Patent Application
20130337461
The present invention relates to a method to diagnose, and/or monitor the efficacy of a therapy and/or screen for a therapeutic agent for multiple sclerosis based on the measurement of expression or localization of specific proteins, involved in human oligodendrocytes differentiation.
Multiple sclerosis (MS) is characterized by inflammation, demyelination and gliosis that damage the central nervous system (CNS) (Noseworthyet al., 2000; Trapp et al., 1998). MS is the most common cause of disability in young adults and is one of the most debilitating medical conditions, not only physically, but also on psycho-social levels. As in other chronic inflammatory diseases, the manifestations of MS change from a benign to a rapidly progressive and debilitating form. Some indirect data suggest an autoimmune etiology for multiple sclerosis, perhaps triggered by a viral infection, in a genetically susceptible individual (Marrie, 2004). Despite the number of studies on the disease and a multidisciplinary approach to the problem, the exact pathogenic mechanisms of multiple sclerosis are still obscure and the aetiology is unknown.
MS has no definitive diagnostic tests rendering necessary to use different diagnostic tools: clinical (Trojano and Paolicelli, 2001), laboratory (Luqueand Jaffe, 2007) and instrumental investigations (Young et al., 1981; Achtenand Deblaere, 2008).
The MS therapies currently available (beta-interferon, steroids, symptomatic therapies) act on the symptomatology and are aimed to slow the progression of the disease (Murdoch and Lyseng-Williamson, 2005). Thus, the early diagnosis of MS is needed because an earliest possible therapeutic intervention would be most effective in the long term.
Previous studies have shown that in MS patients chronic demyelinating lesions, there is a small number of pro-oligodendrocyte (pro-OL) and an increased number of oligodendrocyte progenitor cells (OPCs). This suggests that in pathological conditions, the OPCs maturation process is slower (Chang et al., 2002; Wolswijk, 1998).
This data is actually based on immunocytochemistry and immunohistochemistry investigations of the brain tissue. Therefore, there is the need to develop tools that evaluate the differentiation mechanisms at a molecular level and not at the organ level.
Previous studies showed that oligodendrocytes and their precursors are very sensitive to oxidative stress and this sensitivity is inversely correlated to the maturation level (Fragosoet al., 2004). The authors evaluated the effects of chronic stimulation of MO3-13 cells with low doses of hydrogen peroxide on the expression of oligodendrocyte differentiation markers.
The patent application WO 2008/090213 discloses that P-ERK1/2 or α-SMA may be used in the diagnosis of multiple sclerosis.
The present invention relates to a method to diagnose and/or monitor the efficacy of a therapy and/or for the and/or screen for a therapeutic agent for multiple sclerosis based on the measurement of expression or localization of specific proteins, involved in human oligodendrocytes differentiation.
Changes of expression or localization of specific proteins involved in oligodendrocyte differentiation were measured after incubation of differentiating cells with cerebrospinal fluid, immunoglobulins extracted from blood serum or blood of patients with multiple sclerosis.
On this basis, it is possible, by using acellular system: i) to diagnose multiple sclerosis; ii) to screen drugs, for treatment of multiple sclerosis, already used in clinical therapy or in preclinical development; iii) to follow up treatment with drugs already used to treat multiple sclerosis.
Human oligodendrocyte MO3-13 cells differentiate when grown in the absence of serum and in the presence of PhorbolMyristate Acetate (PMA), also known as 12-O-Tetradecanoylphorbol-13-acetate (TPA). Expression or localization of specific differentiation markers was evaluated at different periods by western blotting, flow cytometry, real time PCR and immunofluorescence confocal microscopy.
The authors found that differentiation of these cells is accompanied by significant changes in specific intracellular markers. In addition, they have set up a new protocol to differentiate human oligodendrocytes. The cells are incubated for 30 min-4 days in complete medium in the presence of low doses (200 μM) of hydrogen peroxide. Then, expression or localization of specific differentiation markers is evaluated. H2O2 produces the same changes of differentiation markers as PMA treatment.
In human oligodendrocyte incubated with CSF from multiple sclerosis (MS) patients, the authors found an inhibition of the differentiation induced by PMA, as indicated by differential expressions and/or cell localization of specific intracellular differentiation markers.
The authors obtained the same results, i.e. inhibition of differentiation, incubating the human oligodendrocyte with IgGs from blood serum from MS patients.
The authors found that in mouse cortical primary neurons and in HEK293 cells, the incubation with CSF and IgG from blood serum from MS patients does not produce any significant effect on the intracellular differentiation markers. This demonstrates that the effects of biological fluids from MS patients specifically target oligodendrocyte cells.
The results obtained testing 63 neurologic and 48 multiple sclerosis patients show that this assay, performed by measuring the intracellular differentiation markers phospho-Cyclic AMP Response Element Binding Protein (P-CREB), Extracellular Signal-Regulated Kinases 1/2 (P-ERK1/2), α-Smooth Muscle Actin (α-SMA), Myelin Basic Protein (MBP), Oligodendrocyte Transcription Factor 2 (olig-2) and muscarinic Achetylcholine Receptor M1 (mAchR M1) has a good sensitivity and specificity and is very reproducible.
