07-678 Sigma-AldrichAnti-LINGO-1 Antibody

Genetic polymorphisms in Solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2) have been linked with essential tremor. SLC1A2 encodes excitatory amino acid transporter type 2 (EAAT2), which clears glutamate from the synaptic cleft. One postulated mechanism for essential tremor is the over-excitation of glutamatergic olivo-cerebellar climbing fibers, leading to excitotoxic death of Purkinje cells. Other glutamatergic excitatory signals are transmitted to Purkinje cells via parallel fibers of cerebellar granule neurons. Therefore, the expression level of glutamate transporters could be important in essential tremor pathogenesis. Using Western blotting, we compared the expression levels of the two main glutamate transporters in the cerebellar cortex, EAAT1 and EAAT2, in postmortem tissue from 16 essential tremor cases and 13 age-matched controls. We also studied the localization of EAAT1 and EAAT2 using immunohistochemistry in 10 essential tremor cases and 12 controls. EAAT1 protein levels were similar in cases and controls (1.12 ± 0.83 vs. 1.01 ± 0.69, p =0.71) whereas EAAT2 protein levels in essential tremor cases were only 1/3 of that in controls (0.35 ± 0.23 vs. 1.00 ± 0.62, p less than 0.01). Interestingly, EAAT2, but not EAAT1, was expressed in astrocytic processes surrounding the Purkinje cell axon initial segment, a region of previously observed pathological changes in essential tremor. Our main finding, a significant reduction in cerebellar cortical EAAT2 protein levels in essential tremor, suggests that Purkinje cells in essential tremor might be more vulnerable to excitotoxic damage than those of controls.

The Lingo-1 sequence variant has been associated with essential tremor (ET) in several genome-wide association studies. However, the role that Lingo-1 might play in pathogenesis of ET is not understood. Since Lingo-1 protein is a negative regulator of axonal regeneration and neurite outgrowth, it could contribute to Purkinje cell (PC) or basket cell axonal pathology observed in postmortem studies of ET brains. In this study, we used Western blotting and immunohistochemistry to examine Lingo-1 protein in ET vs. control brains. In Western blots, Lingo-1 protein expression level was significantly increased in cerebellar cortex (1.56 ± 0.46 in ET cases vs. 0.99 ± 0.20 in controls, p = 0.002), but was similar in the occipital cortex (p = 1.00) of ET cases vs. controls. Lingo-1 immunohistochemistry in cerebellum revealed that Lingo-1 was enriched in the distal axonal processes of basket cells, which formed a "pinceau" structure around the PC axon initial segment (AIS). We found that some Lingo-1-positive pinceau had abnormally elongated processes, targeting PC axon segments distal to the AIS. In ET cases, the percentage of Lingo-1-positive pinceau that were ≥30 or ≥40 μm in length was increased 2.4- to 4.1-fold, respectively, vs. pinceau seen in control brains (p less than 0.0001). Elongated Lingo-1-positive pinceau strongly correlated with number of PC axonal torpedoes and a rating of basket cell axonal pathology. The increased cerebellar Lingo-1 expression and elongated Lingo-1-positive pinceau processes could contribute to the abnormal PC and basket cell axonal pathology and cerebellar dysfunction observed in ET.

Nogo constitutes a family of neurite outgrowth inhibitors contributing to a failure of axonal regeneration in the adult central nervous system (CNS). Nogo-A is expressed exclusively on oligodendrocytes where Nogo-66 segment binds to Nogo receptor (NgR) expressed on neuronal axons. NgR signalling requires a coreceptor p75(NTR) or TROY in combination with an adaptor LINGO-1. To characterize the cell types expressing the NgR complex in the human CNS, we studied demyelinating lesions of multiple sclerosis (MS) brains by immunohistochemistry. TROY and LINGO-1 were identified in subpopulations of reactive astrocytes, macrophages/microglia and neurones but not in oligodendrocytes. TROY was up-regulated, whereas LINGO-1 was reduced in MS brains by Western blot. These results suggest that the ternary complex of NgR/TROY/LINGO-1 expressed on astrocytes, macrophages/microglia and neurones, by interacting with Nogo-A on oligodendrocytes, might modulate glial-neuronal interactions in demyelinating lesions of MS.

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.

Axonal regeneration after injury can be limited in the adult CNS by the presence of inhibitory proteins such as Nogo. Nogo binds to a receptor complex that consists of Nogo receptor (NgR), p75NTR, and Lingo-1. Nogo binding activates RhoA, which inhibits axonal outgrowth. Here we assessed Lingo-1 and NgR mRNA levels after delivery of BDNF into the rat hippocampal formation, Lingo-1 mRNA levels in rats subjected to kainic acid (KA) and running in running wheels. Lingo-1 mRNA was not changed by running. However, we found that Lingo-1 mRNA was strongly up-regulated while NgR mRNA was down-regulated in the dentate gyrus in both the BDNF and the KA experiments. Our data demonstrate inverse regulation of NgR and Lingo-1 in these situations, suggesting that Lingo-1 up-regulation is one characteristic of activity-induced neural plasticity responses.

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.

In the adult mammalian central nervous system (CNS), growth of neuronal fibers is actively inhibited by myelin. The proteins myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgP), and Nogo-66 have been identified as inhibitory components present in CNS myelin. All three proteins exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor (NgR) and the neurotrophin (NT) receptor p75NTR. In their recent publication, Mi et al. identify the novel protein Lingo-1 as an interactor of p75NTR and NgR. The Lingo-1-NgR-p75NTR complex is shown to confer the inhibitory effects on nerve cell regeneration of Nogo-66, OMgP, and MAG by activating the small guanosine triphosphatase (GTPase) RhoA. Together with the recent finding that p75NTR interacts with the transmembrane protein sortilin to form a different receptor complex with cell death-promoting activity, the results of Mi et al. indicate that p75NTR exerts its diverse cellular functions by associating with function-specific co-receptors.

Signalling by the p75 neurotrophin receptor has been implicated in diverse neuronal responses, including increased differentiation or survival, inhibition of regeneration, and initiation of apoptotic cell death. These numerous roles are matched by, but are not yet correlated with, a multiplicity of extracellular ligands and intracellular interactors. Membrane proteins such as sortilin, a member of the Vps10p family of sorting receptors, and the glycosylphosphatidylinositol-linked Nogo receptor (NgR) and the associated adaptor lingo 1 have recently been added to the list of p75-interacting modulators. Other studies have described intramembranal cleavage of p75 and the potential nuclear targeting of cleavage fragments or of the complete receptor after it has been internalized into a putative signalling endosome. These findings suggest that some of the diversity in p75 activities might be due to differential subcellular localization and transport of p75 receptor complexes. We therefore argue that cell-biology-driven approaches are now required to make sense of p75 signalling.