Jean-Christophe Devedjian

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AIMS Joint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs). MAIN METHODS In MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct. KEY FINDINGS TNF-alpha (from 0.1 to 10 ng/ml) and IL-1beta (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-alpha-treated cultures did not reverse TNF-alpha effects, indicating that IL-1 was not involved in TNF-alpha-stimulated TNAP activity. Both TNF-alpha and IL-1beta decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures. SIGNIFICANCE The different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.

Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC.

Parkinsons disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

Effects of the PPAR-α Agonist Fenofibrate on Acute and Short-Term Consequences of Brain Ischemia

In stroke, there is an imperative need to develop disease-modifying drugs able to (1) induce neuroprotection and vasculoprotection, (2) modulate recovery and brain plasticity, and (3) limit the short-term motor and cognitive consequences. We hypothesized that fenofibrate, a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, could exert a beneficial effect on immediate and short-term poststroke consequences related to its pleiotropic mechanisms. Rats or mice were subjected to focal ischemia to determine the effects of acute treatment by fenofibrate on (i) motor and memory impairment, (2) both cerebral and vascular compartments, (3) inflammation, (4) neurogenesis, and (5) amyloid cascade. We show that fenofibrate administration results in both neuronal and vascular protection and prevents the short-term motor and cognitive poststroke consequences by interaction with several mechanisms. Modulation of PPAR-α generates beneficial effects in the immediate poststroke consequences by mechanisms involving the interactions between polynuclear neutrophils and the vessel wall, and microglial activation. Fenofibrate modulates mechanisms involved in neurorepair and amyloid cascade. Our results suggest that PPAR-α agonists could check the key points of a potential disease-modifying effect in stroke.

Identification of CBFA1-regulated genes on SaOs-2 cells.

Current knowledge about mechanisms controlling osteoblast-specific gene expression has led to the identification of Cbfa1 as a key regulator of osteoblast differentiation. Several essential questions about this transcription factor remain to be addressed, e.g., the nature of stimuli that may modulate its own expression, as well as the genetic repercussions following alterations in Cbfa1 levels. To identify such Cbfa1-responsive genes, the SaOs-2 cell line was stably transfected with a dominant negative mutant of Cbfa1 (ΔCbfa1). Comparison of gene expression patterns by differential display on selected SaOs-2 clones allowed the identification of four new genes that may be under the control of Cbfa1. Three of them, SelM, elF-4AI, and RPS24, seemed to be linked to a global change in cellular metabolism and cell growth. The fourth, the CD99/MIC2 gene, was strongly overexpressed (around tenfold) in cells presenting high levels of Δcbfa1. This observation adds evidence to show that this marker of Ewing family tumors is linked to the osteoblast lineage. The exact function of CD99 remains largely undefined, and this is the first time that its regulation by an essential transcription factor involved in osteoblast differentiation has been observed.

Magnetic Resonance Imaging Features of the Nigrostriatal System: Biomarkers of Parkinson’s Disease Stages?

Introduction Magnetic resonance imaging (MRI) can be used to identify biomarkers in Parkinson’s disease (PD); R2* values reflect iron content related to high levels of oxidative stress, whereas volume and/or shape changes reflect neuronal death. We sought to assess iron overload in the nigrostriatal system and characterize its relationship with focal and overall atrophy of the striatum in the pivotal stages of PD. Methods Twenty controls and 70 PD patients at different disease stages (untreated de novo patients, treated early-stage patients and advanced-stage patients with L-dopa-related motor complications) were included in the study. We determined the R2* values in the substantia nigra, putamen and caudate nucleus, together with striatal volume and shape analysis. We also measured R2* in an acute MPTP mouse model and in a longitudinal follow-up two years later in the early-stage PD patients. Results The R2* values in the substantia nigra, putamen and caudate nucleus were significantly higher in de novo PD patients than in controls. Early-stage patients displayed significantly higher R2* values in the substantia nigra (with changes in striatal shape), relative to de novo patients. Measurements after a two-year follow-up in early-stage patients and characterization of the acute MPTP mouse model confirmed that R2* changed rapidly with disease progression. Advanced-stage patients displayed significant atrophy of striatum, relative to earlier disease stages. Conclusion Each pivotal stage in PD appears to be characterized by putative nigrostriatal MRI biomarkers: iron overload at the de novo stage, striatal shape changes at early-stage disease and generalized striatal atrophy at advanced disease.

