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Keywords:

  • experimental autoimmune encephalomyelitis;
  • glial cells;
  • Locus coeruleus;
  • noradrenaline;
  • tyrosine hydroxylase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2012) 121, 206–216.

Abstract

The endogenous neurotransmitter noradrenaline (NA) plays several roles in maintaining brain homeostasis, including exerting anti-inflammatory and neuroprotective effects. The primary source of NA in the CNS are tyrosine hydroxylase (TH)-positive neurons located in the Locus coeruleus (LC) which send projections throughout the brain and spinal cord. We recently demonstrated that dysregulation of the LC:Noradrenergic system occurs in experimental autoimmune encephalomyelitis as well as in MS patients, associated with damage occurring to LC neurons. Vindeburnol, a structural analog of the cerebral vasodilator vincamine, was previously reported to increase TH expression and activity in LC neurons. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, and treated with vindeburnol at the first appearance of clinical signs. Clinical signs continued to increase for about 1 week, at which point mice in the vehicle group continued to worsen while vindeburnol-treated mice showed improvement. Pro-inflammatory cytokine production from splenic T cells was not reduced by vindeburnol suggesting primarily central actions of treatment. In the cerebellum, vindeburnol decreased astrocyte activation and reduced the number of demyelinated regions. Vindeburnol reduced astrocyte activation in the LC, reduced TH+ neuronal hypertrophy, increased expression of several genes involved in LC survival and maturation, and increased NA levels in the spinal cord. These results suggest that treatments with drugs such as vindeburnol which target LC survival or function could be of benefit in MS patients.

Abbreviations used
AD

Alzheimer's disease

COMT

catecholamine-O-methyl transferase

DBH

dopamine beta-hydroxylase

EAE

experimental autoimmune encephalomyelitis

GFAP

glial fibrillary acidic protein

LC

Locus coeruleus

MAO

monoamine oxidase

MOG

myelin oligodendrocyte glycoprotein

NA

noradrenaline

PBS

phosphate-buffered saline

TH

tyrosine hydroxylase

The endogenous neurotransmitter noradrenaline (NA) plays several important roles in maintaining normal CNS physiology. NA acting at β2-adrenergic receptors can suppress glial as well as neuronal inflammatory responses (Frohman et al. 1988; Minghetti et al. 1997; Szabo et al. 1997; Galea and Feinstein 1999), and in vivo selective NA reuptake inhibitors can reduce CNS cytokine and chemokine expression (O’Sullivan et al. 2010), and increase anti-inflammatory cytokine expression (McNamee et al. 2010). NA acting directly on neurons can reduce neurotoxicity elicited by inflammatory (Madrigal et al. 2005) or excitotoxic (Madrigal et al. 2007) stimuli, both in vitro and in vivo (Marien et al. 2004; Traver et al. 2005). NA can increase neurotrophin expression in glial cells (Debeir et al. 2004), including that of brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) (Remy et al. 2001), important for maintenance of neuronal integrity and synaptic plasticity. NA also has beneficial effects on neural progenitor cells (Ghiani et al. 1999), including on the maturation of oligodendrocyte progenitor cells which is of particular relevance within the context of demyelinating diseases. Disturbances in NA homeostasis could therefore influence the course of neurological diseases such as MS.

Evidence that NA plays a role in MS is suggested by studies showing alterations in CNS NA levels. In experimental autoimmune encephalomyelitis (EAE), CSF and white matter NA levels were found increased before clinical signs appeared, but were reduced at later times (Khoruzhaia and Saakov 1975). Similarly, NA levels were found reduced in the brainstem and spinal cords of EAE rats (White et al. 1983; Krenger et al. 1986). There is also evidence that dysregulation of CNS NA occurs in MS, for example NA levels were higher in CSF samples of MS patients compared with controls (Barkhatova et al. 1998); and CSF levels of the NA metabolite methoxyhydroxyphenylglycol were inversely correlated to the duration of illness and number of relapses (Markianos et al. 2009).

