Agatha Schlüter

Antioxidants halt axonal degeneration in a mouse model of X-adrenoleukodystrophy†

Axonal degeneration is a main contributor to disability in progressive neurodegenerative diseases in which oxidative stress is often identified as a pathogenic factor. We aim to demonstrate that antioxidants are able to improve axonal degeneration and locomotor deficits in a mouse model of X‐adrenoleukodystrophy (X‐ALD).

PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome

Peroxisomes are essential organelles that play a key role in redox signalling and lipid homeostasis. They contain a highly diverse enzymatic network among different species, mirroring the varied metabolic needs of the organisms. The previous PeroxisomeDB version organized the peroxisomal proteome of humans and Saccharomyces cerevisiae based on genetic and functional information into metabolic categories with a special focus on peroxisomal disease. The new release (http://www.peroxisomeDB.org) adds peroxisomal proteins from 35 newly sequenced eukaryotic genomes including fungi, yeasts, plants and lower eukaryotes. We searched these genomes for a core ensemble of 139 peroxisomal protein families and identified 2706 putative peroxisomal protein homologs. Approximately 37% of the identified homologs contained putative peroxisome targeting signals (PTS). To help develop understanding of the evolutionary relationships among peroxisomal proteins, the new database includes phylogenetic trees for 2386 of the peroxisomal proteins. Additional new features are provided, such as a tool to capture kinetic information from Brenda, CheBI and Sabio-RK databases and more than 1400 selected bibliographic references. PeroxisomeDB 2.0 is a freely available, highly interactive functional genomics platform that offers an extensive view on the peroxisomal metabolome across lineages, thus facilitating comparative genomics and systems analysis of the organelle.

A key role for the peroxisomal ABCD2 transporter in fatty acid homeostasis.

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimers and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders.

Neuroinflammatory signals in alzheimer disease and APP/PS1 transgenic mice: Correlations with plaques, tangles, and oligomeric species

Abstract To understand neuroinflammation-related gene regulation during normal aging and in sporadic Alzheimer disease (sAD), we performed functional genomics analysis and analyzed messenger RNA (mRNA) expression by quantitative reverse transcription–polymerase chain reaction of 22 genes involved in neuroinflammation-like responses in the cerebral cortex of wild-type and APP/PS1 transgenic mice. For direct comparisons, mRNA expression of 18 of the same genes was then analyzed in the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 of middle-aged human subjects lacking Alzheimer disease–related pathology and in older subjects with sAD pathology covering Stages I–II/0(A), III–IV/A–B, and V–VI/C of Braak and Braak classification. Modifications of cytokine and immune mediator mRNA expression were found with normal aging in wild-type mice and in middle-aged individuals and patients with early stages of sAD-related pathology; these were accompanied by increased protein expression of certain mediators in ramified microglia. In APP/PS1 mice, inflammatory changes coincided with &bgr;-amyloid (A&bgr;) deposition; increased levels of soluble oligomers paralleled the modified mRNA expression of cytokines and mediators in wild-type mice. In patients with sAD, regulation was stage- and region-dependent and not merely acceleration and exacerbation of mRNA regulation with aging. Gene regulation at first stages of AD was not related to hyperphosphorylated tau deposition in neurofibrillary tangles, A&bgr; plaque burden, concentration of A&bgr;1–40 (A&bgr;40) and A&bgr;1–42 (A&bgr;42), or fibrillar A&bgr; linked to membranes but rather to increased levels of soluble oligomers. Thus, species differences and region- and stage-dependent inflammatory responses in sAD, particularly at the initial stages, indicate the need to identify new anti-inflammatory compounds with specific molecular therapeutic targets.

