A monoclonal natural human IgM protects axons in the absence of remyelination

Abstract

Background: Whereas demyelination underlies early neurological symptoms in multiple sclerosis (MS), axonal damage is considered critical for permanent chronic deficits. Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in chronic induced demyelinating disease (TMEV-IDD) with progressive axonal loss and neurologic dysfunction similar to progressive forms of MS. We previously reported that treatment of chronic TMEV-IDD mice with a neurite outgrowth-promoting natural human antibody, HIgM12, improved brainstem NAA concentrations and preserved functional motor activity. In order to translate this antibody toward clinical trial, we generated a fully human recombinant form of HIgM12, rHIgM12, determined the optimal in vivo dose for functional improvement in TMEV-IDD, and evaluated the functional preservation of descending spinal cord axons by retrograde labeling.

Findings: SJL/J mice at 45 to 90 days post infection (dpi) were studied. A single intraperitoneal dose of 0.25 mg/kg of rHIgM12 per mouse is sufficient to preserve motor function in TMEV-IDD. The optimal dose was 10 mg/kg. rHIgM12 treatment protected the functional transport in spinal cord axons and led to 40 % more Fluoro-Gold-labeled brainstem neurons in retrograde transport studies. This suggests that axons are not only present but also functionally competent. rHIgM12-treated mice also contained more mid-thoracic (T6) spinal cord axons than controls.

Conclusions: This study confirms that a fully human recombinant neurite outgrowth-promoting monoclonal IgM is therapeutic in a model of progressive MS using multiple reparative readouts. The minimum effective dose is similar to that of a remyelination-promoting monoclonal human IgM discovered by our group that is presently in clinical trials for MS.

Keywords: Activity monitoring; Axons; Brainstem; Multiple sclerosis; Retrograde labeling; Theiler’s murine encephalomyelitis virus.

Fig. 1

Purification of rHIgM12 to >97 %. IgM was isolated from culture supernatant using a three-step purification protocol. a shows a flow diagram of the three-step purification protocol. b shows the final eluate from the third step, i.e., separation on a Sephacryl S-300 HR column in the presence of 10 mM NaOH and 150 mM NaCl (pH 7.0). c shows an SDS-PAGE gel of the following: lane 1—molecular weight markers, lanes 2 and 3—20-μl samples of the peak fraction showed in b. The IgM heavy chain (H, 75 kDa) and light chain (L, 25 kDa) are shown

Fig. 2

rHIgM12 treatment improves both horizontal and vertical activity in SJL mice during established demyelinating disease. Groups of SJL mice (N = 10 per treatment group) at 45 dpi were placed in activity monitoring boxes. Baseline measurements were collected over 8 days. Beginning at treatment (time zero), mice were monitored continuously over an additional 8 weeks. a, c correspond to horizontal activity and b, d correspond to vertical activity. a, b Original, unfiltered recordings for horizontal and vertical activity normalized to baseline; c, d third-order polynomial fitting of standardized z values normalized to baseline revealed improvement in both horizontal and vertical activity of the rHIgM12-treated group compared to the control IgM-treated group. Horizontal (e) and vertical (f) nocturnal activity of rHIgM12-treated mice compared to control IgM-treated mice significantly diverged above the x-axis (more activity) (p < 0.05) post-treatment. The pointwise lower 95 % confidence bands for the nocturnal activity are represented on the y-axis scale

Fig. 3

rHIgM12 treatment improves the number of retrograde-labeled brainstem neurons and preserves spinal cord axons, but does not affect spinal cord demyelination. a Fluoro-Gold-labeled neurons were counted in brain stem sections. The panel shows an example of a cluster of fluorescently labeled neurons in the brainstem. Extensive labeling of cell bodies as well as axons and dendrites can be easily appreciated. b rHIgM12 treatment increased the number of retrograde-labeled brainstem neurons compared to the saline-treated group (p = 0.009, Mann-Whitney rank sum test). The number of labeled neurons in uninfected positive control mice (N = 3, circles) is shown for reference. Forest plots show the average number of retrograde-labeled brainstem neurons ± SEM per treatment group: rHIgM12 (red triangles) and saline (blue boxes). Mice from both treatment groups had similar levels of spinal cord c demyelination and d inflammation pathology. Pathology analysis was performed blinded. e When the number of myelinated mid-thoracic-level spinal cord axons was compared between treatment groups, rHIgM12-treated mice contained 14.7 % more axons than the saline-treated group (p = 0.038, Mann-Whitney rank sum test). f The number of fluorescent retrograde-labeled brain stem neurons in each mouse correlated positively and significantly with the number of thoracic-level myelinated spinal cord axons (p = 0.016, R ² = 0.28)