The major source of NA in the CNS are large tyrosine hydroxylase (TH)-expressing neurons of the Locus coeruleus (LC) located in the rostral portion of the pons. The LC provides noradrenergic input to most regions of the brain including cortex, cerebellum, brainstem, and spinal cord. Substantial loss of LC neurons (Mann et al. 1980; Tomlinson et al. 1981; Bondareff et al. 1987) and reduced levels of NA (Adolfsson et al. 1979; Mann and Yates 1983) are well known hallmarks of several diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The consequences of LC loss are not fully understood, but increasing reports demonstrate the experimental lesion of the LC exacerbates inflammatory responses and the progression of neurodegeneration in mouse models of AD (Heneka et al. 2006; Kalinin et al. 2007; Jardanhazi-Kurutz et al. 2010). In view of reports suggesting that NA levels are disturbed in EAE and MS, we examined LC physiology in MOG peptide immunized mice and in sections of the LC from MS patients and healthy controls (Polak et al. 2011). These findings demonstrated the presence of LC stress and damage in both EAE and MS, that was associated with increased glial inflammation and reduced levels of NA. A recent study showing perturbation of the LC:Noradrenergic system in a mouse model of Down syndrome (Salehi et al. 2009), and of LC damage in veterans with post-traumatic stress disorder (Bracha et al. 2005), suggests that LC damage may be a commonality to several neurological conditions and diseases.

To address LC damage and the perturbation of NA levels that occurs, several methods to increase CNS NA levels have been tested and shown to provide benefit. Treatment with α2-antagonists, which increase NA release from LC neurons, reduced damage caused by intraparenchymal injection of Αβ1-42 (Kalinin et al. 2006), and that treatment with droxidopa, a synthetic precursor of NA, reduced pathology in a robust mouse model of AD (Kalinin et al. 2011), and reduced behavioral deficits in a mouse model of Down’s syndrome (Salehi et al. 2009). More recently, we showed that treatment of EAE mice with droxidopa stabilized disease progression in MOG peptide induced EAE while co-treatment with a NA reuptake inhibitor led to clinical improvement (Simonini et al. 2010).

While methods to increase CNS NA levels may provide some benefit in neurological diseases including MS, they do not address a primary cause of NA dysfunction, namely LC stress and damage. In this regard, drugs which selectively increase LC neuronal viability or activity might be of benefit. Vindeburnol (14,15-dihydro-20,21-dinoreburnamenine-14-ol, Fig. 1a) is a semi-synthetic derivative of the plant alkaloid vincamine (Fig. 1b), and structurally related to vinpocetine (Fig. 1c), recently shown to inhibit the NFkB signaling system (Jeon et al. 2010). Vincamine is a peripheral vasodilator isolated from the plant Vinca minor; originally characterized for possible effects on cognition and memory caused by its ability to increase blood flow to the brain (Vas and Gulyas 2005). Observations that vincamine could activate noradrenergic firing in the LC (Olpe and Steinmann 1982) suggested vindeburnol might influence LC physiology. Vindeburnol injection caused a rapid depletion of brain NA levels in rats (Euvrard and Boissier 1981), and increased TH activity and protein expression in the LC, but not in other dopaminergic nuclei (Weissmann et al. 1988). Although mechanistic studies have not been done, immunohistochemical studies following treatment in adult rats suggest that vindeburnol induces re-activation of TH in a set of LC neurons that transiently express TH during early development (Marcel et al. 1998; Bezin et al. 2000). In this study, we demonstrate that vindeburnol reduces clinical scores in mice immunized with MOG peptide to develop chronic disease, and is associated with improvements in LC physiology and function.

image

Figure 1.  Chemical structures of related vincamine derivatives: (a) vindeburnol; (b) vincamine; and (c) vinpocetine.

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Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents

General chemicals and reagents were from Sigma (St Louis, MO, USA). Secondary antibodies were from Jackson Immunoresearch (West Grove, PA, USA). The MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Anaspec (San Jose, CA, USA). Vindeburnol was provided by Pierre Fabres (Toulouse, France).

Mice

Female C57BL/6 mice aged 6–8 weeks were purchased from Charles River Breeding (Cambridge, MA, USA). Mice were housed five per cage, and kept in a controlled 12 : 12 h light/dark environment and provided food ad libitum. All animal procedures were approved by the local IACUC.