The present data provide evidence for the development of a test to detect, at an early stage, the changes in intracellular signalling involved in oligodendrocytes differentiation, opening a new scenario for the development of an early and specific functional diagnosis for multiple sclerosis.
It is therefore an object of the invention a method for diagnosis and/or prognosis of multiple sclerosis in a subject, comprising the steps of:
It is a further object of the invention a method for monitoring the efficacy of a therapeutic agent and/or screening for a candidate therapeutic agent for multiple sclerosis, comprising the steps of:
Preferably, the molecular species is P-CREB.
Still preferably, the amount of at least two molecular species is measured.
In a preferred embodiment the amount of at least three molecular species is measured.
In a still preferred embodiment the amount of all of molecular species is measured.
In a yet preferred embodiment the detectable marker is a luciferase or GFP.
Preferably the method of the invention further comprises measuring the amount of at least another molecular species belonging to the group of: P-ERK1/2 or α-SMA.
Preferably the biological sample is cerebrospinal fluid, blood sample or serum sample or Ig-comprising derivatives thereof.
Still preferably the differentiation stimulus consists of incubating oligodendrocyte cells in the presence of: a) Phorbol Myristate Acetate (PMA); b) hydrogen peroxide; c) low serum medium; d) cyclic adenosine monophosphate (cAMP) analogs; e) adenylate cyclase activators; f) thyroid hormones as 3,5,3′-L-triiodothyronine (T3) and thyroxin (T4); g) ERB B inhibitors; h) nuclear receptor ligand; or i) nucleoside analogs.
It is a further object of the invention a kit for the diagnosis and/or prognosis of multiple sclerosis or to monitor the efficacy of a therapy for multiple sclerosis comprising means to measure P-CREB and one or more molecular species belonging to the following group: mAChR M1, olig-2, MBP.
Preferably, the kit further comprises means to measure P-ERK1/2 and/or α-SMA.
In the present invention in the case of a method for diagnosis and/or prognosis of multiple sclerosis, the control amount or localization may be the amount or localization measured or assessed in oligodendrocyte cells incubated with a sample taken from a healthy patient or from a patient affected by another neurological disease.
In the case of a method for monitoring the efficacy of a therapeutic agent, the control amount or localization may be the amount or localization measured or assessed in oligodendrocyte cells incubated with a sample taken from the same subject before the start of the therapy or at various time point throughout the course of the therapeutic agent.
In the case of a method to screen for a candidate therapeutic agent for multiple sclerosis, the control amount or localization may be the amount or localization measured or assessed in oligodendrocyte cells incubated with a sample taken from a positive (multiple sclerosis) patient without candidate therapeutic agent or with a reference treatment. In the present invention, two markers may be measured in the same incubated oligodendrocyte cells (simultaneously or subsequently), said markers being selected from the group of: P-CREB, P-ERK1/2, α-SMA, MBP, muscarinic acethylcholine receptor (mAchR) M1 or olig-2 and/or assessing the cellular localization of olig-2. Any combination is suitable for the purpose of the invention, for instance measuring P-CREB and assessing the cellular localization of olig-2, or measuring P-ERK1/2 and α-SMA or measuring MBP and assessing the cellular localization of olig-2 or measuring P-ERK1/2 and P-CREB, or measuring MBP and P-CREB etc. . . .
In the present invention, three markers may be measured in the same incubated oligodendrocyte cells (simultaneously or subsequently), said markers being selected from the group of: P-CREB, P-ERK1/2, α-SMA, MBP, muscarinic acethylcholine receptor (mAchR) M1 or olig-2 and/or assessing the cellular localization of olig-2. Any combination is suitable for the purpose of the invention, for instance measuring P-CREB and α-SMA and assessing the cellular localization of olig-2, or measuring P-ERK1/2 and MBP and α-SMA, etc. . . .
In the present invention, four markers may be measured in the same incubated oligodendrocyte cells (simultaneously or subsequently), said markers being selected from the group of: P-CREB, P-ERK1/2, α-SMA, MBP, muscarinic acethylcholine receptor (mAchR) M1 or olig-2 and/or assessing the cellular localization of olig-2. Any combination is suitable for the purpose of the invention, for instance measuring P-CREB, MBP, α-SMA and assessing the cellular localization of olig-2, or measuring P-ERK1/2 and MBP and α-SMA and P-CREB, ect. . . .
In the present invention, all markers may be measured in the same incubated oligodendrocyte cells (simultaneously or subsequently), said markers being selected from the group of: P-CREB, P-ERK1/2, α-SMA, MBP, muscarinic acethylcholine receptor (mAchR) M1 or olig-2 and/or assessing the cellular localization of olig-2.
It is a further object of the invention an in vitro method to differentiate oligodendrocyte precursor cells in oligodendrocyte cells comprising exposing said oligodendrocyte precursor cells to an effective amount of hydrogen peroxide. Preferably the amount of hydrogen peroxide is 100-200 μM.
