Jane Relton

LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex

Axon regeneration in the adult CNS is prevented by inhibitors in myelin. These inhibitors seem to modulate RhoA activity by binding to a receptor complex comprising a ligand-binding subunit (the Nogo-66 receptor NgR1) and a signal transducing subunit (the neurotrophin receptor p75). However, in reconstituted non-neuronal systems, NgR1 and p75 together are unable to activate RhoA, suggesting that additional components of the receptor may exist. Here we describe LINGO-1, a nervous system-specific transmembrane protein that binds NgR1 and p75 and that is an additional functional component of the NgR1/p75 signaling complex. In non-neuronal cells, coexpression of human NgR1, p75 and LINGO-1 conferred responsiveness to oligodendrocyte myelin glycoprotein, as measured by RhoA activation. A dominant-negative human LINGO-1 construct attenuated myelin inhibition in transfected primary neuronal cultures. This effect on neurons was mimicked using an exogenously added human LINGO-1-Fc fusion protein. Together these observations suggest that LINGO-1 has an important role in CNS biology.

Blockade of Nogo-66, Myelin-Associated Glycoprotein, and Oligodendrocyte Myelin Glycoprotein by Soluble Nogo-66 Receptor Promotes Axonal Sprouting and Recovery after Spinal Injury

The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind to the Nogo-66 receptor (NgR) and inhibit axonal growth in vitro. Transgenic or viral blockade of NgR function allows axonal sprouting in vivo. Here, we administered the soluble function-blocking NgR ectodomain [aa 27-310; NgR(310)ecto] to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.

Powerful beneficial effects of macrophage colony-stimulating factor on β-amyloid deposition and cognitive impairment in Alzheimer's disease

Alzheimers disease is a major cause of dementia in humans. The appearance of cognitive decline is linked to the overproduction of a short peptide called beta-amyloid (Abeta) in both soluble and aggregate forms. Here, we show that injecting macrophage colony-stimulating factor (M-CSF) to Swedish beta-amyloid precursor protein (APP(Swe))/PS1 transgenic mice, a well-documented model for Alzheimers disease, on a weekly basis prior to the appearance of learning and memory deficits prevented cognitive loss. M-CSF also increased the number of microglia in the parenchyma and decreased the number of Abeta deposits. Senile plaques were smaller and less dense in the brain of M-CSF-treated mice compared to littermate controls treated with vehicle solution. Interestingly, a higher ratio of microglia internalized Abeta in the brain of M-CSF-treated animals and the phagocytosed peptides were located in the late endosomes and lysosomes. Less Abeta(40) and Abeta(42) monomers were also detected in the extracellular protein enriched fractions of M-CSF-treated transgenic mice when compared with vehicle controls. Finally, treating APP(Swe)/PS1 mice that were already demonstrating installed Abeta pathology stabilized the cognitive decline. Together these results provide compelling evidence that systemic M-CSF administration is a powerful treatment to stimulate bone marrow-derived microglia, degrade Abeta and prevent or improve the cognitive decline associated with Abeta burden in a mouse model of Alzheimers disease.

LINGO-1 antagonist promotes functional recovery and axonal sprouting after spinal cord injury

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.

Release of Bradykinin and Expression of Kinin B2 Receptors in the Brain: Role for Cell Death and Brain Edema Formation After Focal Cerebral Ischemia in Mice

Pharmacological studies using bradykinin B2 receptor antagonists suggest that bradykinin, an early mediator of inflammation and the main metabolite of the kallikrein-kinin system, is involved in secondary brain damage after cerebral ischemia. However, the time-course of bradykinin production and kinin receptor expression as well as the conclusive role of bradykinin B2 receptors for brain damage after experimental stroke have not been elucidated so far. C57/Bl6 mice were subjected to 45 mins of middle cerebral artery occlusion (MCAO) and 2, 4, 8, 24, and 48 h later brains were removed for the analysis of tissue bradykinin concentration and kinin B2 receptor mRNA and protein expression. Brain edema, infarct volume, functional outcome, and long-term survival were assessed in WT and B2−/− mice 24 h or 7 days after MCAO. Tissue bradykinin was maximally increased 12 h after ischemia (three-fold), while kinin B2 receptor mRNA upregulation peaked 24 to 48 h after MCAO (10- to 12-fold versus naïve brain tissue). Immunohistochemistry revealed that kinin B2 receptors were constitutively and widely expressed in mouse brain, were upregulated 2 h after ischemia in cells showing signs of ischemic damage, and remained upregulated in the penumbra up to 24 h after ischemia. B2−/− mice had improved motor function (P<0.05), smaller infarct volumes (–38%; P<0.01), developed less brain edema (–87%; P<0.05), and survived longer (P<0.01) as compared with wild-type controls. The current results show that bradykinin is produced in the brain, kinin B2 receptors are upregulated on dying cells, and B2 receptors are involved in cell death and brain edema formation after experimental stroke.