Induction of EAE

EAE was actively induced using synthetic myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35-55) as described (Feinstein et al. 2002). Mice were injected subcutaneously (s.c., two 100 μL injections into adjacent areas in one hind limb) with an emulsion of 300 μg MOG35-55 dissolved in 100 μL phosphate-buffered saline (PBS), mixed with 100 μL complete Freund’s adjuvant containing 500 μg of Mycobacterium tuberculosis (Difco, Detroit, MI, USA). Immediately after MOG35-55 injection, the animals received an intraperitoneal (i.p.) injection of pertussis toxin (200 ng in 200 μL PBS). Two days later the mice received a second pertussis toxin injection, and 1 week later they received a booster injection of MOG35-55. Vindeburnol (or vehicle) was prepared by suspending 4 mg vindeburnol in 1.0 mL PBS containing 1% Tween-20, administered either three or six times per week at 20 mg/kg i.p. Clinical signs were scored on a five-point scale: grade 0, no clinical signs; 1, limp tail; 2, impaired righting; 3, paresis of one hind limb; 4; paresis of two hind limbs; 5, death. When a mouse died, it was assigned a score of 5 and which was carried through for the rest of the study. Scoring was done at the same time each day by a blinded investigator.

T-cell isolation and cytokine measurements

Splenocytes were isolated from MOG35-55 immunized EAE mice. After lysis of red blood cells, splenocytes were plated into 24 well plates at a density of 2 × 105 cells per well in 400 μL RPMI media containing 10% fetal calf serum (FCS), and incubated with 0 or 20 μg/mL MOG35-55 peptide, or with rat monoclonal anti-CD3 (0.20 μg/mL) and anti-CD28 (0.5 μg/mL) antibodies. After 1 day, aliquots of the media were assayed for levels of IL-17 and IFNγ by ELISA following recommended procedures (eBioscience, San Diego, CA, USA).

Tissue preparation and immunohistochemistry

Mouse brains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.6 overnight at 4°C. Dehydration, embedding, paraffin removal, and sectioning were done using standard protocols as described (Sharp et al. 2008). Serial sagittal sections (8 μm) were taken through to include the cerebellum and the rostral part of the brainstem containing the LC. Following paraffin removal, antigen retrieval was accomplished by boiling in 10 mM citrate buffer for 10 min, then blocking with 5% normal donkey serum. Sections were incubated 4°C overnight with primary antibodies diluted in 1% normal donkey serum: rat monoclonal anti-human glial fibrillary acidic protein (GFAP) B2.210 at 1 : 300 (Trojanowski et al. 1986); rabbit polyclonal anti-tyrosine hydroxylase (TH) at 1 : 300 (Pel-Freeze, Rogers, AK, USA); rabbit polyclonal anti-proteolipid protein at 1 : 800 (a gift of Dr Robert Skoff). After washing, sections were incubated 1 h at 37°C with donkey anti-rabbit RRX conjugated and donkey anti-rat conjugated with FITC secondary antibodies. Sections were washed, fixed with 3.7% formaldehyde in PBS, quenched in 50 mM ammonium chloride in PBS for 15 min, then final washes done in PBS with 400 ng/mL DAPI included in the second wash. Vectashield mounting fluid (Vector Laboratories Inc.) was placed on sections prior to cover slips.

Image analysis

Images were obtained on a Zeiss Axioplan 2 microscope using an MRm Axiocam for image acquisition and densitometric analysis conducted using Axiovision version 4.5 software (Carl Zeiss Inc. Thornwood, NY, USA). Image acquisition was from sections stained simultaneously and exposed for identical amounts of time. Quantitation of GFAP staining was done using an object area cutoff of 10 μm2 to include cell bodies and processes. The data were analyzed to determine the total number of positively stained objects per field of view, and the total area covered by positively stained objects presented as a percentage of the total field of view. Quantitation of TH stained cell bodies was accomplished using an object area cutoff of 60 μm2 to exclude counting of processes. TH data were analyzed to determine the total number of positively stained cell bodies per field of view, and the average cell body area.