The invention will be now illustrated by means of non-limiting examples referring to the following figures.
(A1) Increase of P-CREB and P-ERK1/2 levels in M03-13 cells stimulated with PMA. The not differentiated (N.D.) cells were incubated for 16 h in medium containing 0.2% of FBS and then stimulated with 100 nM PMA for 30 min (Differentiated, Diff.) before harvesting them for Western blotting analysis of P-CREB and P-ERK1/2 levels. (A2) The histogram shows the values (means±SEM) relative to control obtained by densitometric analysis of protein bands normalized for α-tubulin of three independent experiments. *p<0.01 vs N.D. (B1) Western blotting analysis of P-CREB, P-ERK 1/2, α-SMA and Olig-2 levels in M03-13 cells after 24 h of differentiation of M03-13 cells with 100 nM PMA in medium without serum. (B2) The histogram shows the values (means±SEM) relative to control obtained by densitometric analysis of protein bands normalized for α-tubulin of three independent experiments. *p<0.01 vs N.D. **p<0.001 vs N.D. (C1) MBP protein increased levels when compared to not differentiated cells (N.D.) at different days (1 day or 4 days) of differentiation (Diff.) of M03-13 cells with 100 nM PMA in medium without serum. Immunoreactivity for MBP was evidenced by indirect immunofluorescence and flow cytometry using primary antibodies against MBP and CY3-conjugated anti rabbit IgG as secondary antibodies. Negative (Neg.) was treated with secondary antibodies alone. Ten thousands cells were counted for each sample. (C2) The histogram shows the means±SEM of three independent experiments. N.D. indicates cells growing in complete medium. *p<0.01 vs N.D. (D) Translocation of Olig-2 transcription factor from nucleus to cytosol after 24 h of differentiation of M03-13 cells with 100 nM PMA in medium without serum (Diff.). Cells were stained with anti-human Olig-2 antibodies and CY3-conjugated anti rabbit IgG as secondary antibodies and analyzed by confocal microscopy. Neg. was treated with secondary antibodies alone. N.D. indicates cells growing in complete medium. (E1) mAchR M1 protein increased levels when compared to N.D. at different days of differentiation of MO3-13 cells with PMA 100 nM in medium without serum. mAchR M1 levels were measured by Western blotting analysis. (E2) The hystogram shows the values (means±SEM) relative to N.D., obtained by densitometric analysis of protein bands normalized for α-tubulin of three independent experiments. *p<0.05 vs N.D.
(A1) Western blotting analysis of α-SMA, P-CREB and P-ERK1/2 levels on M03-13 cells treated for 24 h with 200 μM H2O2 in the absence or presence of N-Acetyl-Cysteine (NAC, 10 mM) or 4-(2-aminoethyl)benzene sulfonyl fluoride (AEBSF, 40 μM). (A2) The histogram shows means±SEM values relative to control of three independent experiments. *p<0.01 vs control; **p<0.01 vs. H2O2 treatment. (B1) Increase of MBP protein levels in M03-13 cells treated for 4d with 200 μM H2O2. Immunoreactivity for MBP was evidenced by indirect immunofluorescence and flow cytometric analysis as described in
Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence of 30% CSF from Control (Ctr.) or multiple sclerosis (MS) patients and then harvested for Western blotting analysis of P-ERK1/2 levels. The histogram (A) shows the mean±SEM of P-ERK1/2 values relative to Diff. samples obtained by densitometric analysis of P-ERK1/2 band normalized for α-tubulin. (B) the scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiment is shown. *p<0.05 vs CSF Ctr.
Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence of 30% CSF from Ctr. or MS patients and then harvested for Western blotting analysis of α-SMA levels. The histogram (A) shows the mean±SEM of α-SMA values relative to Diff. samples obtained by densitometric analysis of α-SMA band normalized for α-tubulin. (B) the scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiment is shown. *p<0.05 vs CSF Ctr.
Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence of 30% CSF from Control (Ctr.) or multiple sclerosis (MS) patients and then harvested for Western blotting analysis of Olig-2 levels. The histogram (A) shows the mean±SEM of Olig-2 values relative to Diff. samples obtained by densitometric analysis of Olig-2 band normalized for α-tubulin. (B) the scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiment is shown. *p<0.05 vs. CSF Ctr.
(A) Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence of 30% CSF from Ctr. or MS patients and then immune reactivity for MBP was evidenced by indirect immunofluorescence and flow cytometry using primary antibodies against MBP and CY3-conjugated anti rabbit IgG as secondary antibodies. Neg. was treated with secondary antibodies alone. Ten thousands cells were counted for each sample. The histogram (A) shows the means±SEM fluorescence values relative to not stimulated samples; (B) the scatter plot with the single relative values for each patient is shown. *p<0.05 vs CSF Ctr. The panel (C) shows the flow cytometric histograms of a representative experiment.
Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence of 30% CSF from Ctr. or MS patients and then MBP mRNA levels were analysed by RT-PCR. The histogram (A) shows the mean±SEM values relative to Diff. sample. (B) the scatter plot with the single values for each patient. *p<0.05 vs CSF Ctr.
The staining for Olig-2 in N.D. cells is shown in panel (A). Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (B) (Diff. 24 h) and presence of 30% CSF from Ctr. (C) or MS (D) patients and then stained with anti-human Olig-2 antibodies and CY3-conjugated anti rabbit IgG as secondary antibodies and analyzed by confocal microscopy.
(A) The cells were stained with anti-MBP and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff. was treated only with PMA 100 nM in serum-free medium for 1 day; CSF N indicates cells stimulated by CSF from control patients; CSF MS indicates cells stimulated with CSF of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-Olig-2 and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. For each image are shown three panels: on the left Olig-2 (red); on the center nuclei (blue); on the right the merged image. Diff. was treated only with PMA 100 nM in serum-free medium for 1 day; CSFN indicates cells stimulated by CSF from control patients; CSFMS indicates cells stimulated with CSF of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-α-SMA and anti-mouse secondary antibody conjugated to CY2 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff was treated only with PMA100 nM in serum-free medium for 4 days; CSFN indicates cells stimulated by CSF from control patients; CSFMS indicates cells stimulated with CSF of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-MBP and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff was treated only with PMA 100 nM in serum-free medium for 4 days; CSFN indicates cells stimulated by CSF from control patients; CSFMS indicates cells stimulated with CSF of patients with MS. (B) the table shows the fluorescence values (mean±SD, arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-Olig-2 and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. For each image are shown three panels: on the left Olig-2 (red); on the center nuclei (blue); on the right the merged image. Diff. was treated only with PMA100 nM in serum-free medium for 4 days; CSFN indicates cells stimulated by CSF from control patients; CSFMS indicates cells stimulated with CSF of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
Cells were differentiated for 4 days with 100 nM PMA in medium without serum, stimulated for 30 min with 30% CSF from Ctr. or MS patients and then P-CREB levels were evaluated by Western blotting analysis. The histogram (A) shows the mean±SEM levels relative to cells not stimulated with CSF (Diff.), obtained by densitometric analysis of protein bands normalized for α-tubulin. (B) the scatter plot with the single values for each patient. (C) a representative Western blotting experiment is shown. *p<0.05 vs CSF Ctr.
Cells were cultured in medium containing 0.2% FBS for 16 h, preincubated for 30 min in the absence (Diff.) or presence of 200 μg/ml of serum IgG and then stimulated with 100 nM PMA for 30 min before harvesting them for Western blotting analysis of P-ERK1/2 levels. The histogram (A) shows the mean±SEM of P-ERK1/2 values relative to Diff sample obtained by densitometric analysis of protein bands normalized for α-tubulin. (B) scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiment is shown. *p<0.05 vs Ig Ctr.
Cells were treated for 24 h with 100 nM PMA in medium without serum in the absence (Diff.) or presence of 200 μg/ml of serum IgG from Ctr. or MS patients and then P-ERK1/2 levels were evaluated by Western blotting analysis. The histogram (A) shows the mean±SEM of P-ERK1/2 values relative to Diff. samples obtained by densitometric analysis of P-ERK1/2 band normalized for α-tubulin. (B) scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiment is shown. *p<0.05 vs Ig Ctr.
Cells were treated as in
Cells were treated as in
Cells were treated as in
Cells were treated for 24 h with 100 nM PMA in medium without serum in absence (Diff.) and presence 200 μg/ml of serum IgG from Ctr. or MS patients and then α-SMA mRNA levels were evaluated by RT-PCR analysis. The histogram (A) shows the mean±SEM values relative to Diff. samples. (B) scatter plot with the single values for each patient. *p<0.05 vs Ig Ctr.
(A) The cells were stained with anti-α-SMA and anti-mouse secondary antibody conjugated to CY2 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff. was treated only with PMA 100 nM in serum-free medium for 4 days; Ig N indicates cells stimulated by IgG of control patients; Ig MS indicates cells stimulated with IgG of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
Cells were treated as in
Cells were treated as in
(A) The cells were stained with anti-MBP and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff. was treated only with PMA 100 nM in serum-free medium for 1 day; Ig N indicates cells stimulated by IgG of control patients; Ig MS indicates cells stimulated with IgG of patients with MS. (B) The table shows the fluorescence values (mean±SD, arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-MBP and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. Diff. was treated only with PMA100 nM in serum-free medium for 4 days; Ig N indicates cells stimulated by IgG of control patients; Ig MS indicates cells stimulated with IgG of patients with MS. (B) The table shows the fluorescence values (mean±SD, arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
Cells were treated for 24 h with 100 nM PMA in medium without serum in the absence or presence of 200 μg/ml of serum IgG from Control or MS patients and then stained with Olig-2 antibodies and analyzed by confocal microscopy. The staining for Olig-2 in N.D. cells is also shown.
