas applications of immunomodulatory and immunosuppressive drugs have failed to prevent progression of cerebral neuroinflammation. The use of Lorenzos oil and the inefficacy of lovastatin to normalize very‐long‐chain fatty acids in clinical trials as well as currently experimental and therefore possible future therapeutic strategies are reviewed. The latter include pharmacological gene therapy mediated by targeted upregulation of ABCD2, the closest homolog of ABCD1, antioxidative drug treatment, small molecule histone deacetylase inhibitors such as butyrates and valproic acid, and other neuroprotective attempts.

Leukodystrophies: recent developments in genetics, molecular biology, pathogenesis and treatment.

The combined application of recently developed techniques for genetic and biochemical analysis, neuroimaging and the ability to create animal models has led to remarkable advances in the field of leukodystrophy research. The present review focuses on recent developments in X-linked adrenoleukodystrophy, Alexanders disease, Canavans disease, metachromatic leukodystrophy, globoid cell leukodystrophy (Krabbes disease) and Pelizaeus-Merzbacher disease, and briefly discusses new data on six other rare inherited leukodystrophies. Of the leukodystrophies, 12 can now be diagnosed precisely using noninvasive techniques, and the molecular defect has been identified in nine of these. Disease incidence can be reduced through genetic counselling. Presymptomatic diagnosis provides an opportunity for therapeutic intervention. Study of animal models facilitates elucidation of pathogenic mechanisms and identifies pathways that could be targeted by future therapies.

Impaired very long-chain acyl-CoA β-oxidation in human X-linked adrenoleukodystrophy fibroblasts is a direct consequence of ABCD1 transporter dysfunction.

Background: ABCD1 is a peroxisomal ABC transporter whose dysfunction causes X-linked adrenoleukodystrophy (X-ALD). Results: β-Oxidation of C26:0 and C22:0 acyl-CoA esters is impaired in X-ALD. ABCD3 accounts for residual β-oxidation activity in X-ALD fibroblasts. Conclusion: ABCD1 mediates very long-chain acyl-CoA ester β-oxidation without need for additional re-esterification by an acyl-CoA synthetase. Significance: Our study provides proof of deficient acyl-CoA ester β-oxidation in X-ALD. X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.

Peroxisomes in brain development and function

Peroxisomes contain numerous enzymatic activities that are important for mammalian physiology. Patients lacking either all peroxisomal functions or a single enzyme or transporter function typically develop severe neurological deficits, which originate from aberrant development of the brain, demyelination and loss of axonal integrity, neuroinflammation or other neurodegenerative processes. Whilst correlating peroxisomal properties with a compilation of pathologies observed in human patients and mouse models lacking all or individual peroxisomal functions, we discuss the importance of peroxisomal metabolites and tissue- and cell type-specific contributions to the observed brain pathologies. This enables us to deconstruct the local and systemic contribution of individual metabolic pathways to specific brain functions. We also review the recently discovered variability of pathological symptoms in cases with unexpectedly mild presentation of peroxisome biogenesis disorders. Finally, we explore the emerging evidence linking peroxisomes to more common neurological disorders such as Alzheimer’s disease, autism and amyotrophic lateral sclerosis. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.

Liver X Receptor α Interferes with SREBP1c-mediated Abcd2 Expression NOVEL CROSS-TALK IN GENE REGULATION

The peroxisomal ATP binding cassette (ABC) transporter adrenoleukodystrophy-related protein, encoded by ABCD2, displays functional redundancy with the X-linked adrenoleukodystrophy-associated protein, making ABCD2 up-regulation of therapeutic value. Cholesterol lowering activates human ABCD2 in cultured cells. To investigate in vivo regulation by sterols, we first characterized a sterol regulatory element (SRE) in the murine Abcd2 promoter that is directly bound by SRE-binding proteins (SREBPs). Intriguingly, this element overlaps with a direct repeat 4, which serves as binding site for liver X receptor (LXR)/retinoid X receptor heterodimers, suggesting novel cross-talk between SREBP and LXR/retinoid X receptor in gene regulation. Using fasting-refeeding and cholesterol loading, SREBP accessibility to the SRE/direct repeat 4 was tested. Results suggest that adipose Abcd2 is induced by SREBP1c, whereas hepatic Abcd2 expression is down-regulated by concurrent activation of LXRα and SREBP1c. In cell culture, SREBP1c-mediated Abcd2 induction is counteracted by ligand-activated LXRα. Finally, hepatic Abcd2 expression in LXRα,β-deficient mice is inducible to levels vastly exceeding wild type. Together, we identify LXRα as negative modulator of Abcd2, acting through a novel regulatory mechanism involving overlapping SREBP and LXRα binding sites.

cDNA cloning and mRNA distribution of a mouse very long-chain acyl-CoA synthetase

The interaction of the adrenoleukodystrophy protein (ALDP), mutated in the peroxisomal disorder X‐linked adrenoleukodystrophy, and the very long‐chain acyl‐CoA synthetase (VLACS), the enzyme whose function is missing in this disease, remains obscure. As a first step to studying this interaction in wild type versus ALDP‐deficient mice, we have cloned a VLACS cDNA from mouse liver. The 1860 bp open reading frame encodes a 620 amino acid protein with a predicted molecular mass of 70.3 kDa. By Northern blot analysis, a 2.6 kbp VLACS mRNA was highly abundant in liver and kidney and present at low levels in brain and testes. By RT‐PCR VLACS mRNA was also detected in heart and lung but remained undetectable in skeletal muscle and spleen. In contrast to the peroxisomal β‐oxidation marker acyl‐CoA oxidase, whose mRNA level steadily increases during brain development, the VLACS transcript was found at a constant low level from embryo through adulthood, suggesting that additional isoforms may exist in brain.