Measurements of noradrenaline levels

Cell lysates were prepared from lumbar spinal cords of EAE and non-EAE mice. Tissues were homogenized on ice in 40 volumes of 0.01 N HCl, 1 mM EDTA and 4 mM sodium metabisulfite. ELISA for NA was performed per manufacturer’s instructions (Rocky Mountain Diagnostics Inc., Colorado Springs, CO, USA).

mRNA analysis

Total RNA from the LC of control and EAE mice was isolated using TRIZOL reagent (Invitrogen/GIBCO, Carlsbad, CA, USA). Real-time quantitative PCR (qPCR) was carried out using Taqman 384-well Micro Fluidic cards (Applied Biosystems, Carlsbad, CA, USA) with primers designed to span an intron and amplify alternatively spliced transcripts. The qPCR was carried out in a 7900 HT cycler, and analyzed using DataAssist V2.0. Calculated Ct (cycle take off) values were used to determine relative mRNA levels normalized to values measured for β-actin in the same samples. Significant differences (p < 0.050) were determined by unpaired t-tests.

Data analysis

Comparison of clinical signs over time in one group was done by one-way, non-parametric anova (Kruski–Wallis test) followed by Dunn’s multiple comparison tests. Comparison of the effect of treatment versus control on the development of clinical signs was done by two-way repeated measures anova. Two group comparisons were done by Mann–Whitney non-parametric unpaired t-tests. In all cases, significance was taken at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Vindeburnol reduces clinical scores in MOG peptide induced EAE

In the first set of studies, we tested the effects of 20 mg/kg vindeburnol administered intraperitoneally 3 times per week (Monday, Wednesday, and Friday). Treatment with vindeburnol or vehicle was started 10 days after the booster immunization with MOG peptide, at which time about 80% of the mice in each group were exhibiting clinical signs and the average clinical score was the same. In both the vehicle-treated (n = 25) and the vindeburnol-treated (n = 23) groups, the incidence of disease reached 100% by day 12 (Fig. 2a). In the vehicle group, there was a significant increase in clinical scores versus baseline at day 13 (one-way anova), and the maximum average clinical score (3.8 ± 0.10) was reached on day 19 after which time it remained unchanged (Fig. 2b). From day 10 onward, 3/25 mice showed clinical improvement in the vehicle group, while 4/25 mice died.

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Figure 2.  Vindeburnol ameliorates clinical signs of EAE. C57BL/6 female mice aged 6–8 weeks were immunized with MOG 35–55 peptide and treated with vindeburnol (20 mg/kg i.p.) 3 times per week. The data shown are mean ± SE and are derived from two separate studies. Data in panel (a) show the average daily incidence of disease; and in panel (b) the average daily clinical scores in vehicle (filled circles, n = 25) and vinderburnol-treated (open circles, n = 23) mice. In both groups, scores were significantly different from baseline values by day 12 post-MOG booster (one-way anova). There was a significant effect of vindeburnol on clinical scores from day 10 onwards [two-way repeated measures anova, F(14,1) = 3.52, p < 0.0001]. Data in panel (c) show the daily average clinical scores for the group of vindeburnol-treated mice who showed improvement (‘responders’, open circles, n = 11, two-way repeated measures anovaF(14,1) = 7.68, p < .0001] and for those that did not show any improvement (‘non-responders’, filled circles, n = 11).

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In the vindeburnol group, clinical scores were significantly different from baseline at day 13, and the maximal average clinical score (3.6 ± 0.16) was reached on day 17, after which it began to decrease reaching 3.0 ± 0.20 at the end of the study. From day 10 onward, 11/23 mice showed clinical improvement, while one mouse died. There was a statistically significant treatment x time effect between vehicle and vindeburnol groups, using data from treatment start (day 10) onwards [F(14,1) = 3.52, p < 0.0001; two-way repeated measures anova]. Inspection of the development of clinical scores in the 11 mice who responded to treatment (Fig. 2c) shows that the average clinical score decreased from 3.8 ± 0.10 on day 14 to 2.4 ± 0.11 at the end of the study.