PeroxisomeDB: a database for the peroxisomal proteome, functional genomics and disease

Peroxisomes are essential organelles of eukaryotic origin, ubiquitously distributed in cells and organisms, playing key roles in lipid and antioxidant metabolism. Loss or malfunction of peroxisomes causes more than 20 fatal inherited conditions. We have created a peroxisomal database () that includes the complete peroxisomal proteome of Homo sapiens and Saccharomyces cerevisiae, by gathering, updating and integrating the available genetic and functional information on peroxisomal genes. PeroxisomeDB is structured in interrelated sections ‘Genes’, ‘Functions’, ‘Metabolic pathways’ and ‘Diseases’, that include hyperlinks to selected features of NCBI, ENSEMBL and UCSC databases. We have designed graphical depictions of the main peroxisomal metabolic routes and have included updated flow charts for diagnosis. Precomputed BLAST, PSI-BLAST, multiple sequence alignment (MUSCLE) and phylogenetic trees are provided to assist in direct multispecies comparison to study evolutionary conserved functions and pathways. Highlights of the PeroxisomeDB include new tools developed for facilitating (i) identification of novel peroxisomal proteins, by means of identifying proteins carrying peroxisome targeting signal (PTS) motifs, (ii) detection of peroxisomes in silico, particularly useful for screening the deluge of newly sequenced genomes. PeroxisomeDB should contribute to the systematic characterization of the peroxisomal proteome and facilitate system biology approaches on the organelle.

Oxidative Damage Compromises Energy Metabolism in the Axonal Degeneration Mouse Model of X-Adrenoleukodystrophy

AIMS Chronic metabolic impairment and oxidative stress are associated with the pathogenesis of axonal dysfunction in a growing number of neurodegenerative conditions. To investigate the intertwining of both noxious factors, we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits axonal degeneration in spinal cords and motor disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. Oxidative stress due to VLCFA excess appears early in the neurodegenerative cascade. RESULTS In this study, we demonstrate by redox proteomics that oxidative damage to proteins specifically affects five key enzymes of glycolysis and TCA (Tricarboxylic acid) cycle in spinal cords of Abcd1(-) mice and pyruvate kinase in human X-ALD fibroblasts. We also show that NADH and ATP levels are significantly diminished in these samples, together with decrease of pyruvate kinase activities and GSH levels, and increase of NADPH. INNOVATION Treating Abcd1(-) mice with the antioxidants N-acetylcysteine and α-lipoic acid (LA) prevents protein oxidation; preserves NADH, NADPH, ATP, and GSH levels; and normalizes pyruvate kinase activity, which implies that oxidative stress provoked by VLCFA results in bioenergetic dysfunction, at a presymptomatic stage. CONCLUSION Our results provide mechanistic insight into the beneficial effects of antioxidants and enhance the rationale for translation into clinical trials for X-adrenoleukodystrophy.

Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain.

Parkinson disease (PD) is no longer considered a complex motor disorder but rather a systemic disease with variable nonmotor deficits that may include impaired olfaction, depression, mood and sleep disorders, and altered cortical function. Increasing evidence indicates that multiple metabolic defects occur in regions outside the substantia nigra, including the cerebral cortex, even at premotor stages of the disease. We investigated changes in gene expression in the frontal cortex in PD patient brains using a transcriptomics approach. Functional genomics analysis indicated that cortical olfactory receptors (ORs) and taste receptors (TASRs) are altered in PD patients. Olfactory receptors OR2L13, OR1E1, OR2J3, OR52L1, and OR11H1 and taste receptors TAS2R5 and TAS2R50 were downregulated, but TAS2R10 and TAS2R13 were upregulated at premotor and parkinsonian stages in the frontal cortex area 8 in PD patient brains. Furthermore, we present novel evidence that, in addition to the ORs, obligate downstream components of OR function adenylyl cyclase 3 and olfactory G protein (Gαolf), OR transporters, receptor transporter proteins 1 and 2 and receptor expression enhancing protein 1, and OR xenobiotic removing UDP-glucuronosyltransferase 1 family polypeptide A6 are widely expressed in neurons of the cerebral cortex and other regions of the adult human brain. Together, these findings support the concept that ORs and TASRs in the cerebral cortex may have novel physiologic functions that are affected in PD patients.