(A) The cells were stained with anti-Olig-2 and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. For each image are shown three panels: on the left Olig-2 (red); on the center nuclei (blue); on the right the merged image. Diff. was treated only with PMA 100 nM in serum-free medium for 1 day; Ig N indicates cells stimulated by IgG of control patients; Ig MS indicates cells stimulated with IgG of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
(A) The cells were stained with anti-Olig-2 and anti-rabbit secondary antibody conjugated to CY3 and the nuclear dye DAPI, and analyzed by confocal microscopy. For each image are shown three panels: on the left Olig-2 (red); on the center nuclei (blue); on the right the merged image. Diff. was treated only withPMA100 nM in serum-free medium for 4 days; Ig N indicates cells stimulated by IgG of control patients; Ig MS indicates cells stimulated with IgG of patients with MS. (B) The table shows the values (mean±SD, fluorescence arbitrary units) obtained by the quantitative analysis of 25 cells for each sample.
Cells were treated for 4 days with 100 nM PMA in medium without serum in the absence (Diff.) or presence of 200 μM of serum IgG from Ctr. or MS patients and then mAchR M1 levels were evaluated by Western blotting analysis. The histogram (A) shows the mean±SEM of mAchR M1 levels relative to Diff. samples, obtained by densitometric analysis of mAchR M1 bands normalized for α-tubulin. (B) scatter plot with the single values for each patient is shown. (C) a representative Western blotting experiments is shown. *p<0.05 vs Ig ctr.
(A) Mouse cortical neurons were treated for 30 min with 30% CSF from Ctr. or MS patients and then a harvested for Western blotting analysis of P-CREB levels. Mouse cortical neurons were treated for 30 min with 200 μg/ml of serum IgG from Control or MS patients and then harvested for Western blotting analysis of (B) P-CREB, (C) α-SMA or (D) P-ERK1/2 levels. The histograms (A1, B1, C1, D1) show the mean±SEM values relative to not stimulated (NS) samples obtained by densitometric analysis of protein bands normalized for α-tubulin. In the lower part of the figures (A2, B2, C2, D2) the representative Western blotting experiments are shown.
M03-13 or HEK293 cells were treated for 24 h with 200 μg/ml of serum IgG from Ctr. or MS patients in the presence of 100 nM PMA in medium without serum and then P-ERK1/2 levels were analyzed by Western blotting analysis. The histogram (A) shows the values relative to NS samples of 1 Control and 2 MS patients obtained by densitometric analysis of protein bands normalized for α-tubulin. In the lower part of the figures (B, C) the representative Western blotting experiments are shown.
M03-13 cells—The M03-13 cells are an immortal human-human hybrid cell line (CELLutionBiosystem Inc., Canada) with the phenotypic characteristics of primary oligodendrocytes, derived from the fusion of human rhabdomyosarcoma with oligodendrocytes obtained from adult human brain. They were grown in Dulbecco's Modified Eagles Medium (DMEM), containing 4.5 g/L glucose, supplemented with 10% FBS, 100 U/ml penicillin and 100 m/ml streptomycin.
HEK293—The HEK293 derived from human embryonic kidney cells (American Type Culture Collection, ATCC, USA), were grown in Dulbecco's Modified Eagles Medium (DMEM), containing 4.5 g/L glucose, supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.
Primary cultures of mouse cortical neurons—Cortical neuronal cultures were prepared from foetal mice (14-16 days gestation). Embryos were decapitated, and the neocortices were dissected, dissociated and plated in Eagle's minimal essential medium (Gibco, Auckland, New Zealand) supplemented with 20 mmol glucose (Sigma Chemical), 2 mmol glutamine (Gibco), 5% foetal bovine serum (Gibco) and 5% horse serum (Gibco) on 96-well culture plates, which were prepared by incubation overnight with 40 g/mL poly-d-lysine (Sigma-Aldrich) and subsequent rinsing with water. Glial cell replication was halted at 5 days in vitro (DIV), by 2 days exposure to 10 mol cytosine arabinoside. Cultures were kept in a 5% CO2 and 95% air atmosphere at 37° C.; at 7 DIV they were shifted into a growth medium identical to the plating medium, but lacking foetal serum. Cultures were used for experiments at 13-14 DIV.
The cells were kept in a 5% CO2 and 95% air atmosphere at 37° C.
Authors generated clones of human oligodendrocytes, expressing the firefly luciferase (de Wet et al., 1997), or the Green Fluorescent Protein (GFP) gene (Chalfie et al., 1994) under the control of anyone of MBP, α-SMA, Olig-2 or mAChR M1 promoters (see below for gene accession numbers).
The DNA sequences corresponding to 1 kb 5′ flanking region (promoter) of the MBP, Olig-2, or mAChR M1 genes (see below) were inserted in the firefly luciferase reporter vector pGL3.0 Basic (Promega) or GFP reporter vector. The MO3-13 cells were transfected with recombinant vectors using Lipofectamine 2000 (Invitrogen).