To determine if increased drug exposure could provide further benefit, we carried out a second study (Fig. 3) in which a dosing regimen of 6 days per week (only once per weekend) was directly compared with dosing every other day, because limited solubility of vindeburnol prevented using higher daily doses. Similar to the first study, the incidence of disease reached 100% in all groups by day 13. Clinical scores in the vehicle group peaked on day 17 at 2.6 ± 0.20, and only 1/11 mice showed any clinical improvement till the end of the study (day 20). As in study 1, in the group treated with vindeburnol 3 times per week, 3/6 (50%) showed improvement during treatment, and the maximal average score decreased from 2.5 ± 0.6 on day 11 to 1.8 ± 0.2 on day 20 [F(11,1) = 2.25, p = 0.014]. In mice treated with vindeburnol six times per week, the maximal average score was 2.4 ± 0.3 on day 11, after which it began to decrease reaching 1.1 ± 0.3 at the end of the study. In this group, from day 10 onward, six of eight mice showed clinical improvement and two mice did not respond to treatment. The overall incidence in this group decreased to 87.5% (7/8) at day 17 because one animal showed full recovery. There was a statistically significant treatment × time effect between the vehicle and 6× vindeburnol groups, using data from treatment start onwards [F(11,1) = 8.58, p < 0.0001; two-way repeated measures anova].

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Figure 3.  Effect of dosing schedule on clinical scores. C57BL/6 female mice were immunized with MOG 35–55 peptide, then treated with vindeburnol (20 mg/kg i.p.) either 3× or 6× per week beginning on day 9. The data in panel (a) show the average daily incidence of disease; and in panel (b) the average daily clinical scores (mean ± SE) in mice treated with vehicle 6× per week (filled circles, n = 11), vinderburnol 3× per week (open circles, n = 6), or vindeburnol 6× per week (filled squares, n = 8). In both treatment groups, there was a significant effect on clinical scores from day 11 onwards as compared with vehicle-treated mice [two-way repeated measures anova; F(11,1) = 2.25, p = 0.014 for 3× per week; F(11,1) = 8.58, p < 0.0001 for 6× per week].

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Although vindeburnol is expected to act primarily on noradrenergic neurons present in the LC, possible effects on peripheral immune function could contribute to observed beneficial effects on clinical scores. To test this possibility, splenic T cells were isolated from vehicle and vindeburnol-treated EAE mice, and re-stimulated ex vivo with MOG35-55 peptide or with antibody to the T-cell receptor CD3 (Fig. 4). ELISA for cytokines in conditioned media generated after 24 h incubation did not reveal any differences in levels of IL-17a or of IFNγ between the vehicle-treated mice and the vindeburnol-treated mice that showed clinical improvement. Interestingly, media levels of both IL-17 and IFNγ were higher in T cells isolated from mice that did not respond to vindeburnol treatment. Further characterization of vindeburnol effects are therefore reported only for those mice that responded to treatment.

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Figure 4.  Vindeburnol does not reduce T cell cytokine production. At the end of the study shown in Fig. 2, splenic T cells were isolated from vehicle-treated mice and from mice provided vindeburnol who responded (Resp) or did not respond (Non) to treatment. The cells were cultured in media alone, or in the presence of MOG35-55 peptide (20 μg/mL), or with antibody to CD3 receptor. After 24 h, the levels of (a) IL-17 and (b) IFNγ were measured in the media. Data are mean ± SE of the fold activation compared to media alone, n = 4 mice per group; *p < 0.05 versus vehicle.

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Vindeburnol reduces glial activation and lesion numbers in the cerebellum

Immunohistochemical staining was used to determine if treatment reduced astrocyte activation (Fig. 5). In the cerebellum, there was a significant increase in GFAP staining for both the number of objects stained (Fig. 5b) and the total area covered by staining (Fig. 5c) in EAE mice compared with non-immunized mice, in both the white matter as well as the molecular layers. GFAP staining was significantly less in both regions in the vindeburnol-treated mice.

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Figure 5.  Vindeburnol reduces astrocyte activation in cerebellum. At the end of the study shown in Fig. 2, mice were killed, and serial cerebellar sections stained for GFAP. (a) Representative images from non-immunized (n = 6); vehicle-treated EAE (n = 5); and vindeburnol-treated responder EAE (n = 5) mice are shown. Quantitative analysis of 3–5 sections per mouse shows a significant reduction in total number of GFAP+ stained objects (b) and % area covered (c) in vindeburnol versus vehicle-treated groups. **p < 0.01 versus non-EAE; §< 0.005 versus vehicle. Scale bar is 500 μm.

