To date, limited studies have examined locus coeruleus pathophysiology or the role of central noradrenaline in multiple sclerosis or its animal model EAE. Our data confirm that there are significant reductions in central levels of noradrenaline in both the brains and spinal cords of EAE mice, and that locus coeruleus neuronal damage is present as indicated by tyrosine hydroxylase positive neuronal cell shrinkage. Our data provide evidence for inflammation occurring in and near to the locus coeruleus in human multiple sclerosis samples, for reduced noradrenaline levels in the tissue surrounding the locus coeruleus and for hypertrophy of tyrosine hydroxylase positive stained neurons. Although several possible explanations could account for reduced noradrenaline levels (decreased synthesis, increased metabolism, increased re-uptake) our findings of inflammation in the locus coeruleus, as well as stress in tyrosine hydroxylase positive neurons argues for loss of noradrenaline synthesis as a contributing cause.
Additional evidence for neuronal damage comes from findings that in EAE, there is a significant reduction in expression of Ear2 in the locus coeruleus, but not the spinal cord. Ear2 is an orphan nuclear receptor expressed during early development in the area where the locus coeruleus develops, and in Ear2 null mice over 70% of locus coeruleus neurons are absent in the adult (Warnecke et al., 2005
). While the role of Ear2 in the adult is largely unknown, its ability to repress lymphocyte expression of IL-17 (Hermann-Kleiter et al., 2008
), a proinflammatory cytokine implicated in EAE and multiple sclerosis disease pathogenesis (Segal, 2010
), suggests that decreased Ear2 could allow for increased IL-17 expression and increased inflammation.
Our data demonstrate locus coeruleus tyrosine hydroxylase positive neuronal atrophy in EAE but not neuronal loss, suggesting that focal inflammatory lesions, which might be expected to cause neuronal death, are relatively sparse in the locus coeruleus in this model. An alternative explanation for increased locus coeruleus neuronal stress are reduced levels of necessary trophic factors or receptors, as suggested by our findings that messenger RNA levels of BDNF are decreased in EAE spinal cord. Locus coeruleus neurons express high levels of the BDNF receptor TrkB during normal development, and a localization, synthesis and anterograde transport of BDNF within noradrenergic neurons has been described (Fawcett et al., 1998
). TrkB deficient mice have 30% fewer locus coeruleus neurons (Holm et al., 2003
), and more recently, BDNF and NT4 were shown to be potent co-inducers of noradrenergic phenotype in primary locus coeruleus cultures (Traver et al., 2006
). Since noradrenaline increases expression of several neurotrophins including BDNF (Zafra et al., 1992
), diminished locus coeruleus function and lower noradrenaline levels could contribute to damage by reducing neurotrophic support from glial cells as well as from locus coeruleus neurons themselves (Fawcett et al., 1998
The above changes are consistent with our findings of reduced cortical and spinal cord levels of noradrenaline, which derive from locus coeruleus afferent fibres. This does not appear to be due to loss of noradrenaline neurons, since locus coeruleus tyrosine hydroxylase positive cell numbers were not reduced. However, the decrease in average cell size, similar to that reported for TgAPP mice (German et al., 2005
), suggests that the locus coeruleus neurons may be compromised in their ability to synthesize or store noradrenaline. Further studies to examine other structural or functional markers of noradrenaline neuronal integrity in the cortex (e.g. fibre density, noradrenaline release or transporter expression) could address that question. Consistent with the decrease of spinal cord noradrenaline levels, we observed increased astrocyte activation in the ventral portion of the locus coeruleus as well as in the area immediately beneath, which contains the dorsal portion of the subcoeruleus neurons, two areas that send noradrenergic afferents primarily to the spinal cord (Holstege and Bongers 1991
; Proudfit and Clark 1991
; Tanaka et al., 1997
). Since spinal cord pathology is a hallmark of myelin oligodendrocyte glycoprotein peptide induced EAE, this raises the possibility that locus coeruleus inflammation or damage may be due in part to retrograde signals originating in the cord, as postulated to occur to cholinergic (Pearson et al., 1983
) and adrenergic (German et al., 1987
) neurons in Alzheimer’s disease. It is not clear why a similar loss was detected in the frontal cortex, since there are few ventral projections to this area. However, this could suggest the presence of more subtle perturbations of dorsally located tyrosine hydroxylase positive neurons that were not detected by our assays. Alternatively, this could be due to increased noradrenaline metabolism in the cortex, rather than reduced noradrenaline production, for example by the enzyme COMT1, whose expression is significantly increased in the brain under inflammatory conditions (Helkamaa et al., 2007
Our findings regarding locus coeruleus damage in patients with multiple sclerosis also point to locus coeruleus neuronal stress or damage. We observed a statistically significant decrease in noradrenaline levels in the central pons area immediately adjacent to the locus coeruleus through which locus coeruleus neurons send projections; to our knowledge this is the first direct demonstration of reduced noradrenaline levels in multiple sclerosis brain. We also measured a statistically significant increase in GFAP staining in the locus coeruleus and the adjacent dorsal tegmental nucleus, similar to the increase observed in the EAE mice. As for EAE, GFAP staining was not always associated with tyrosine hydroxylase positive stained neurons suggesting selective stress or damage to locus coeruleus and dorsal tegmental nucleus neurons.