Bioavailable Trace Metals in Neurological Diseases

Opinion statementMedical treatment in Wilson’s disease includes chelators (d-penicillamine and trientine) or zinc salts that have to be maintain all the lifelong. This pharmacological treatment is categorised into two phases; the first being a de-coppering phase and the second a maintenance one. The best therapeutic approach remains controversial, as only a few non-controlled trials have compared these treatments. During the initial phase, progressive increase of chelators’ doses adjusted to exchangeable copper and urinary copper might help to avoid neurological deterioration. Liver transplantation is indicated in acute fulminant liver failure and decompensated cirrhosis; in cases of neurologic deterioration, it must be individually discussed. During the maintenance phase, the most important challenge is to obtain a good adherence to lifelong medical therapy. Neurodegenerative diseases that lead to a mislocalisation of iron can be caused by a culmination of localised overload (pro-oxidant siderosis) and localised deficiency (metabolic distress). A new therapeutic concept with conservative iron chelation rescues iron-overloaded neurons by scavenging labile iron and, by delivering this chelated metal to endogenous apo-transferrin, allows iron redistribution to avoid systemic loss of iron.

Leptin Receptors and β2-Adrenergic Receptor mRNA Expression in Brain Injury-Related Heterotopic Ossification

Heterotopic ossification (HO) frequently occurs after brain injury. Recently, we found that leptin levels were decreased in the serum of patients with HO. Data suggest two mechanisms mediating leptin effects: a central suppressive mechanism acting via the β2-adrenergic system and a direct stimulatory action starting when leptin binds to its receptors in osteoblastic cells. In this study, we analyzed leptin and β2-adrenergic receptors mRNA expression in osteocytes originated from normal or heterotopic bone biopsies to investigate whether direct or indirect pathway signaling might be implicated in this pathological bone formation. We report for the first time the mRNA expression of the leptin receptor isoforms in osteocytes isolated from all biopsies. Moreover, quantitative reverse transcription-polymerase chain reaction allowed us to measure a significant decrease in the level of β2-adrenergic receptor mRNA in cells isolated from heterotopic bone biopsies. These results could suggest an association between hypothalamic leptin signaling and brain injury-related HO.

Continuous cerebroventricular administration of dopamine: A new treatment for severe dyskinesia in Parkinson's disease?

In Parkinsons disease (PD) depletion of dopamine in the nigro-striatal pathway is a main pathological hallmark that requires continuous and focal restoration. Current predominant treatment with intermittent oral administration of its precursor, Levodopa (l-dopa), remains the gold standard but pharmacological drawbacks trigger motor fluctuations and dyskinesia. Continuous intracerebroventricular (i.c.v.) administration of dopamine previously failed as a therapy because of an inability to resolve the accelerated dopamine oxidation and tachyphylaxia. We aim to overcome prior challenges by demonstrating treatment feasibility and efficacy of continuous i.c.v. of dopamine close to the striatum. Dopamine prepared either anaerobically (A-dopamine) or aerobically (O-dopamine) in the presence or absence of a conservator (sodium metabisulfite, SMBS) was assessed upon acute MPTP and chronic 6-OHDA lesioning and compared to peripheral l-dopa treatment. A-dopamine restored motor function and induced a dose dependent increase of nigro-striatal tyrosine hydroxylase positive neurons in mice after 7days of MPTP insult that was not evident with either O-dopamine or l-dopa. In the 6-OHDA rat model, continuous circadian i.c.v. injection of A-dopamine over 30days also improved motor activity without occurrence of tachyphylaxia. This safety profile was highly favorable as A-dopamine did not induce dyskinesia or behavioral sensitization as observed with peripheral l-dopa treatment. Indicative of a new therapeutic strategy for patients suffering from l-dopa related complications with dyskinesia, continuous i.c.v. of A-dopamine has greater efficacy in mediating motor impairment over a large therapeutic index without inducing dyskinesia and tachyphylaxia.

