D Grant

Axonal damage accumulates in the progressive phase of multiple sclerosis: three year follow up study

Background: Neurofilament phosphoforms (Nf) are principal components of the axoskeleton released during axonal injury. Cerebrospinal fluid (CSF) levels of Nf phosphoforms might be useful surrogate markers for disability in multiple sclerosis (MS), aid in distinguishing clinical subtypes, and provide valuable prognostic information. Method: Thirty four patients with MS were included in a three year follow up study along with 318 controls with other non-inflammatory neurological diseases. CSF levels of two Nf heavy chain (NfH) phosphoforms (NfHSMI35, NfHSMI34) were quantified at baseline and three year follow up using new ELISA techniques. Levels of NfH phosphoforms, the degree of phosphorylation (NfHSMI34:NfHSMI35 ratio), and changes in NfH levels between baseline and follow up (ΔNfH) were related to the clinical phenotype (RR or SP/PP), to three clinical scales (Kurtzke’s EDSS, ambulation index (AI), and nine hole peg test (9HPT)), and to progression of disability. Results: A significantly higher proportion (59%) of patients with SP/PPMS experienced an increase in NfHSMI35 levels between baseline and follow up compared with those with RRMS (14%, p<0.05). CSF NfHSMI34 levels at baseline were higher in patients with SP/PP (11 pg/ml) compared with RR (7 pg/ml, p<0.05) and NfHSMI35 levels were higher at follow up in SP/PP (129 pg/ml) compared with levels below assay sensitivity in RR (p<0.05). NfHSMI35 correlated with the EDSS (rs = 0.54, p<0.01), the AI (rs = 0.42, p<0.05), and the 9HPT (rs = 0.59, p<0.01) at follow up. Conclusion: The increase in NfH during the progressive phase of the disease together with the correlation of NfHSMI35 with all clinical scales at follow up suggests that cumulative axonal loss is responsible for sustained disability and that high NfHSMI35 levels are a poor prognostic sign.

Acute axonal damage predicts clinical outcome in patients with multiple sclerosis

The objectives of this study were (1) to determine how cerebrospinal fluid (CSF) neurofilament heavy chain (NfHSMI34 and NfHSMI35) levels relate to clinical outcome in optic neuritis (ON) and multiple sclerosis (MS) relapse patients treated with high dose oral methylprednisolone; and (2) to correlate neurofilament and myelin basic protein (MBP) concentrations, particularly as the latter was previously associated with clinical disability. Fifty subjects participated in two double-blind, randomized, placebo-controlled clinical trials. Eight/18 patients in the ON trial and 15/32 subjects in the MS attack trial were treated with oral methylprednisolone. In the MS attack trial group, CSF NfHSMI34 and NfHSMI35 measured at week 3 and DCSF NfHSMI34 levels from baseline to week 3 were predictive of clinical outcome at week 8 and 52. In the ON group, no such association was seen. When both groups were combined, baseline CSF NfHSMI34 and NfHSMI35 correlated positively with baseline enhancing lesion volume (ELV) (rs=0.50, p<0.01 and rs=0.53, p<0.01, respectively). Levels of NfHSMI35 at baseline and week 3 also strongly correlated with the MBP concentration. This study supports the view that acute inflammation in ON and MS results in axonal pathology and that the latter has a role in determining functional impairment.

CSF neurofilament levels: A potential prognostic marker in Guillain–Barré syndrome

Long-term morbidity from Guillain–Barré syndrome (GBS) is caused by axonal damage. This prospective study demonstrated that neurofilaments (NfHs), a biomarker for axonal damage, were of prognostic value in GBS. CSF NfH levels correlated with the F score and Medical Research Council summed score and were higher in patients with neurophysiologic evidence of axonal degeneration compared to those without. Pathologically high CSF NfH levels (>0.73 ng/mL) predicted worse motor and functional outcome.

