Axonal Damage in Multiple Sclerosis - CLINICAL DATA SUPPORTING NEURONAL DAMAGE IN MS

CLINICAL DATA SUPPORTING NEURONAL DAMAGE IN MS

Clinically, MS disease progression is measured by the Expanded Disability Status Scale (EDSS).52 Progressive exacerbation of the clinical symptoms is seen over time in SPMS patients and correlates most significantly with axonal damage.4 In addition, PPMS disease progression correlates with axonal loss and exhibits less-prominent inflammation than in SPMS.53,54 One of the most striking clinical observations is the fact that immunomodulatory therapies have been able to control the number of relapses,55 but patients continue to worsen clinically56 and show signs of cortical atrophy.57

Axonal damage in MS patients, as defined by spectroscopic measurements of metabolites, has also been correlated also with the progressive worsening of numerous symptoms including fatigue,58 cognitive dysfunction,59 and memory impairment.60 Very recently, cognitive impairment in MS patients was reported to be associated with GM atrophy,61 especially in the CA1 and CA3 region of the hippocampus.62 Together these data contribute to support the concept that the pathogenesis of neurodegeneration in MS may be independent of the immune system and blood-brain barrier permeability and involve other mechanisms and players.

Whereas immunomodulatory therapies have been able to control the number of relapses, patients continue to worsen clinically and show signs of cortical atrophy. The pathogenesis of neurodegeneration in multiple sclerosis may thus be independent of the immune system and blood-brain barrier permeability and involve other mechanisms and other players.

BIOCHEMICAL DATA SUPPORTING NEURONAL DAMAGE IN MULTIPLE SCLEROSIS

Further supporting the notion that axonal injury occurs in MS, increased levels of axonal cytoskeletal proteins are found in the cerebral spinal fluid (CSF) of MS patients, including tubulin, actin, NFL,63 and tau.64 Interestingly, the detection of NFL in the CSF of MS patients positively correlates with EDSS score65 and its presence is not limited to the late stages of disease progression, but it is observed throughout the disease course, thereby suggesting that axonal damage may not only occur as the consequence of long-term demyelination.66 One intriguing hypothesis is that a primary neurodegenerative event generates axonal debris and possibly myelin degradation products that may then elicit the production of reactive autoantibodies that appear in the CSF, including those to myelin-specific proteins.67 However, we cannot exclude the possibility that the detection of autoantibodies against neuronal components could be the consequence of axonal damage and transections and thereby could be considered as marker of axonal integrity and disease progression.

IMAGING DATA SUPPORTING NEURONAL DAMAGE IN MULTIPLE SCLEROSIS

Magnetic Resonance Imaging Studies

Magnetic resonance imaging (MRI) is widely used to diagnose MS and monitor disease progression by evaluating focal abnormalities in the CNS. Conventional MRI techniques have utility for the noninvasive measurement of WM lesions, including global and regional brain volume determinations.68 Advanced, quantitative MRI methods have the ability to detect early changes in the NAWM of MS patients, which provides clues to early disease pathogenesis.69 Longitudinal MRI studies in RRMS patients show that brain atrophy correlates positively with subsequent disability status.70,71 Thus, several studies correlate cortical thinning with MS disease severity but do not define whether thinning is just the consequence of axonal damage following long-term demyelination or occurring independently from it.57 New studies, reporting cortical and subcortical GM volume loss since the earliest stages of the disease, suggest that axonal damage may occur independently of demyelination.72 In a recent longitudinal study, Fisniku et al.38 evaluated tissue-specific atrophy in a cohort of 73 MS patients initially presenting with a clinically isolated syndrome (CIS) who were followed for 20 years, with clinical and MRI evaluations. The investigators noted that the extent of atrophy in the GM of MS patients was greater than that in WM areas, and that GM atrophy proved to be a stronger predictor of disability than focal WM lesion load and WM atrophy.38

New studies, reporting cortical and subcortical gray matter volume loss since the earliest stages of the disease, suggest that axonal damage may occur independently of demyelination.

With the advancement of powerful MRI techniques, the early occurrence of GM lesions is being further appreciated,73 although MRI techniques currently underestimate their number.74,75 Regional volume measurements by MRI have revealed smaller hippocampal CA1 regions in MS brains, and a subset of MS patients with depression had a smaller CA2-3/dentate gyrus volume.62

Another quantitative technique, diffusion MRI, measures the microscopic Brownian motion of water molecules. This motion is hindered by cellular structures such as cell membranes and the axonal cytoskeleton.76 Using diffusion MRI and applying field gradients in multiple directions, it is possible to infer the orientation of axons and reconstruct the pathways of the major WM bundles.77 Abnormalities in diffusivity patterns have been detected in focal MS lesions, NAWM, and GM. These abnormalities have been shown to correlate with physical disability78 and cognitive impairment in MS.79 Magnetic resonance imaging measurement of sodium ions in the plaques and NAWM of MS patients supports the hypothesis that demyelination results in eventual axonal damage. Sodium ion concentrations are higher in both acute and chronic lesions and in NAWM.80 This supports the idea that, following a demyelination event, sodium channel expression is increased and they redistribute along the axon in order to maintain nerve conduction,81 suggesting axons remain intact following demyelination.

