Cytosolic persistence of mouse brain CYP1A1 in chronic heme deficiency

Previous work has demonstrated that the function of extrahepatic cytochrome P450 CYP1A1 is dependent on the availability of heme. CYP1A1 is involved in the activation of polyaromatic hydrocarbons. In the present study we used a transgenic mouse model with chronic impairment of heme synthesis - female porphobilinogen deaminase-deficient (PBGD-/-) mice - to investigate the effects of limited heme in untreated and β-naphthoflavone (β-NF)-treated animals on the function of CYP1A1 in brain. The heme content of PBGD-/- mice was diminished in the liver and brain compared to wild types. In the liver, partial heme deficiency led to less potent induction of CYP1A1 mRNA after β-NF treatment. In the brain, CYP1A1 protein was detected not only at the endoplasmic reticulum (ER), but also in the cytosol of PBGD-/- mice. Furthermore, 7-deethylation of ethoxyresorufin, an indicator of CYP1A1 metabolic activity, could be restored by heme in cytosol of PBGD-/- mouse brain. Independent of the genotype, we found only one cyp1a1 gene product, indicating that the cytosolic appearance of CYP1A1 most likely did not originate from mutant alleles. We conclude that heme deficiency in the brain leads to incomplete heme saturation of CYP1A1, which causes its improper incorporation into the ER membrane and persistence in the cytosol. It is suggested that diseases caused by relative heme deficiency, such as hepatic porphyrias, may lead to impaired hemoprotein function in brai

Neutralizing antibodies against IFN‐β in multiple sclerosis: antagonization of IFN‐β mediated suppression of MMPs

Neutralizing antibodies (NAb) against interferon‐β (IFN‐β) develop in about a third of treated multiple sclerosis patients and are believed to reduce therapeutic efficacy of IFN‐β on clinical and MRI measures. The expression of the interferon acute‐response protein, myxovirus resistance protein A (MxA) is a sensitive measure of the biological activity of therapeutically applied IFN‐β and of its reduced bioavailability due to NAb. However, MxA may not be operative in the pathogenesis of multiple sclerosis or the therapeutic effect of IFN‐β. Instead, matrix metalloproteinases (MMPs) are increased in brain tissue, CSF and blood circulation of multiple sclerosis patients and function as effector molecules in several steps of multiple sclerosis pathogenesis. One of the molecular mechanisms by which IFN‐β exerts its beneficial effect in multiple sclerosis is reduction of MMP‐9 expression and increase of its endogenous tissue inhibitor, TIMP‐1. Quantitative PCR measurements of MMP‐2 and MMP‐9, TIMP‐1 and TIMP‐2, and MxA were performed in peripheral mononuclear cells from clinically stable multiple sclerosis patients with relapsing remitting disease course after short‐term and long‐term treatment with IFN‐β. IFN‐β therapy down‐regulated the expression of MMP‐9 and abolished that of MMP‐2 in long‐term, but not short‐term treated multiple sclerosis, while levels of MxA were increased in both instances. The presence of NAb reversed these effects, i.e. led to reduced MxA and increased MMP‐2/MMP‐9 expression levels compared with NAb- patients. In contrast, expression of TIMPs in peripheral blood mononuclear cells remained unaffected by IFN‐β therapy and the presence of NAb. While MxA is able to detect the biological action and reduced bioavailability of IFN‐β on the basis of single injections, only MMP‐9 shows quantitative correlation with the NAb titre. Together with evidence that an imbalance between MMP and TIMP expression is a crucial pathogenetic feature in multiple sclerosis, these findings support the concept of a significant role of NAb in reducing the therapeutic efficacy of IFN‐

Genome-wide association analysis of susceptibility and clinical phenotype in multiple sclerosis

Multiple sclerosis (MS), a chronic disorder of the central nervous system and common cause of neurological disability in young adults, is characterized by moderate but complex risk heritability. Here we report the results of a genome-wide association study performed in a 1000 prospective case series of well-characterized individuals with MS and group-matched controls using the Sentrix® HumanHap550 BeadChip platform from Illumina. After stringent quality control data filtering, we compared allele frequencies for 551 642 SNPs in 978 cases and 883 controls and assessed genotypic influences on susceptibility, age of onset, disease severity, as well as brain lesion load and normalized brain volume from magnetic resonance imaging exams. A multi-analytical strategy identified 242 susceptibility SNPs exceeding established thresholds of significance, including 65 within the MHC locus in chromosome 6p21.3. Independent replication confirms a role for GPC5, a heparan sulfate proteoglycan, in disease risk. Gene ontology-based analysis shows a functional dichotomy between genes involved in the susceptibility pathway and those affecting the clinical phenotyp

Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis

Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. In the context of neuro-inflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system. This review focuses on the role of MMPs in multiple sclerosis (MS) and bacterial meningitis (BM), two neuro-inflammatory diseases where current therapeutic approaches are insufficient to prevent severe disability in the majority of patients. Inhibition of enzymatic activity may prevent MMP-mediated neuronal damage due to an overactive or deviated immune response in both diseases. Downregulation of MMP release may be the molecular basis for the beneficial effect of IFN-beta and steroids in MS. Instead, synthetic MMP inhibitors offer the possibility to shut off enzymatic activity of already activated MMPs. In animal models of MS and BM, they efficiently attenuated clinical disease symptoms and prevented brain damage due to excessive metalloproteinase activity. However, the required target profile for the therapeutic use of this novel group of compounds in human disease is not yet sufficiently defined and may be different depending on the type and stage of disease. Currently available MMP inhibitors show little target-specificity within the MMP family and may lead to side-effects due to interference with physiological functions of MMPs. Results from human MS and BM indicate that only a restricted number of MMPs specific for each disease is up-regulated. MMP inhibitors with selective target profiles offer the possibility of a more efficient therapy of MS and BM and may enter clinical trials in the near future

Natalizumab-induced POU2AF1/Spi-B upregulation: A possible route for PML development

OBJECTIVES: To assess messenger RNA (mRNA) expression of POU2AF1 and Spi-B and their potential regulatory microRNAs (miRNAs) in natalizumab-treated patients with multiple sclerosis and in therapy-associated progressive multifocal leukoencephalopathy (PML). METHODS: Expression of POU2AF1/Spi-B was analyzed by using real-time reverse transcription PCR assays on isolated B/CD8(+) T lymphocytes and peripheral blood mononuclear cells (PBMCs) from cohorts of untreated and natalizumab-treated patients with and without PML. Longitudinal expression analysis was performed on CD4(+), CD8(+) T and B cells from 14 patients who interrupted natalizumab therapy for 8 weeks. The miRNA profiling was conducted in PBMCs from 5 untreated and 5 natalizumab-treated patients using low-density arrays followed by validation with single miRNAs assays in untreated and natalizumab-treated patients. RESULTS: POU2AF1 and Spi-B mRNAs were upregulated in B and CD8(+) T cells from natalizumab-treated patients, which was validated in PBMCs from different cohorts of natalizumab-treated patients with and without PML, with a noteworthy higher expression of Spi-B in patients with PML. In contrast, downregulation of POU2AF1/Spi-B expression was measured in B and CD8(+) T cells after natalizumab discontinuation. Seventeen differentially expressed miRNAs including miR-10b, a regulator of POU2AF1 mRNA, were identified in long-term natalizumab-treated patients compared with untreated ones. CONCLUSIONS: Upregulation of POU2AF1 and Spi-B, known transactivators of the JC virus, the causative agent for PML, and its association with occurrence of PML in natalizumab-treated patients, corroborates POU2AF1/Spi-B as potential biomarkers for PML risk, which merits further evaluation

Motor neuropathy in porphobilinogen deaminase–deficient mice imitates the peripheral neuropathy of human acute porphyria

Acute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias’ enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD(–/–)) imitate acute porphyria through massive induction of hepatic δ-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD(–/–) mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD(–/–) mice exhibit a marked decrease in large-caliber (>8 μm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD(–/–) mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor δ-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy

Cytokine profiles show heterogeneity of interferon-β response in multiple sclerosis patients

OBJECTIVE: To evaluate serum cytokine profiles for their utility to determine the heterogeneous responses to interferon (IFN)-beta treatment in patients with multiple sclerosis (MS). METHODS: Patients with relapsing-remitting MS (RRMS) or clinically isolated syndrome receiving de novo IFN-beta treatment were included in this prospective, observational study. Number of relapses and changes in disability were assessed 2 years prior to and 2 years after initiation of treatment. Sera were collected at baseline and after 3 months on therapy. Cytokine levels in sera were assessed by Luminex multiplex assays. Baseline cytokine profiles were grouped by hierarchical clustering analysis. Demographic features, changes in cytokines, and clinical outcome were then assessed in the clustered patient groups. RESULTS: A total of 157 patients were included in the study and clustered into 6 distinct subsets by baseline cytokine profiles. These subsets differed significantly in their clinical and biological response to IFN-beta therapy. Two subsets were associated with patients who responded poorly to therapy. Two other subsets, associated with a good response to therapy, showed a significant reduction in relapse rates and no worsening of disability. Each subset also had differential changes in cytokine levels after 3 months of IFN-beta treatment. CONCLUSIONS: There is heterogeneity in the immunologic pathways of the RRMS population, which correlates with IFN-beta response