Such cells represent a cell system for easy and fast detection of factors or drugs or molecules modifying oligodendrocyte differentiation measured by luminometry or by flow cytometric analysis for luciferase or GFP, respectively.
Differentiation of M03-13 cells was obtained using two different protocols:
Protocol 1—Incubating the cells in medium with 0-0.2% Foetal Bovine Serum (FBS, Sigma) in the presence of 100 nM PMA (Sigma-Aldrich) (time range: 30 min-4 days);
Protocol 2—Incubating the cells with 100-200 μM H2O2 in complete medium (time range: 30 min-4 days).
Anti MBP (Cat. # AB980), anti P-CREB (Cat. #06-519), anti PLP (Cat# AB15454), anti MAG clone 513 (Cat #MAB1567), anti Olig-1 (Cat. # MAB5540) and anti Olig-2 (Cat. # AB9610) antibodies were purchased by Millipore. Anti α-SMA (Cat. # A5228) and anti mAchR (Cat# M9808) antibodies were purchased by SIGMA. Anti P-ERK1/2 (E-4) antibodies were purchased by Santa Cruz Biotechnology (Cat. # sc-7383).
Total cells lysates were obtained in RIPA buffer (50 mM Tris-HCl, pH 7.5, NaCl 150 mM, 1% NP40, 0.5% deoxycholate, 0.1% SDS) containing 2.5 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaF, 0.5 mM PMSF, and a cocktail of protease inhibitors (Roche, USA). The cells were kept for 15 min at 4° C. and disrupted by repeated aspiration through a 21-gauge needle. Cell lysates were centrifuged for 10 min at 11,600×g and the pellets were discarded. Fifty micrograms of total proteins were subjected to SDS-PAGE under reducing conditions. After electrophoresis, the proteins were transferred onto a nitrocellulose filter membrane (Biorad, UK) with a Trans-Blot Cell (Bio-Rad Laboratories, UK) and transfer buffer containing 25 mM Tris, 192 mM glycine, 20% methanol. Membranes were placed in 5% non-fat milk in phosphate-buffered saline, 0.5% Tween 20 (PBST) at 4° C. for 2 hr to block the nonspecific binding sites. Filters were incubated with specific antibodies before being washed three times in PBST and then incubated with a peroxidase-conjugated secondary antibody (GE-Healthcare, UK). After washing with PBST, peroxidase activity was detected with the ECL system (GE-Healthcare, UK).
The filters were also probed with an anti α-tubulin antibody (Sigma, USA). Protein bands were revealed by ECL and, when specified, quantified by densitometry using Scionlmage software. Densitometric values were normalized to α-tubulin.
Cells were grown to semiconfluency in 60-mm culture dishes. After trypsin detachment, 5×105 cells are suspended in 1 mL of phosphate buffered saline (PBS) and fixed overnight with 1% formaldehyde at room temperature. Next, cells were permeabilized with 0.1% Triton X-100 for 40 min at 4° C., washed 4× with PBS containing 2% FBS, 0.01% NaN3, 0.1% Triton X-100 (buffer A), and incubated for 45 min at 4° C. with 1:50 dilution of rabbit polyclonal anti-human MBP Ig. The cells were then washed twice with the same buffer and incubated for 45 min at 4° C. with Cy3-conjugated anti-(rabbit IgG) Ig at 1:50 dilution. Control cells were incubated with Cy3-conjugated anti-(rabbit IgG) Ig alone. After two washes in buffer A, cells were resuspended in PBS and analyzed by flow cytometry using FAC SCAN (BD, Heidelberg, Germany) and WINMDI software.
Cells, grown to semiconfluence in 100-mm culture dishes in complete medium, were collected by scraping them into a buffer containing 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 10 mM Pipes 2 mM NaVO4, 10 mM phenylarsine oxide, 5 mM NaF, and the cocktail of protease inhibitors. Cells were then disrupted by sonication (2-10 sec pulses at 100 W) and centrifuged at 600×g for 10 min. Next, the supernatants were centrifuged at 100,000×g for 45 min. Fifty micrograms of cytosol proteins were subjected to Western blotting analysis for myelin basic protein (MBP) as described above.
RNA isolation and real-time PCR were performed as follow: total RNA was extracted using TRI-reagent according to the protocol provided by the manufacturer (Sigma, USA). Total RNA (4 μg) was reverse transcribed with Omniscript Reverse Transcriptase (Quiagen, USA) by oligo-dT primers for 60 min at 37° C. in 40 μl reaction volumes. Real-time PCR was performed with an ABI 5700 or ABI PRISM-7900HT Sequence Detection System (Applied Biosystems Inc., USA). Reactions were carried out in 96-well optical reaction plates in a 50 μA final volume containing 25 μl of the SYBR-Green (Applied Biosystems Inc., USA) PCR master mix, 1.25 μl of each gene-specific primer, 40 ng of sample cDNA. Gene-specific primers were designed to selectively amplify α-SMA and MBP and relative expression values were normalized using glucose-6-phosphate dehydrogenase (G6PD). The SYBRGreen (Applied Biosystems Inc., USA) fluorescence was measured at each extension step. The threshold cycle (Ct) value reflects the cycle number at which the fluorescence measurement reached an arbitrary threshold. Melting curve analysis was performed to determine the specificity of the reaction. Real-time PCR was conducted in triplicate for each sample and the mean value was calculated. Primers for human α-SMA and MBP, and G6PD used are the following:
M03-13 cells were grown on glass coverslip under culture conditions described in the specific experiments. Then, the medium was removed and cells immediately fixed in 3.7% paraformaldehyde in PBS with 2% sucrose for 5 min at 22° C.