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Immunostaining for proteolipid protein, an abundant membrane-associated protein component of the myelin membrane expressed throughout the CNS in oligodendrocytes, revealed the presence of demyelinated areas in the cerebellum of most EAE mice (Fig. 6a) that was reduced in mice who responded to treatment (Fig. 6b). Quantitation of the number of demyelinated regions showed that treatment with vindeburnol significantly reduced the average number of demyelinated areas by about 50% (Fig. 6c) compared with vehicle treatment.

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Figure 6.  Vindeburnol reduces lesions in cerebellum. Serial cerebellar sections from vehicle (n = 5) and vindeburnol (n = 5)-treated EAE mice as described above were stained for proteolipid protein. (a) Representative images from vehicle-treated and vindeburnol-treated mice showing the presence of large lesions (indicated by an *) in the vehicle-treated section. (b) Quantitative analysis of six sections per mouse revealed a significant reduction in the total number of large lesions in the vindeburnol-treated group. Data are mean ± SE total lesions per mouse; *p < 0.05.

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Vindeburnol reduces glial activation in the LC

As previously demonstrated (Polak et al. 2011), there was a significant increase in astrocyte activation assessed by GFAP staining in and around the LC of EAE mice as compared with non-immunized controls (Fig. 7a). In vindeburnol-treated EAE mice, there was an almost significant (p = 0.06) decrease in the total number of objects (cells and processes) that were stained for GFAP (Fig. 7b), and a significant decrease of approximately 30% in the area covered by GFAP staining (Fig. 7c).

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Figure 7.  VIndeburnol reduces astrocyte activation in the LC. (a) Representative images from immunostaining of serial sections prepared from the LC of non-EAE, vehicle-treated, and vindeburnol-treated responder mice co-stained for GFAP (green) and TH (red). Quantitative analysis showed an almost significant reduction (p = 0.06) in the number of GFAP+ stained objects (b), and a significant reduction in the % area stained (c) in the vindeburnol versus vehicle-treated animals. Data are mean ± SE of n = 5 mice per group, n = 3 sections per mouse. *p < 0.05 versus EAE-vehicle treated.

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Vindeburnol increases markers of LC maturation and survival

To determine if vindeburnol modified LC neuronal physiology, we stained serial sections through the LC for the noradrenergic marker TH (Fig. 8). In agreement with previous findings, we did not observe a significant decrease in either total TH+ cell numbers or the area stained by TH in EAE mice compared with controls; and treatment with vindeburnol did not modify these values (Fig. 8a and b). Measurements of the average TH+ neuronal cell size did not show any significant difference between the EAE mice and controls (Fig. 8c); but did show significantly reduced cell size in the vindeburnol-treated samples compared with vehicle-treated samples. A frequency distribution of cell sizes (Fig. 8d) shows a significant, approximately 2-fold increase in the relative number of TH+ cells in the size range 125–225 μm2 in the vindeburnol-treated versus the vehicle-treated EAE mice.

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Figure 8.  Vindeburnol reduces LC TH+ neuronal hypertrophy. The same images as described in Fig. 7 were analyzed for TH+ neurons. Quantitative analysis showed no differences in either (a) the number of TH+ cells; or (b) the % area covered by TH staining. (c) There was a significant reduction in average TH+ cell body size in the vindeburnol versus vehicle-treated samples. (d) Histogram showing relative frequency distribution of TH+ stained cell sizes. Each bin includes cells of that size ± 25 μm2, and shows an increase in the number of cells in the 125–225 μm2 range in the vindeburnol-treated samples. Data are mean ± SE of n = 5 mice per group, n = 3 sections per mouse. *p < 0.05 versus EAE-vehicle treated.

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As a second index of LC TH+ neuronal health, we measured levels of several mRNAs involved in LC maturation and survival (Fig. 9a). The relative mRNA levels of the Noradrenergic transcription factor Mash1, the NA transporter NET1, and the enzymes dopamine decarboxylase and TH were significantly reduced in EAE mice compared with non-EAE mice. In vindeburnol-treated mice, these levels were significantly increased as compared with vehicle-treated mice. In addition, vindeburnol significantly increased the relative mRNA levels of the enzyme dopamine beta-hydroxylase (DBH) and the α2-adrenergic receptor compared with vehicle-treated samples. Levels of the noradrenergic transcription factor Phox2a were not significantly altered in EAE, or by vindeburnol treatment. These findings suggest that vindeburnol prevented losses, or increased expression of genes involved in LC maturation and function. Interestingly, the increases in DBH and TH levels were only observed in those mice that responded to treatment (Fig. 9b).