Intracerebroventricular dopamine for Parkinson’s disease

Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder worldwide. The depletion of dopamine in the nigro-striatal pathway is a main pathological hallmark that requires continuous and focal restoration. Since dopamine does not cross the digestive mucosa or the blood brain barrier, its lipophilic precursor L-dopa has been employed and remains the pivotal oral medication. However, after 5-7 years of L-dopa, many pharmacokinetic drawbacks contribute to the occurrence of severe motor and non-motor complications (i.e. under and overdoses) [1]. Indeed L-dopa has a short half-life, limited and variable reabsorption through the digestive and blood brain barriers and potentially harmful peripheral distribution. Moreover, L-dopa requires the aromatic L-amino acid decarboxylase for the synthesis of dopamine, which declines in the striatum with disease progression [2]. A viable optimal therapeutic regime would be to continuously compensate the deficit in dopamine in order to prevent oscillations in neurotransmitter concentration [5]. A continuous intracerebroventricular (i.c.v.) administration better mimics the physiological released of dopamine caused by the tonic background activity of dopaminergic neurons. The i.c.v administration of dopamine can be performed through an abdominal pump with subcutaneous catheter from the pump to the lateral ventricle. The latter is directly upstream to the third ventricle, being very close to the bilateral striatum (Figure). De Yebenes et al. [4] previously demonstrated that i.c.v. administered dopamine with an anti-oxidant adjuvant (sodium metabisulfite; SMBS) transiently improved motor handicap and increased dopamine in rat brains with unilateral neurotoxin 6-hydroxydopamine (6-OHDA)induced damage as well as 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) intoxicated monkeys. The clinical feasibility of this administrative route has been supported by two PD patient case reports of dopamine infusion to the frontal ventricle, whereby a reduction in motor handicap was observed [6, 8]. The first human case report [8] described a good tolerance to dopamine infusion over 1 year. A subsequent human case report [6] showed that long-term i.c.v. dopamine provides a smooth control of motor symptoms. However, both preclinical and clinical reports also highlight two overriding problems that prevented further development; (i) occurrence of tachyphylaxia and (ii) oxidation of dopamine causing enhanced dopamine metabolism and oxidative stress. Indeed, PD patients from previous studies received dopamine prepared aerobically (O-dopamine) and at the same dose throughout a 24 hours cycle. Prior experience obtained from the use of an apomorphine pump and duodopa® has identified the need to differentiate between diurnal and nocturnal minimum efficient dose in order to avoid worsening motor fluctuations [3]. Recently, the authors have overcome these challenges [7]. Indeed, deleterious effect of dopamine oxidation was avoided by anaerobic preparation of dopamine (A-dopamine: prepared, stored and administered in very low oxygen conditions). A-dopamine did not need a conservator (sodium metabisulfite, SMBS), which has detrimental effects. A-dopamine restored motor function (i.e. actimetry analysis) of the mouse mice, 7 days after MPTP insult, and had a broader therapeutic index than peripheral L-dopa treatment. A-dopamine demonstrated good diffusion into the striatum. Concomitantly, A-dopamine induced a dose dependent increase of nigrostriatal dopaminergic neurons that was not evident with either O-dopamine or L-dopa. A-dopamine and L-dopa treatments were equally protective in regard to the redox state of dopaminergic neurons. In addition, greater advances in programmable pumps now minimize tachyphylaxia by allowing administration of a lower effective dopamine dose in accordance with the circadian cycle. In a chronic rat model using 6-OHDA-lesioning, continuous circadian i.c.v injection of A-dopamine (16h/day) over 30 days improved motor activity (i.e. ‘stepping’ and ‘cylinder’ tests and actimetry analysis) without occurrence of tachyphylaxia. Editorial

Comments on the publication “A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone”

Dear Editor: We readwith interest the paper by A. E. Handschin et al. published ahead online on the web page of Langenbeck’s Archives of Surgery [1]. The authors characterized the expression of osteoblast-specific genes in cells originating from normal and heterotopic bone. The analysis was made on a 7-day primary human osteoblast cell culture using reverse transcription polymerase chain reaction, real-time PCR, and immunohistochemistry. The results were then compared with those from three other studies. These studies included the works from Kaysinger et al. [2] and Sell et al. [3], using human cultured cells, and our work [4] performed directly on human biopsies without culturing step. However, the authors did not clearly define that our study was not a cell culture study, which was even mentioned twice in the text. In the beginning of the discussion, there is a statement concerning our work and the one from Sell et al. [3], “Other studies have also found collagen expression and alkaline phosphatase expression to be similar in both cell culture types.”And in their Table 1, it was indicated that our culture time was not reported. Moreover, we would like to point out another error in their discussion. Concerning type 1 collagen expression, we clearly showed a significant upregulation in heterotopic ossification specimen contrary to what was mentioned in the paper of Handschin et al. [1]. We think that these comments should be taken into consideration in this comparative analysis and in the conclusions deduced from this work.