Functional genomic analysis unravels a metabolic-inflammatory interplay in adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder characterized by axonopathy and demyelination in the central nervous system and adrenal insufficiency. Main X-ALD phenotypes are: (i) an adult adrenomyeloneuropathy (AMN) with axonopathy in spinal cords, (ii) cerebral AMN with brain demyelination (cAMN) and (iii) a childhood variant, cALD, characterized by severe cerebral demyelination. Loss of function of the ABCD1 peroxisomal fatty acid transporter and subsequent accumulation of very-long-chain fatty acids (VLCFAs) are the common culprits to all forms of X-ALD, an aberrant microglial activation accounts for the cerebral forms, whereas inflammation allegedly plays no role in AMN. How VLCFA accumulation leads to neurodegeneration and what factors account for the dissimilar clinical outcomes and prognosis of X-ALD variants remain elusive. To gain insights into these questions, we undertook a transcriptomic approach followed by a functional-enrichment analysis in spinal cords of the animal model of AMN, the Abcd1− null mice, and in normal-appearing white matter of cAMN and cALD patients. We report that the mouse model shares with cAMN and cALD a common signature comprising dysregulation of oxidative phosphorylation, adipocytokine and insulin signaling pathways, and protein synthesis. Functional validation by quantitative polymerase chain reaction, western blots and assays in spinal cord organotypic cultures confirmed the interplay of these pathways through IkB kinase, being VLCFA in excess a causal, upstream trigger promoting the altered signature. We conclude that X-ALD is, in all its variants, a metabolic/inflammatory syndrome, which may offer new targets in X-ALD therapeutics.

Deregulation of purine metabolism in Alzheimer's disease

The neuroprotective role of adenosine and the deregulation of adenosine receptors in Alzheimers disease (AD) have been extensively studied in recent years. However, little is known about the involvement of purine metabolism in AD. We started by analyzing gene expression in the entorhinal cortex of human controls and AD cases with whole-transcript expression arrays. Once we identified deregulation of the cluster purine metabolism, messenger RNA expression levels of 23 purine metabolism genes were analyzed with qRT-PCR in the entorhinal cortex, frontal cortex area 8, and precuneus at stages I-II, III-IV, and V-VI of Braak and Braak and controls. APRT, DGUOK, POLR3B, ENTPD3, AK5, NME1, NME3, NME5, NME7, and ENTPD2 messenger RNAs were deregulated, with regional variations, in AD cases when compared with controls. In addition, liquid chromatography mass spectrometry based metabolomics in the entorhinal cortex identified altered levels of dGMP, glycine, xanthosine, inosine diphosphate, guanine, and deoxyguanosine, all implicated in this pathway. Our results indicate stage- and region-dependent deregulation of purine metabolism in AD.

Oxidative stress regulates the ubiquitin–proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy

Oxidative damage is a pivotal aetiopathogenic factor in X-linked adrenoleukodystrophy. This is a neurometabolic disease characterized by the accumulation of very-long-chain fatty acids owing to the loss of function of the peroxisomal transporter Abcd1. Here, we used the X-linked adrenoleukodystrophy mouse model and patients fibroblasts to detect malfunctioning of the ubiquitin-proteasome system resulting from the accumulation of oxidatively modified proteins, some involved in bioenergetic metabolism. Furthermore, the immunoproteasome machinery appears upregulated in response to oxidative stress, in the absence of overt inflammation. i-Proteasomes are recruited to mitochondria when fibroblasts are exposed to an excess of very-long-chain fatty acids in response to oxidative stress. Antioxidant treatment regulates proteasome expression, prevents i-proteasome induction and translocation of i-proteasomes to mitochondria. Our findings support a key role of i-proteasomes in quality control in mitochondria during oxidative damage in X-linked adrenoleukodystrophy, and perhaps in other neurodegenerative conditions with similar pathogeneses.