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Figure 9.  Vindeburnol modifies LC mRNA expression and function. (a) Total RNA samples were prepared from the LC of non-immunized mice, vehicle-treated EAE mice, and vindeburnol-treated responder EAE mice at the end of treatment shown in Fig. 2 (n = 3 per group). The data are mean ± SE of relative mRNA levels normalized to values measured for β-actin in the same samples. *p < 0.05 versus non-EAE; #p < 0.05 versus vehicle. (b) Relative levels for DBH and TH mRNAs were measured in RNA samples prepared from a second cohort of non-immunized mice, EAE mice treated with vehicle, and EAE mice treated with vindeburnol who did (responders) or did not (non-responders) show any clinical improvement. Data are mean ± SE n = 4 per group. *p < 0.05 versus non-EAE; #p < .05 versus vehicle treatment. (c) The levels of NA were measured by specific ELISA in tissue lysates prepared from the lumbar spinal cords of vehicle and vindeburnol-treated non-EAE, and from EAE mice treated with vehicle, or with vindeburnol who responded (R) or did not respond (NR) to treatment. The data are mean ± SE pg NA per mg wet weight tissue, n = 3–4 per group, *p < 0.05 versus vehicle-treated EAE.

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To test if the vindeburnol dependent increases in LC-related mRNAs had a functional outcome, we measured NA levels in the lumbar spinal cord, the region where NA levels were found most reduced in EAE mice (Polak et al. 2011). As previously observed, there was a significant decrease in NA in the spinal cord (Fig. 9c) that was attenuated in those mice that responded to vindeburnol treatment. In contrast, NA levels were not significantly increased in mice that did not respond to vindeburnol treatment.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we show that vindeburnol provides benefit in a chronic mouse model of MS, associated with improvements in LC physiology. Early studies using vindeburnol, also known as RU-24722, were based on observations that the structurally related compound vincamine increases cerebral blood flow and metabolism (Tesseris et al. 1975) and activates noradrenergic neurons in the LC of the rat (Olpe and Steinmann 1982). Vindeburnol was originally shown to enhance post-ischemic recovery in gerbils and rats (Barzaghi et al. 1985), and found to selectively deplete CNS NA levels (Euvrard and Boissier 1981). Subsequently, it was found that vindeburnol (a single dose of 10 mg/kg, i.p.) caused a rapid (within 1 h) decrease of NA levels in mouse brain at the same time increasing levels of several NA metabolites, indicating an increase in NA turnover (Takeda et al. 1984). Vindeburnol’s mechanism of action appears to differ from that of vincamine which had little effect on brain monoamine levels when used at higher doses, and differs from that of reserpine, an inhibitor of the vesicular monoamine transporter which blocks storage of monoamines into vesicles.

The above studies led to the hypothesis that vinderburnol influenced the LC: noradrenergic signaling system. This was confirmed in studies using adult rats, where single injections of vindeburnol were shown to selectively increase TH protein levels in the LC (Labatut et al. 1988) but not in other dopaminergic nuclei (Weissmann et al. 1988). Within the LC, the specific cells that are induced to increase TH expression are not located throughout, but are present in certain sub-regions. Using quantitative immunostaining methods for TH (Debure et al. 1992), it was shown that in normal adult rat LC, there is a heterogeneous distribution of TH+ cells and TH protein expression with a significantly greater number and greater expression in the posterior compared with the anterior region of the LC. Following treatment with vindeburnol, the total numbers of TH+ neurons and levels of TH expression were significantly increased across the entire LC, due to an increase of TH expression in the anterior located cells, and to de novo appearance of TH expression in the posterior region. These cells were subsequently shown to be Phox2A+/TH− neurons that are capable of re-expressing TH (Bezin et al. 2000). Our data showing that vindeburnol increased levels of several mRNAs that are involved in LC maturation (Mash1), NA synthesis (DBH, dopamine decarboxylase), or NA signaling (NET1, α2AR) could therefore be due to reduced damage occurring to existing TH+ neurons, or to the appearance of new TH+ cells.