For olig-2 staining, after 2 washes in PBS with 2% sucrose, cells were permeabilized for 5 min at 4° C. with 0.1% Triton X-100 in 20 mM Hepes, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2. The cells, after blocking with 20% goat serum in PBS for 45 min at 4° C., were labelled with primary rabbit polyclonal anti human olig-2 antibody. The cells were washed and labelled with secondary Cy3-conjugated anti rabbit IgG (Jackson ImmunoResearch, USA).
For MBP and α-SMA staining, after fixation, the cells were permeabilized for 10 min at 4° C. with 0.01% Saponin (Sigma-Aldrich, from quillaj a bark) in PBS. The cells, after blocking with 20% FBS in PBS with 0.01% Saponin for 30 minutes at 4° C., were labelled with primary rabbit-polyclonal anti human MBP antibody or with primary mouse-polyclonal anti human α-SMA antibody. The cells were washed and labelled with secondary Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, USA) or with secondary Cy2-conjugated anti-mouse IgG (Jackson ImmunoResearch, USA).
For all staining, controls were incubated with secondary antibodies alone. After treatment with nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI), the coverslips were briefly washed first, in PBS and then in distilled water, and finally mounted on glass slides for microscopy examination. Cells were analyzed with a Zeiss LSM 510 Meta laser scanning confocal microscope. Images were analysed using the ImageJ software: the threshold was set on the maximum fluorescence value of the sample treated only with secondary antibody and, for each sample, were quantify 25 cells.
The purification of IgG fractions from serum of MS and control subjects was carried out by affinity chromatography on A/G Sepharose columns (Pierce, Rockford, Ill.). The protein concentration of immunoglobulin fractions thus prepared was assessed spectrophotometrically and used in oligodendrocyte differentiation cell models.
In the study were included men and women between 15 and 50 years of age who meet all the following criteria:
All the patients were subjected to CSF collection by lumbar puncture to execute the routine laboratory analysis in the hospital where they were hospitalized. A quantity of 1-2 ml of CSF was sent to our laboratories to carry out the investigations of our interest. At the same time a blood sample, from each patients, was collected to purify the IgG fractions from blood serum. Patients gave written informed consent before any study-related procedures was performed.
Statistical differences were evaluated using a Student's t-test for unpaired samples.
The authors have developed 2 different protocols to differentiate human oligodendrocyte M03-13 cells. Cells were grown i) in medium with 0-0.2% serum in presence of 100 nM PMA or ii) in complete medium in presence of low doses (200 μM) of hydrogen peroxide. Then, the expression or the localization of specific differentiation markers was evaluated at different times.
The authors first evaluated the effects of 30 min exposure to 100 nM PMA on P-CREB and P-ERK1/2 levels in cells pre-incubated for 16 h in medium containing 0.2% FBS. It is known that both proteins are signalling molecules involved in oligodendrocyte differentiation (Afshari et al., 2001; Chandran et al., 2003).
Different experimental evidences suggest that α-Smooth Muscle Actin (α-SMA) can be considered a negative oligodendrocyte differentiation marker; in Schwann cell-like cells derived from the bone marrow, the withdrawal of differentiation stimulus is accompanied with the generation of SMA expressing cells (Shea et al., 2010); moreover, the treatment of neural stem cells derived from rat spinal cord with Bone Morphogenetic Protein (BMP) inhibits oligodendrocyte formation and generate SMA expressing cells (Chandran, 2003). Therefore, the authors evaluated the expression of P-CREB, P-ERK1/2, α-SMA and Olig-2 protein levels after 24 h of differentiation with 100 nM PMA in absence of serum. As can be shown in
Moreover, the authors evaluated the effects of PMA on the translocation of the specific oligodendrocyte differentiation marker olig-2 from nucleus to cytosol. This protein is located in the nucleus during the early stage of oligodendocyte maturation steps and then migrates from the nucleus to the cytoplasm, becoming inactive.
The authors used the second protocol to differentiate the oligodendrocytes, stimulating M03-13 cells with low doses of hydrogen peroxide for 30 min-4 days, then they evaluated the expression of oligodendrocyte differentiaton markers.
