Roman J. Giger

Semaphorin 5A Is a Bifunctional Axon Guidance Cue Regulated by Heparan and Chondroitin Sulfate Proteoglycans

The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.

Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS

This study evaluates the expression of the chemorepellent semaphorin III (D)/collapsin-1 (sema III) following lesions to the rat CNS. Scar tissue, formed after penetrating injuries to the lateral olfactory tract (LOT), cortex, perforant pathway, and spinal cord, contained numerous spindle-shaped cells expressing high levels of sema III mRNA. The properties of these cells were investigated in detail in the lesioned LOT. Most sema III mRNA-positive cells were located in the core of the scar and expressed proteins characteristic for fibroblast-like cells. Neuropilin-1, a sema III receptor, was expressed in injured neurons with projections to the lesion site, in a subpopulation of scar-associated cells and in blood vessels around the scar. In contrast to lesions made in the mature CNS, LOT transection in neonates did not induce sema III mRNA expression within cells in the lesion and was followed by vigorous axonal regeneration. The concomitant expression of sema III and its receptor neuropilin-1 in the scar suggests that sema III/neuropilin-1-mediated mechanisms are involved in CNS scar formation. The expression of the secreted chemorepellent sema III following CNS injury provides the first evidence that chemorepulsive semaphorins may contribute to the inhibitory effects exerted by scars on the outgrowth of injured CNS neurites. The vigorous regrowth of injured axons in the absence of sema III following early neonatal lesions is consistent with this notion. The inactivation of sema III in scar tissue by either antibody perturbation or by genetic or pharmacological intervention could be a powerful means to promote long-distance regeneration in the adult CNS.

NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1−/−; Ngr2−/−; Ngr3−/−; which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1−/−; Ngr3−/−), but not Ngr1 and Ngr2 (Ngr1−/−; Ngr2−/−), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1−/−; Ngr3−/− mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

The Nogo-66 Receptor Homolog NgR2 Is a Sialic Acid-Dependent Receptor Selective for Myelin-Associated Glycoprotein

The Nogo-66 receptor (NgR1) is a promiscuous receptor for the myelin inhibitory proteins Nogo/Nogo-66, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). NgR1, an axonal glycoprotein, is the founding member of a protein family composed of the structurally related molecules NgR1, NgR2, and NgR3. Here we show that NgR2 is a novel receptor for MAG and acts selectively to mediate MAG inhibitory responses. MAG binds NgR2 directly and with greater affinity than NgR1. In neurons NgR1 and NgR2 support MAG binding in a sialic acid-dependent Vibrio cholerae neuraminidase-sensitive manner. Forced expression of NgR2 is sufficient to impart MAG inhibition to neonatal sensory neurons. Soluble NgR2 has MAG antagonistic capacity and promotes neuronal growth on MAG and CNS myelin substrate in vitro. Structural studies have revealed that the NgR2 leucine-rich repeat cluster and the NgR2 “unique” domain are necessary for high-affinity MAG binding. Consistent with its role as a neuronal MAG receptor, NgR2 is an axonassociated glycoprotein. In postnatal brain NgR1 and NgR2 are strongly enriched in Triton X-100-insoluble lipid rafts. Neural expression studies of NgR1 and NgR2 have revealed broad and overlapping, yet distinct, distribution in the mature CNS. Taken together, our studies identify NgRs as a family of receptors (or components of receptors) for myelin inhibitors and provide insights into how interactions between MAG and members of the Nogo receptor family function to coordinate myelin inhibitory responses.

Synaptic Function for the Nogo-66 Receptor NgR1: Regulation of Dendritic Spine Morphology and Activity-Dependent Synaptic Strength

In the mature nervous system, changes in synaptic strength correlate with changes in neuronal structure. Members of the Nogo-66 receptor family have been implicated in regulating neuronal morphology. Nogo-66 receptor 1 (NgR1) supports binding of the myelin inhibitors Nogo-A, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), and is important for growth cone collapse in response to acutely presented inhibitors in vitro. After injury to the corticospinal tract, NgR1 limits axon collateral sprouting but is not important for blocking long-distance regenerative growth in vivo. Here, we report on a novel interaction between NgR1 and select members of the fibroblast growth factor (FGF) family. FGF1 and FGF2 bind directly and with high affinity to NgR1 but not to NgR2 or NgR3. In primary cortical neurons, ectopic NgR1 inhibits FGF2-elicited axonal branching. Loss of NgR1 results in altered spine morphologies along apical dendrites of hippocampal CA1 neurons in vivo. Analysis of synaptosomal fractions revealed that NgR1 is enriched synaptically in the hippocampus. Physiological studies at Schaffer collateral–CA1 synapses uncovered a synaptic function for NgR1. Loss of NgR1 leads to FGF2-dependent enhancement of long-term potentiation (LTP) without altering basal synaptic transmission or short-term plasticity. NgR1 and FGF receptor 1 (FGFR1) are colocalized to synapses, and mechanistic studies revealed that FGFR kinase activity is necessary for FGF2-elicited enhancement of hippocampal LTP in NgR1 mutants. In addition, loss of NgR1 attenuates long-term depression of synaptic transmission at Schaffer collateral–CA1 synapses. Together, our findings establish that physiological NgR1 signaling regulates activity-dependent synaptic strength and uncover neuronal NgR1 as a regulator of synaptic plasticity.

