Jens O. Watzlawik

PDGF is Required for Remyelination-Promoting IgM Stimulation of Oligodendrocyte Progenitor Cell Proliferation

Background Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair. Methods We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots. Results rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2. Conclusion Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.

Importance of oligodendrocyte protection, BBB breakdown and inflammation for remyelination

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. A better understanding of why remyelination fails in MS is necessary to improve remyelination strategies. Remyelination is mediated by oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, it is still unclear whether OPCs detectable in MS lesions survive the inflammatory response but are unable to myelinate or whether OPC and oligodendrocyte death is primarily responsible for remyelination failure and detectable OPCs enter demyelinated areas from adjacent tissue as the lesion evolves. Remyelination strategies should, therefore, focus on stimulation of differentiation or prevention of apoptosis, as well as establishment of a supportive environment for OPC-mediated remyelination, which may be especially important in chronically demyelinated lesions.

High-affinity binding of remyelinating natural autoantibodies to myelin-mimicking lipid bilayers revealed by nanohole surface plasmon resonance.

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (K(D) ≈ 0.9 nM) for natural autoantibodies. This is approximately one to 2 orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair.

A patterned recombinant human IgM guides neurite outgrowth of CNS neurons

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

Cellular targets and mechanistic strategies of remyelination-promoting IgMs as part of the naturally occurring autoantibody repertoire.

Immunoglobulins with germline sequences occur in invertebrates and vertebrates and are named naturally occurring autoantibodies (NAbs). NAbs may target foreign antigens, self- or altered self-components and are part of the normal immunoglobulin repertoire. Accumulating evidence indicates that naturally occurring antibodies can act as systemic surveillance molecules, which tag, damaged or stressed cells, invading pathogens and toxic cellular debris for elimination by the immune system. In addition to acting as detecting molecules, certain types of NAbs actively signal in different cell types with a broad range of responses from induction of apoptosis in cancer cells to stimulation of remyelination in glial cells. This review emphasizes functions and characteristics of NAbs with focus on remyelination-promoting mouse and human antibodies. Human remyelination-promoting NAbs are potential therapeutics to combat a wide spectrum of disease processes including demyelinating diseases like multiple sclerosis. We will highlight the identified glycosphingolipid (SL) antigens of polyreactive remyelination-promoting antibodies and their proposed mechanism(s) of action. The nature of the identified antigens suggests a lipid raft-based mechanism for remyelination-promoting antibodies with SLs as most essential raft components. However, accumulating evidence also suggests involvement of other antigens in stimulation of remyelination, which will be discussed in the text.

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

A single dose of a neuron-binding human monoclonal antibody improves brainstem NAA concentrations, a biomarker for density of spinal cord axons, in a model of progressive multiple sclerosis

BackgroundIntracerebral infection of susceptible mouse strains with Theiler’s murine encephalomyelitis virus (TMEV) results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis (MS). We previously showed that as the disease progresses, a marked decrease in brainstem N-acetyl aspartate (NAA; metabolite associated with neuronal integrity) concentrations, reflecting axon health, is measured. We also demonstrated stimulation of neurite outgrowth by a neuron-binding natural human antibody, IgM12. Treatment with either the serum-derived or recombinant human immunoglobulin M 12 (HIgM12) preserved functional motor activity in the TMEV model. In this study, we examined IgM-mediated changes in brainstem NAA concentrations and central nervous system (CNS) pathology.Findings1H-magnetic resonance spectroscopy (MRS) showed that treatment with HIgM12 significantly increased brainstem NAA concentrations compared to controls in TMEV-infected mice. Pathologic analysis demonstrated a significant preservation of axons in the spinal cord of animals treated with HIgM12.ConclusionsThis study links drug efficacy of slowing deficits with axon preservation and NAA concentrations in the brainstem in a model of progressive MS. HIgM12-mediated changes of NAA concentrations in the brainstem are a surrogate marker of axon injury/preservation throughout the spinal cord. This study provides proof-of-concept that a neuron-reactive human IgM can be therapeutic and provides a biomarker for clinical trials.

Naturally Occurring Monoclonal Antibodies and Their Therapeutic Potential for Neurologic Diseases

IMPORTANCE Modulating the immune system does not reverse long-term disability in neurologic disorders. Better neuroregenerative and neuroprotective treatment strategies are needed for neuroinflammatory and neurodegenerative diseases. OBJECTIVE To review the role of monoclonal, naturally occurring antibodies (NAbs) as novel therapeutic molecules for treatment of neurologic disorders. EVIDENCE REVIEW Peer-reviewed articles, including case reports, case series, retrospective reviews, prospective randomized clinical trials, and basic science reports, were identified in a PubMed search for articles about NAbs and neurologic disorders that were published from January 1, 1964, through June 30, 2015. We concentrated our review on multiple sclerosis, Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis. FINDINGS Many insults, including trauma, ischemia, infection, inflammation, and neurodegeneration, result in irreversible damage to the central nervous system. Central nervous system injury often results in a pervasive inhibitory microenvironment that hinders regeneration. A common targeted drug development strategy is to identify molecules with high potency in animal models. Many approaches often fail in the clinical setting owing to a lack of efficacy in human diseases (eg, less than the response demonstrated in animal models) or a high incidence of toxic effects. An alternative approach is to identify NAbs in humans because these therapeutic molecules have potential physiologic function without toxic effects. NAbs of the IgG, IgA, or IgM isotype contain germline or close to germline sequences and are reactive to self-components, altered self-components, or foreign antigens. Our investigative group developed recombinant, autoreactive, natural human IgM antibodies directed against oligodendrocytes or neurons with therapeutic potential for central nervous system repair. One such molecule, recombinant HIgM22, directed against myelin and oligodendrocytes completed a successful phase 1 clinical trial without toxic effects with the goal of promoting remyelination in multiple sclerosis. CONCLUSIONS AND RELEVANCE Animal studies demonstrate that certain monoclonal NAbs are beneficial as therapeutic agents for neurologic diseases. This class of antibodies represents a unique source from which to develop a new class of disease-modifying therapies.

ABCA7 loss-of-function variants, expression, and neurologic disease risk

Objective: To investigate and characterize putative “loss-of-function” (LOF) adenosine triphosphate–binding cassette, subfamily A member 7 (ABCA7) mutations reported to associate with Alzheimer disease (AD) risk. Methods: We genotyped 6 previously reported ABCA7 putative LOF variants in 1,465 participants with AD, 381 participants with other neuropathologies (non-AD), and 1,043 controls and assessed the overall mutational burden for association with different diagnosis groups. We measured brain ABCA7 protein and messenger RNA (mRNA) levels using Western blot and quantitative PCR, respectively, in 11 carriers of the 3 most common variants, and sequenced all 47 ABCA7 exons in these participants to screen for other coding variants. Results: At least one of the investigated variants was identified in 45 participants with late-onset Alzheimer disease, 12 participants with other neuropathologies, and 11 elderly controls. Association analysis revealed a significantly higher burden of these variants in participants with AD (p = 5.00E-04) and those with other neuropathologies (p = 8.60E-03) when compared with controls. Concurrent analysis of brain ABCA7 mRNA and protein revealed lower protein but not mRNA in p.L1403fs carriers, lower mRNA but not protein in p.E709fs carriers, and additional deleterious mutations in some c.5570+5G>C carriers. Conclusions: Our results suggest that LOF may not be a common mechanism for these ABCA7 variants and expand the list of neurologic diseases enriched for them.

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long‐term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody‐mediated cell adhesion, and neurite outgrowth on HIgM12‐coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)‐positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro‐motor disorders including multiple sclerosis.