The authors found that stimulation of cells with 200 μM H2O2 for 4 days in complete medium decreasesα-SMA and induces P-CREB levels; the P-ERK1/2 levels were, instead, not affected. The generic antioxidant N-Acetyl-Cysteine (NAC) reverted hydrogen peroxide effects, while the inhibitor of the membrane NADPH oxidase AEBSF, did not (
The effects of 100 nM PMA in serum-free medium or of 200 μM hydrogen peroxide in complete medium for 1 and 4 days on α-SMA, MBP or Olig-2 protein levels were also evaluated by confocal microscopy analysis.
Overall these data demonstrate that stimulation of cells with low doses of hydrogen peroxide induces the differentiation of oligodendrocyte precursors to differentiated cells.
In conclusion, the authors have studied the oligodendrocyte differentiation markers P-CREB, P-ERK1/2, α-SMA, MBP, mAchR M1 and olig-2 and have observed that:
Therefore, such markers are useful for diagnosis, prognosis, to follow up efficacy of a therapy and for the development of treatment for multiple sclerosis.
The following examples demonstrate that incubation of human oligodendrocyte cells with biological fluids from MS patients (CSF or Serum IgG) inhibits cell differentiation.
Oligodendrocytes were treated for 24 h with 100 nM PMA in medium without serum in absence (Differentiated, Diff) and presence of 30% CSF from Control (Ctr) or MS patients and then subjected to Western blotting analysis of P-ERK1/2 (
In differentiated oligodendrocytes in presence of CSF from MS patients, the levels of P-ERK1/2 and Olig-2 were significantly lower and the levels of α-SMA significantly higher compared to Controls (
Oligodendrocytes were treated for 4 days with 100 nM PMA in medium without serum in absence (Differentiated, Diff.) and presence of 30% CSF from Control (N) or MS patients and then subjected to confocal analysis of α-SMA, MBP, and Olig-2. In oligodendrocytes differentiated in presence of CSF from MS patients the levels of α-SMA (
It is known that CREB is a mediator of signal transduction pathways that operates at different stages of oligodendrocyte development and differentiation. In particular, the expression pattern of CREB protein suggests a role of this protein at the later stages of differentiation. For this reason, the authors have also evaluated the effects of CSF from MS patients on P-CREB protein levels in oligodendrocytes differentiated for 4 days. Thirty min exposure of the cells with CSF from MS patients reduces P-CREB protein levels compared to CSF from Controls (
The authors have analyzed the effects of serum IgG from MS patients on P-ERK1/2 levels.
Serum IgG from MS patients reduced P-ERK1/2 levels compared to those found in samples incubated in the presence of IgG of control patients either at 30 min (
The authors have analyzed the effects of serum IgG from MS patients on P-CREB levels after 30 min of differentiation. Serum IgG from MS patients reduced P-CREB levels compared to those in Controls (
The authors have analyzed the effects of serum IgG from MS patients on Olig-2 levels. Serum IgG from MS patients reduced Olig-2 levels compared to those found in samples incubated in the presence of serum IgG of control patients at 24 h of differentiation (
The authors have analyzed the effects of serum IgG from MS patients on α-SMA protein and mRNA levels after 24 h of differentiation.
Serum IgG from MS patients increased α-SMA protein levels after 1d (
The authors have analyzed the effects of serum IgG from MS patients on total (by flow cytometry and confocal analysis) and cytosolic MBP protein levels by Western blotting analysis, after 24 h of differentiation.
Either total or cytosolic MBP protein levels were significantly lower in cells treated with serum IgG from MS compared to those in samples incubated with serum IgG of control patients (
In addition, the effects of serum IgG from MS patients on total MBP protein levels by confocal analysis, after 4 days of differentiation were measured. MBP protein levels were significantly lower in cells treated with serum IgG from MS compared to those in cells treated with IgG of control patients (
The authors have analyzed the effects of serum IgG from MS patients on Olig-2 localization by confocal microscopy. Serum IgG from MS patients inhibits the translocation of Olig-2 from nucleus to cytosol after 24 h of differentiation (
In addition, the effects of serum IgG from MS patients on Olig-2 protein levels by confocal analysis, after 1 and 4 days of differentiation were measured.
Olig-2 protein levels were significantly lower in cells treated with serum IgG from MS compared to those in cells treated with serum IgG from control patients after 1d (
The authors have analyzed the effects of serum IgG from MS patients on mAchR M1 protein levels after 4 days of differentiation.
Serum IgG from MS patients decreased mAchR M1 protein levels compared to those found in samples from serum IgGs of control patients (
To ascertain whether the effects of biological fluids from MS patients on several differentiation markers were specific for oligodendrocyte cells, the authors carried out experiments on mouse cortical primary neurons and on HEK293 cells.
The mouse neurons were incubated for 30 min with CSF from MS or Control patients and P-CREB levels were analyzed (
The authors also tested the effects of serum IgGs from one Control and from two MS patients on P-ERK1/2 levels after 24 h of differentiation in both M03-13 cells and HEK293 cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| RM2010A000654 | Dec 2010 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/072491 | 12/12/2011 | WO | 00 | 8/9/2013 |