The mechanisms of action of vindeburnol are not yet well known. In vivo studies showed that its effects on TH maturation could be prevented by co-treatment with clonidine, an α2-adrenergic receptor antagonist (Labatut et al. 1988), suggesting that vindeburnol actions involve binding to these receptors. As LC neurons express α2-adrenergic receptors, binding of vindeburnol to those autoreceptors could lead to a sustained increase in NA release and ultimately a requirement for de novo NA synthesis and increased TH expression. The related compound vinpocetine has been tested in several models of neurological diseases, is used in Europe to treat age related memory deficits, and has been considered for treatment of AD and Parkinson's disease (Szatmari and Whitehouse 2003). Vinpocetine was shown to inhibit certain phosphodiesterases and modulate calcium channels (Vas and Gulyas 2005), and recently to inhibit the NFkB signaling system (Jeon et al. 2010). Whether vindeburnol has any of these properties remains to be determined.

Our results using splenic T cells show no reduction because of vindeburnol treatment on pro-inflammatory cytokine production; and is consistent with the premise that the actions of vindeburnol occur within the CNS. However, it is unclear why the effects of vindeburnol on clinical scores in EAE were not observed in all the treated mice. After 3 weeks treatment with vindeburnol given 3× per week, we observed significant improvement of clinical signs in about 50% of the vindeburnol-treated mice. When the dosing regimen was increased to 6× per week (only given once on weekends), the number of responders increased to 75% (6/8). The observations that IFNγ and IL17 levels were greater in splenic T cells prepared from non-responder mice offers a possible explanation to help account for the apparent lack of clinical improvement, because larger peripheral inflammatory responses could have caused increased inflammation within the LC, thereby reducing the efficacy of vindeburnol to prevent LC neuronal damage. That these mice showed similar clinical signs as the mice who responded to vindeburnol could be due to the fact that treatment was started at day 10, the time when clinical signs first appear and before the consequences of increased immune responses could be detected by the scoring methods used. Other possible reasons for lack of response to low dosing of vindeburnol, for example differences in expression levels of enzymes that metabolize NA, could also contribute to this observation and are currently being explored.

Regardless of the precise reasons why some mice did not respond, our findings that increases in LC TH and DBH mRNA levels, and in spinal cord NA levels, occurred in responders supports the premise that beneficial effects of vindeburnol are due, at least in part, to improvement of LC function. In those mice, measurements of spinal cord NA showed an approximate 50% increase over levels measured in vehicle-treated mice. However, these values (300 pg/mg wet weight) are still significantly reduced as compared with levels measured in non-immunized mice. One possible explanation is that during EAE, there is not only reduced production of NA, but increased metabolism. NA (as well as other catecholamines) are metabolized by a variety of enzymes including catecholamine-O-methyl transferase (COMT) and monoamine oxidase (MAO). Both MAO and COMT are ubiquitous enzymes expressed throughout the CNS including in the spinal cord (Hong et al. 1998; Samantaray et al. 2008), and both have been shown to be increased in neurological diseases including amyotrophic lateral sclerosis (Ekblom et al. 1993). MAO inhibitors have shown efficacy in MOG peptide induced EAE (Musgrave et al. 2011), while COMT inhibitors are now used in Parkinson’s disease as adjuncts to treatment with levodopa (Marin and Obeso 2010). Treatments to increase CNS NA levels, either by directly targeting the LC, or by use of NA precursors such as droxidopa (Simonini et al. 2010) may therefore benefit by co-administration of MAO or COMT inhibitors.

In conclusion, the current data suggest that treatment with vindeburnol can provide benefit in EAE, and is associated with positive effects on LC physiology. Vindeburnol is now being tested in phase I clinical trials in France for treatment of clinical resistant depression (http://www.biocortech.com/newEvents.php?news=2010), based on its ability to increase blood flow to the brain. Positive findings from those studies together with our current findings could provide the basis for testing of vindeburnol in MS patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by a grant from the National Multiple Sclerosis society.

References

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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