CP-0597, a Selective Bradykinin B2 Receptor Antagonist, Inhibits Brain Injury in a Rat Model of Reversible Middle Cerebral Artery Occlusion

BACKGROUND AND PURPOSE Recent studies demonstrated a significant neuroprotective action of the selective peptide-based bradykinin B2 receptor antagonist CP-0597 after permanent middle cerebral artery (MCA) occlusion (MCAO) in the rat. We therefore evaluated the efficacy of this compound after reversible MCAO in the rat. METHODS Male Wistar rats underwent reversible MCAO by insertion of a nylon monofilament to the origin of the MCA. After 1 hour the filament was retracted and the ischemic tissue reperfused. Immediately after MCAO, primed miniosmotic pumps containing either vehicle or CP-0597 (300 ng/kg per minute) were implanted into the subcutaneous space (n = 14 per group). Twenty-four hours after surgery, animals were killed and brains fixed, and 4-micron sections were taken from five sequential tissue blocks labeled A through E and stained with hematoxylin and eosin. Clinical evaluation of rats was performed by neurological scoring and change in body weight. RESULTS Treatment with CP-0597 significantly reduced percent increase in hemisphere size of the ischemic hemisphere in all brain sections (C section: vehicle, 40.6 +/- 4.3% versus CP-0597, 20.8 +/- 5.3%; P < 0.001), total infarct volume (vehicle, 206.5 +/- 7.7 mm3 versus CP-0597, 94.0 +/- 19.2 mm3; P < .001), cortical infarct volume (vehicle, 145.5 +/- 4.5 mm3 versus CP-0597, 64.0 +/- 15.1 mm3; P < .001), subcortical infarct volume (vehicle, 55.8 +/- 4.1 mm3 versus CP-0597, 27.5 +/- 4.5 mm3; P < .001), and the number of necrotic neurons (vehicle 42.9 +/- 3.8 versus CP-0597, 23.6 +/- 4.7 per field; P < .01). Neurological score (vehicle, 2.78 +/- 0.36 versus CP-0597, 6.29 +/- 0.87 P < .01) and change in body weight (vehicle, -28.7 +/- 2.0 g versus CP-0597, -18.2 +/- 2.8 g; P < .01) were also significantly improved. CONCLUSIONS The present data demonstrate the significant overall efficacy profile of CP-0597 in a rat model of reversible MCAO and provide strong rationale for the use of such bradykinin B2 receptor antagonist in the treatment of stroke.

Inhibition of α4 Integrin Protects Against Transient Focal Cerebral Ischemia in Normotensive and Hypertensive Rats

Background and Purpose—The present study was performed to determine the role of α4 (CD49d), a member of the integrin family of adhesion molecules, in ischemic brain pathology. Methods—Male spontaneously hypertensive rats (SHR) or Sprague-Dawley rats underwent 60-minute middle cerebral artery occlusion (MCAO) followed by 23-hour reperfusion. Animals were injected intravenously with 2.5 mg/kg anti-rat α4 antibody (TA-2) or isotype control antibody (anti-human LFA-3 IgG1, 1E6) 24 hours before MCAO. Infarct volume was quantified by staining of fresh tissue with tetrazolium chloride and myeloperoxidase activity measured in SHR tissue homogenates 24 hours after MCAO. In SHR, mean arterial blood pressure was recorded before and after MCAO in animals treated with TA-2 and 1E6. Fluorescence-activated cell sorting analysis was performed on peripheral blood leukocytes before and after MCAO. Results—TA-2 treatment significantly reduced total infarct volume by 57.7% in normotensive rats (1E6, 84.2±11.5 mm3, n=17; TA-2...

Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival

Colony-stimulating factor 1 and IL-34 protect against and partially reverse neurodegeneration in mice in part via promoting CREB signaling.

Effect of combined treatment with methylprednisolone and soluble Nogo-66 receptor after rat spinal cord injury

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 µm intrathecal, 0.25 µL/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.

Assessment of functional recovery and axonal sprouting in oligodendrocyte-myelin glycoprotein (OMgp) null mice after spinal cord injury

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.