Biomarker Report from the Phase II Lamotrigine Trial in Secondary Progressive MS – Neurofilament as a Surrogate of Disease Progression

Objective Lamotrigine trial in SPMS was a randomised control trial to assess whether partial blockade of sodium channels has a neuroprotective effect. The current study was an additional study to investigate the value of neurofilament (NfH) and other biomarkers in predicting prognosis and/or response to treatment. Methods SPMS patients who attended the NHNN or the Royal Free Hospital, UK, eligible for inclusion were invited to participate in the biomarker study. Primary outcome was whether lamotrigine would significantly reduce detectable serum NfH at 0-12, 12–24 and 0–24 months compared to placebo. Other serum/plasma and CSF biomarkers were also explored. Results Treatment effect by comparing absolute changes in NfH between the lamotrigine and placebo group showed no difference, however based on serum lamotrigine adherence there was significant decline in NfH (NfH 12–24 months p = 0.043, Nfh 0–24 months p = 0.023). Serum NfH correlated with disability: walking times, 9-HPT (non-dominant hand), PASAT, z-score, MSIS-29 (psychological) and EDSS and MRI cerebral atrophy and MTR. Other biomarkers explored in this study were not found to be significantly associated, aside from that of plasma osteopontin. Conclusions The relations between NfH and clinical scores of disability and MRI measures of atrophy and disease burden support NfH being a potential surrogate endpoint complementing MRI in neuroprotective trials and sample sizes for such trials are presented here. We did not observe a reduction in NfH levels between the Lamotrigine and placebo arms, however, the reduction in serum NfH levels based on lamotrigine adherence points to a possible neuroprotective effect of lamotrigine on axonal degeneration.

Cerebrospinal fluid levels of brain specific proteins in optic neuritis

This study evaluates levels of cerebrospinal fluid (C SF) brain-specific proteins (BSP) in subjects with optic neuritis (O N) who are at high risk of progression to multiple sclerosis (MS). Forty-one subjects had acute O N and 17 subjects with other neurological diseases (OND) served as controls. Twenty-o ne subjects with O N had white matter lesions on magnetic resonance imaging (MRI) and intrathecal synthesis of oligoclonal IgG bands (OB) consistent with being at high risk of progression to MS; eight of whom later were diagnosed with clinically definite MS (C DMS). Levels of S100B, ferritin and two neurofilament heavy chain phosphoforms (NfHSMI34 and NfHSMI35) were analysed using ELISA technique. A putative index of ‘axonal health’ was expressed as a ratio of NfHSMI34 to NfHSMI35. NfHSMI34 and the NfHSMI34:SMI35 were significantly elevated in subjects with O N compared to controls. No significant differences in levels of C SF BSP were seen between O N subjects with C DMS plus those at high risk of progression to MS and O N subjects with normal MRI and negative C SF analysis. In conclusion, there is evidence of axonal damage in subjects who present with O N, which is independent of the diagnosis of C DMS.

Serum neurofilament is associated with progression of brain atrophy and disability in early MS

Objective: To investigate a potential effect of riluzole on serum neurofilaments (Nf) compared to placebo and the relationship between longitudinal clinical and MRI outcomes and serum Nf levels. Methods: Serum samples were obtained from participants enrolled in a randomized double-blind trial of neuroprotection with riluzole vs placebo as an add-on to weekly interferon-β (IFN-β)–1a IM initiated 3 months after randomization. Nf measurements were performed by ELISA and electrochemiluminescence immunoassay. Results: Longitudinal serum samples were available from 22 riluzole and 20 placebo participants over 24 months. There was no observed treatment effect with riluzole. Nf light chain (NfL) levels decreased over time (p = 0.007 at 24 months), whereas the Nf heavy chain was unchanged (p = 0.997). Changes in NfL were correlated with EDSS change (p = 0.009) and neuropsychological outcomes. Brain volume decreased more rapidly in patients with high baseline NfL (p = 0.05 at 12 months and p = 0.008 at 24 months) and this relationship became stronger at 24 months (p = 0.024 for interaction). Higher and increasing NfL predicted higher number of gadolinium-enhancing lesions (p < 0.001 for both). Conclusions: Our findings support the potential value of serum NfL as a marker of neuroaxonal injury in early multiple sclerosis. Its reduction over time could represent regression to the mean, or a possible treatment effect of IFN-β-1a. The association with whole brain atrophy and the formation of acute white matter lesions has relevant implications to use serum NfL as a noninvasive biomarker of the overall consequences of brain damage and ongoing disease activity. ClinicalTrials.gov identifier: NCT00501943.

