Dinah W. Y. Sah

LINGO-1 negatively regulates myelination by oligodendrocytes

The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.

TAJ/TROY, an Orphan TNF Receptor Family Member, Binds Nogo-66 Receptor 1 and Regulates Axonal Regeneration

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration.

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.

Structure and axon outgrowth inhibitor binding of the Nogo‐66 receptor and related proteins

The myelin‐derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell‐surface proteins signal through multi‐subunit neuronal receptors that contain a common ligand‐binding glycosylphosphatidylinositol‐anchored subunit termed the Nogo‐66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell‐surface NgR requires the entire leucine‐rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi‐ligand binding, we determined the crystal structure of the biologically active ligand‐binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N‐terminal cap and a small C‐terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.

Multiple actions of systemic artemin in experimental neuropathy

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury–induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

GFRalpha3 is expressed predominantly in nociceptive sensory neurons

Activation of the RET receptor tyrosine kinase by glial‐derived neurotrophic factor family members is dependent on a family of coreceptors, GFRα1–4. GFRα3 preferentially binds the newest member of the glial‐derived neurotrophic factor family of ligands, artemin. The major site of GFRα3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRα3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRα3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene‐related peptide. In addition, a significant subpopulation of GFRα3‐expressing cells also binds the lectin IB4. We demonstrate that GFRα3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF‐responsive population. Our results indicate artemin may exert selective effects on pain sensation.

Neurotrophic factors as novel therapeutics for neuropathic pain.

Neuropathic pain is a chronic condition that is caused by injury to the nervous system. Unlike acute pain, which is protective, neuropathic pain persists and serves no useful purpose, and severely affects quality of life. However, present therapies have modest efficacy in most patients, are palliative rather than curative, and their side effects represent significant limitations. Tremendous progress has been made over the past decade in our understanding of the biology of pain sensory neurons. The recent discovery that neurotrophic factors play an important role in neuropathic pain indicates that these pathways could serve as novel intervention points for therapy. Moreover, neurotrophic factors have the potential to address the underlying pathophysiology of neuropathic pain, thereby halting or reversing the disease process.

Targeting the Nogo Receptor to Treat Central Nervous System Injuries

Axonal damage is a key pathology in many injuries of the central nervous system (CNS), such as spinal cord injury, traumatic brain injury and stroke, as well as in multiple sclerosis. An attractive drug discovery strategy to treat such conditions is to search for agents that promote CNS axonal regeneration. Historically, limited knowledge concerning the basis of poor CNS regeneration has precluded a rational drug discovery approach for promoting axonal regeneration. The recent identification of the Nogo receptor, which interacts with inhibitory myelin protein, established the crucial role of this molecular pathway in mediating the inhibitory effects of CNS myelin. This provides an unprecedented opportunity to manipulate adult CNS axonal regeneration. The development of therapeutics targeting the Nogo receptor has the potential to promote functional recovery and reverse the devastating consequences of CNS injuries.

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin.

The Nogo66 receptor (NgR1) is a neuronal, leucinerich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinsons Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.