ABSTRACT
NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys²66 and Cys³°9 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.
Subject(s)
Disulfides/metabolism , Myelin Proteins/chemistry , Protein Engineering/methods , Receptors, Cell Surface/chemistry , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Animals , Behavior, Animal/drug effects , Crystallography, X-Ray , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , Humans , Male , Molecular Sequence Annotation , Molecular Sequence Data , Myelin Proteins/genetics , Nogo Receptor 1 , Protein Stability , Rats , Rats, Sprague-Dawley , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Spinal Cord Injuries/drug therapy , Spinal Nerve Roots/injuriesABSTRACT
The extent of disability caused by spinal cord injury (SCI) relates to secondary tissue destruction arising partly from an intraspinal influx of neutrophils and monocyte/macrophages after the initial injury. The integrin alpha4beta1, expressed by these leukocytes, is a key to their activation and migration into/within tissue. Therefore, blocking this integrin's functions may afford significant neuroprotection. Rats were treated intravenously with a blocking monoclonal antibody (mAb) to the alpha4 subunit of alpha4beta1 at 2 and 24 h after thoracic clip-compression SCI. Anti-alpha4beta1 treatment significantly decreased neutrophil and monocyte/macrophage influx at 3 d by 47% and 53%, respectively, and decreased neutrophil influx by 61% at 7 d after SCI. Anti-alpha4beta1 treatment also significantly reduced oxidative activity in injured cord homogenates at 3 d. For example, myeloperoxidase activity decreased by 38%, inducible nitric oxide by 44%, dichlorofluorescein (marking free radicals) by 33% and lipid peroxidation (malondialdehyde) by 42%. At 2-8 weeks after SCI, motor function improved by up to 2 points on an open-field locomotor scale. Treated rats supported weight with their hind paws instead of sweeping. At 2-4 weeks after SCI, anti-alpha4beta1 treatment decreased blood pressure responses during autonomic dysreflexia by as much as 43% and, at 2-8 weeks, decreased mechanical allodynia elicited from the trunk and hind paw by up to 54% and 40%, respectively. This improved functional recovery correlated with spared myelin-containing white matter and >10-fold more bulbospinal serotonergic axons below the injury than were in controls. The significant neurological improvement offered by this neuroprotective strategy underscores the potential for an anti-integrin treatment for SCI.
Subject(s)
Antibodies, Monoclonal/pharmacology , Integrin alpha4beta1/antagonists & inhibitors , Neuroprotective Agents/pharmacology , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/immunology , Spinal Cord/immunology , Acute Disease , Animals , Axons/pathology , Axons/physiology , Cell Count , Cell Movement/immunology , Female , Integrin alpha4beta1/immunology , Integrin alpha4beta1/metabolism , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Male , Monocytes/immunology , Monocytes/metabolism , Motor Activity , Neuralgia/drug therapy , Neuralgia/immunology , Neuralgia/pathology , Neutrophils/immunology , Neutrophils/metabolism , Oxidative Stress/drug effects , Oxidative Stress/immunology , Rats , Rats, Wistar , Recovery of Function/drug effects , Recovery of Function/immunology , Serotonin/physiology , Spinal Cord/pathology , Spinal Cord Injuries/pathologyABSTRACT
Oligodendrocyte-myelin glycoprotein (OMgp) is a myelin component that has been shown in vitro to inhibit neurite outgrowth by binding to the Nogo-66 receptor (NgR1)/Lingo-1/Taj (TROY)/p75 receptor complex to activate the RhoA pathway. To investigate the effects of OMgp on axon regeneration in vivo, OMgp(-/-) mice on a mixed 129/Sv/C57BL/6 (129BL6) or a C57BL/6 (BL6) genetic background were tested in two spinal cord injury (SCI) models - a severe complete transection or a milder dorsal hemisection. OMgp(-/-) mice on the mixed 129BL6 genetic background showed greater functional improvement compared to OMgp(+/+) littermates, with increased numbers of cholera toxin B-labeled ascending sensory axons and 5-HT(+) descending axons and less RhoA activation after spinal cord injury. Myelin isolated from OMgp(-/-) mice (129BL6) was significantly less inhibitory to neurite outgrowth than wild-type (wt) myelin in vitro. However, OMgp(-/-) mice on a BL/6 genetic background showed neither statistically significant functional recovery nor axonal sprouting following dorsal hemisection.