Our results show that the average size of locus coeruleus tyrosine hydroxylase positive neurons was increased in the multiple sclerosis samples versus controls, in contrast to neuronal atrophy observed in EAE. The discrepancy may reflect important differences in EAE versus multiple sclerosis disease, or could be due to species differences, or to relative ages and the duration of the disease. In EAE, mice were 4-months old at the end of the study and had clinical signs for 2 months. In contrast, the patients with multiple sclerosis ranged in age from 49 to 82, and their disease was an ongoing condition over several years, during which time locus coeruleus neurons may have undergone changes not present in an acute animal model. There is conflicting evidence as to how locus coeruleus neuronal morphology is effected in other neurodegenerative diseases, with swollen cell bodies described in both Alzheimer’s disease and Parkinson’s disease brains (Chan-Palay, 1991
), but cell atrophy (Mann, 1983
) and selective loss of large neurons (Hoogendijk et al., 1995
) observed in some patients with Alzheimer’s disease and locus coeruleus neuronal shrinkage in TgAPP mice (German et al., 2005
). The basis for these differences remains unclear, but may reflect differences in the proportion of surviving locus coeruleus neurons versus those that are undergoing cell death.
An important contributor to disease progression in multiple sclerosis and EAE is leukocyte infiltration through the blood brain barrier. It is well known that noradrenergic innervation of cerebral vasculature preserves the integrity of the blood brain barrier (Harik and McGunigal, 1984
). Correspondingly, we have shown that locus coeruleus lesion leads to disorganization of tight junctions in cerebral endothelial cells (Kalinin et al., 2006a
). Locus coeruleus damage could therefore increase infiltration of activated lymphocytes and exacerbation of disease.
The precise cause(s) of locus coeruleus stress remain to be determined. In both EAE and multiple sclerosis, diffuse axonal damage or inflammation throughout the CNS could account for our findings in the locus coeruleus. However, this does not appear to be the direct cause since two measurements of multiple sclerosis pathology (increased infiltrates, reduced proteolipid protein staining) were inversely correlated to locus coeruleus neuronal stress. Furthermore, findings that show increased GFAP staining is not diffusely spread throughout the locus coeruleus but is primarily in the dorsal portion suggest a topographically defined location for inflammation and axonal damage. Since this area of the locus coeruleus, as well as the dorsal subcoeruleus, sends projections to the lumbar spinal cord, we propose that focal axonal damage and inflammation occurring in the spinal cord results in loss of necessary trophic support or damage to noradrenergic fibre terminals.
Demonstration of perturbations of noradrenaline levels in both EAE and multiple sclerosis provides a rationale for proposing therapeutic strategies to activate, replace or supplement locus coeruleus-noradrenaline transmission. Such an approach has been validated to some extent by preclinical and clinical investigations. Antidepressants that inhibit noradrenaline reuptake can increase BDNF expression in the hippocampus (Russo-Neustadt et al., 1999
), and similarly increasing CNS noradrenaline levels using the selective noradrenaline reuptake inhibitor atomoxetine reduced chemokine and cell adhesion molecule expression following systemic inflammation (O’Sullivan et al., 2010
). In EAE studies we showed that raising CNS noradrenaline levels with l
-threo-3,4-dihydroxyphenylserine (a direct precursor of noradrenaline) stabilized or improved clinical severity (Simonini et al., 2010
). Suggestions of benefit from raising CNS noradrenaline levels in humans come from a limited number of clinical trials. In a small clinical trial (69 patients with multiple sclerosis per arm), treatment with l
-phenylalanine (required for noradrenaline synthesis) together with the noradrenaline reuptake inhibitor lofepramine reduced total cumulative disability over 24 weeks as compared with the control group (Wade et al., 2002
). In a subset of 15 of those patients, effects of treatment on MRI were observed including a significantly reduced T1
lesion number (Puri et al., 2001
). Together, the preclinical and clinical outcomes of these noradrenaline-based approaches suggest that other pharmacological strategies to increase synaptic noradrenaline transmission may hold promise as alternative or additional therapies in multiple sclerosis.