Spectroscopic Measurement of Metabolites

The neuronal metabolite N-acetyl aspartate (NAA) is a measure of mitochondrial activity, which can be determined by proton magnetic resonance spectroscopy (1H-MRS).82,83 In the adult brain, NAA is present only in neurons and axons,84 so its measurement in vivo by 1H-MRS is useful to determine the extent of axonal damage/loss.85 In MS, NAA is decreased in both lesional areas and NAWM, which suggests that either mitochondrial dysfunction and/or axonal damage occurs also in regions devoid of active demyelination.82,86,87 Importantly, due to the high pathological specificity of NAA, its levels yield a better correlation with the degree of disability occurring in the presence or absence of demyelinating activity.8890 Correlative MRI-histological studies have shown that reduced NAA levels correlate with reduced axonal numbers in lesions of SPMS patients,91 and NAA concentrations are decreased in PPMS.92 Decreased NAA levels reverse as inflammation subsides following anti-inflammatory treatment with glatiramer acetate.93 Furthermore, a combination of NAA measurement, lesion imaging, and genetic analysis of MS patients with the DRB*1501 haplotype has been proposed as a method to stratify patients according to disease severity.94 Increased lactate levels in the CSF of MS patients have also been reported,95,96 whereas others have found decreased lactate levels in early stages of MS97 or throughout the MS disease course,98 suggesting its levels may fluctuate with disease progression or be indicative of disease heterogeneity.

The neuronal metabolite N-acetyl aspartate is a measure of mitochondrial activity, which can be determined by proton magnetic resonance spectroscopy. In multiple sclerosis, N-acetyl aspartate is decreased in both lesional areas and normal appearing white matter, which suggests that either mitochondrial dysfunction and/or axonal damage occurs also in regions devoid of active demyelination.

Importantly, due to the high pathological specificity of N-acetyl aspartate, its levels yield a better correlation with the degree of disability occurring in the presence or absence of demyelinating activity.

Other metabolite changes observed in NAWM of MS patients include the measurement of the excitatory neurotransmitter glutamate and of the glial-enriched metabolite myo-inositol.99 Notably, myo-inositol concentration was found to be elevated in the NAWM of patients with CIS suggestive of MS.100 Because myo-inositol is preferentially concentrated in glial cells,101 its increase may reflect astrocytosis and microglial activation. Because no correlation could be detected between NAWM myo-inositol levels and T2-lesion load, it is conceivable that the early detection of myo-inositol in CIS might reflect a relevant pathogenic process, which occurs independently from inflammatory demyelination.

The elevated levels of the excitatory neurotransmitter glutamate in acute MS lesions and NAWM but not in chronic MS lesions,102 together with the consistently decreased NAA levels in chronic lesions, suggest the possibility that excitotoxicity may trigger or be part of the mechanism leading to neurodegeneration in MS. Although these results will need to be confirmed in longitudinal studies assessing the predictive value of increased glutamate on the development of chronic lesions and brain atrophy, they are supported by studies in cultured neurons, where exposure to glutamate and inflammatory cytokines is sufficient to induce deficits in axonal transport and leads to the formation of localized varicosities and frank transections.103 Excessive glutamate release and impaired clearance may be cytotoxic to either neurons or oligodendrocytes.103105 The correlation of elevated glutamate levels and decreased NAA levels was also associated with the rs794185 noncoding single nucleotide polymorphism in a subset of MS patients with high brain volume loss and severe neurodegeneration.106 Thus, suggesting that glutamate may be part of the mechanisms that, within the context of an inflammatory environment might lead to axonal loss in MS.

The elevated levels of the excitatory neurotransmitter glutamate in acute multiple sclerosis lesions and normal appearing white matter but not in chronic multiple sclerosis lesions, together with the consistently decreased N-acetyl aspartate levels in chronic lesions, suggest the possibility that excitotoxicity may trigger or be part of the mechanism leading to neurodegeneration in multiple sclerosis.

Positron Emission Tomography

Positron emission tomography (PET) is a functional imaging technique which employs detection of radioligand tracers to clinically quantitate molecular processes of disease. In MS, PET imaging is used to correlate metabolic patterns to clinical symptoms, such as fatigue, cognitive impairment, and disability.107 Analysis of brain glucose metabolism in MS patients through PET shows increased glucose utilization, suggesting increased energy demand following demyelination.108 In MS, PET is also used to investigate microglial activation and inflammation as a marker of disease activity.109,110 Furthermore, PET imaging has shown decreased cortical cerebral metabolism in MS.111 Importantly, recent advancements have been made in radioligand development for PET imaging of demyelination/remyelination levels in animal models of MS.112 Therefore, PET imaging of neurodegeneration in relation to demyelination and remyelination holds promise for determining early pathological changes during the disease course of MS.