Subject(s)
Axons/physiology , Myelin-Associated Glycoprotein/deficiency , Recovery of Function/physiology , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathology , Analysis of Variance , Animals , Biotin/analogs & derivatives , Biotin/metabolism , Cholera Toxin/metabolism , Dextrans/metabolism , Exploratory Behavior/physiology , Female , Functional Laterality/genetics , GPI-Linked Proteins , Ganglia, Spinal/pathology , Glial Fibrillary Acidic Protein/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Proteins , Myelin-Oligodendrocyte Glycoprotein , Neurites/physiology , Neurons/pathology , Recovery of Function/genetics , Serotonin/metabolism , Time Factors , rhoA GTP-Binding Protein/metabolismABSTRACT
Neuroinflammation may play a role in the pathogenesis of Parkinson's disease (PD). The present study questioned whether this neuroinflammatory response differs between the olfactory bulb, as an early affected region and the nigrostriatal system. Indeed, increased microgliosis was shown in post-mortem olfactory bulb of PD patients. Also in olfactory bulb of MPTP-treated mice, microgliosis and increased expression of IL-1alpha, IL-1beta and IL-1ra mRNA was observed early after treatment. These observations implicate that neuroinflammation is not restricted to the nigrostriatal system. MPTP-induced microgliosis in striatum and olfactory bulb was reduced in IL-1alpha/beta knockout mice, indicating that IL-1 affects microglia activation. Importantly, MPTP induced differential regulation of IL-1 receptors. mRNA levels of IL-1RI and, to a lesser extent, IL-1RII were increased in striatum. Interestingly, in the olfactory bulb only IL-1RII mRNA was enhanced. We suggest that differential regulation of IL-1 signaling can serve as an important mechanism to modulate neuroinflammatory activity after MPTP treatment and possibly during PD.
Subject(s)
MPTP Poisoning/immunology , MPTP Poisoning/pathology , Olfactory Bulb/immunology , Olfactory Bulb/pathology , Parkinson Disease/immunology , Parkinson Disease/pathology , Parkinsonian Disorders/immunology , Parkinsonian Disorders/pathology , Receptors, Interleukin-1/genetics , Animals , Base Sequence , Corpus Striatum/drug effects , Corpus Striatum/immunology , Corpus Striatum/pathology , DNA Primers/genetics , Gene Expression , Humans , MPTP Poisoning/genetics , Male , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/pathology , Olfactory Bulb/drug effects , Parkinson Disease/genetics , Parkinsonian Disorders/genetics , Receptors, Interleukin-1/classification , Substantia Nigra/drug effects , Substantia Nigra/immunology , Substantia Nigra/pathologyABSTRACT
LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexes mediate the axonal growth inhibitory effects of Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) via RhoA activation. Soluble LINGO-1 (LINGO-1-Fc), which acts as an antagonist of these pathways by blocking LINGO-1 binding to NgR1, was administered to rats after dorsal or lateral hemisection of the spinal cord. LINGO-1-Fc treatment significantly improved functional recovery, promoted axonal sprouting and decreased RhoA activation and increased oligodendrocyte and neuronal survival after either rubrospinal or corticospinal tract transection. These experiments demonstrate an important role for LINGO-1 in modulating axonal outgrowth in vivo and that treatment with LINGO-1-Fc can significantly enhance recovery after spinal cord injury.
Subject(s)
Axons/drug effects , Membrane Proteins/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/therapeutic use , Recovery of Function/drug effects , Spinal Cord Injuries/drug therapy , Analysis of Variance , Animals , Apoptosis/drug effects , Axons/physiology , Caspase 3/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Forelimb/drug effects , Humans , Immunohistochemistry/methods , In Situ Nick-End Labeling/methods , MAP Kinase Kinase 4/metabolism , Membrane Proteins/chemistry , Membrane Proteins/physiology , Nerve Regeneration/drug effects , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Organogenesis/drug effects , Protein Binding/drug effects , RNA-Binding Proteins/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathology , Time Factors , Tubulin/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Methylprednisolone (MP) is a synthetic glucocorticoid used for the treatment of spinal cord injury (SCI). Soluble Nogo-66 receptor (NgR) ectodomain is a novel experimental therapy for SCI that promotes axonal regeneration by blocking the growth inhibitory effects of myelin constituents in the adult central nervous system. To evaluate the potential complementarity of these mechanistically distinct pharmacological reagents we compared their effects alone and in combination after thoracic (T7) dorsal hemisection in the rat. Treatment with an ecto-domain of the rat NgR (27-310) fused to a rat IgG [NgR(310)ecto-Fc] (50 microm intrathecal, 0.25 microL/h for 28 days) or MP alone (30 mg/kg i.v., 0, 4 and 8 h postinjury) improved the rate and extent of functional recovery measured using Basso, Beattie, Bresnahan (BBB) scoring and footprint analysis. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NgR(310)ecto-Fc was not apparent until 2 weeks after SCI. NgR(310)ecto-Fc or MP treatment resulted in increased axonal sprouting and/or regeneration, quantified by counting biotin dextran amine-labeled corticospinal tract axons, and increased the number of axons contacting motor neurons in the ventral horn gray matter caudal to the lesion. Combined treatment with NgR(310)ecto-Fc and MP had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone. The data demonstrate that NgR(310)ecto-Fc and MP act in a temporally and mechanistically distinct manner and suggest that they may have complementary effects.