Neural cell adhesion molecule — Description of a CSF ELISA method and evidence of reduced levels in selected neurological disorders

Neural cell adhesion molecule (NCAM) is important for neuronal growth and repair. Here we describe the development and validation of a sensitive ELISA for NCAM using commercially available reagents. The measurable range of NCAM ELISA is 16-500 ng/mL, with a constant coefficient-of variation and good parallelism between the reference standard curve and CSF. CSF NCAM was measured in 36 benign-intracranial hypertension, 51 multiple sclerosis, 27 neuropathy, 37 Alzheimers disease, 12 cognitive impairment, 15 motoneurone disease, 13 meningitis, 17 encephalitis, and 17 control cases. Significant reductions were found between controls and multiple sclerosis, Alzheimers disease and meningitis.

Neurofilament a biomarker of neurodegeneration in autoimmune encephalomyelitis

Monitoring neuroaxonal loss in multiple sclerosis is an important objective to study the pathogenesis and response to treatment of the disease. The release of neurofilaments is a potential surrogate biomarker of neurodegeneration. One route to explore this aspect further is through the use of animal models that have well-defined, and predictable, disease courses. The release of neurofilaments into plasma across the course of relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis in Biozzi ABH was assessed, as well as measuring anti-neurofilament antibodies using ELISA. It was found that there was an immediate release in neurofilaments into the blood following the initial paralytic attack. Neurofilament release was continuous in relapse and remission but returned towards baseline in chronic disease, as animals entered the post-relapsing progressive phase of the disease. This was mirrored by a loss of neurofilament-specific antibodies. In contrast neurofilament levels increased dramatically as neurodegeneration and clinical disease occurred in the G93A SOD1 transgenic C57BL/6 x SJL mice, model of amyotrophic lateral sclerosis. These data further support neurofilament levels as a good surrogate measure of neurodegeneration and their potential use as a surrogate endpoint in neuroprotective studies.

Progression in multiple sclerosis is associated with low endogenous NCAM

Multiple sclerosis (MS) is a CNS disorder characterized by demyelination and neurodegeneration. Although hallmarks of recovery (remyelination and repair) have been documented in early MS, the regenerative capacity of the adult CNS per se remains uncertain with the wide held belief that it is either limited or non‐existent. The neural cell adhesion molecule (NCAM) is a cell adhesion molecule that has been widely implicated in axonal outgrowth, guidance and fasciculation. Here, we used in vitro and in vivo of MS to investigate the role of NCAM in disease progression. We show that in health NCAM levels decrease over time, but this occurs acutely after demyelination and remains reduced in chronic disease. Our findings suggest that depletion of NCAM is one of the factors associated with or possibly responsible for disease progression in MS.

Growth associated protein (GAP-43): Cloning and the development of a sensitive ELISA for neurological disorders

GAP-43 has been studied in the rodent and mammalian brain and shown to be present specifically in areas undergoing axonal elongation and synapse formation. GAP-43 was cloned using the baculovirus expression system and purified. A sandwich ELISA was developed using the recombinant GAP-43 as standard and validated. CSF GAP-43 levels were analysed in benign intracranial hypertension, movement disorders, multiple sclerosis, neuropathy, CNS infections, motor neuron disease, and headache (neurological controls). GAP-43 levels were low in all disorders analysed (in particular motor neuron disease; p=0.001, and movement disorders and multiple sclerosis; p<0.0001) compared to controls, aside from CNS infections. GAP-43 is preferentially reduced in the CSF of neurological disorders associated with neurodegeneration.