Semaphorin function in neural plasticity and disease

The semaphorins, originally discovered as evolutionarily conserved steering molecules for developing axons, also influence neuronal structure and function in the early postnatal and juvenile nervous systems through several refinement processes. Semaphorins control synaptogenesis, axon pruning, and the density and maturation of dendritic spines. In addition, semaphorins and their downstream signaling components regulate synaptic physiology and neuronal excitability in the mature hippocampus, and these proteins are also implicated in a number of developmental, psychiatric, and neurodegenerative disorders. Significant inroads have been made in defining the mechanisms by which semaphorins regulate dynamic changes in the neuronal cytoskeleton at the molecular and cellular levels during embryonic nervous system development. However, comparatively little is known about how semaphorins influence neuronal structure and synaptic plasticity during adult nervous system homeostasis or following injury and disease. A detailed understanding of how semaphorins function beyond initial phases of neural network assembly is revealing novel insights into key aspects of nervous system physiology and pathology.

Anatomical distribution of the chemorepellent semaphorin III/collapsin-1 in the adult rat and human brain: Predominant expression in structures of the olfactory-hippocampal pathway and the motor system

Alterations in neuronal connectivity of the mature central nervous system (CNS) appear to depend on a delicate balance between growth‐promoting and growth‐inhibiting molecules. To begin to address a potential role of the secreted chemorepulsive protein semaphorin(D)III/collapsin‐1 (semaIII/coll‐1) in structural plasticity during adulthood, we used high‐resolution nonradioactive in situ hybridization to identify neural structures that express semaIII/coll‐1 mRNA in the mature rat and human brain. SemaIII/coll‐1 was expressed in distinct but anatomically and functionally linked structures of the adult nervous system. The olfactory‐hippocampal pathway displayed semaIII/coll‐1 expression in a continuum of neuronal structures, including mitral and tufted cells of the olfactory bulb, olfactory tubercle, and piriform cortex; and distinct nuclei of the amygdaloid complex, the superficial layers of the entorhinal cortex, and the subiculum of the hippocampal formation. In addition, prominent labeling was found in neuronal components of the motor system, particularly in cerebellar Purkinje cells and in subpopulations of cranial and spinal motoneurons. Retrograde tracing combined with in situ hybridization also revealed that the staining of semaIII/coll‐1 within the entorhinal cortex was present in the stellate neurons that project via the perforant path to the molecular layer of the dentate gyrus. Like in the rat, the human brain displayed discrete expression of semaIII/coll‐1. Among the structures examined, the most prominent staining was observed in the cellular islands of the superficial layers of the human entorhinal cortex. The constitutive expression of the chemorepellent semaIII/coll‐1 in discrete populations of neurons in the mature rat and human CNS raises the possibility that, in addition to its function as repulsive axon guidance cue during development, semaIII/coll‐1 might be involved in restricting structural changes that occur in the wiring of the intact CNS. J. Neurosci. Res. 52:27–42, 1998.

Oligodendrocyte-Myelin Glycoprotein and Nogo Negatively Regulate Activity-Dependent Synaptic Plasticity

In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral–CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral–CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB−/− and NgR1−/− single mutants and NgR1−/−;PirB−/− double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1−/−, but not PirB−/−, slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.

Regulation of Semaphorin III/Collapsin-1 Gene Expression during Peripheral Nerve Regeneration ☆

The competence of neurons to regenerate depends on their ability to initiate a program of gene expression supporting growth and on the growth-permissive properties of glial cells in the distal stump of the injured nerve. Most studies on intrinsic molecular mechanisms governing peripheral nerve regeneration have focussed on the lesion-induced expression of proteins promoting growth cone motility, neurite extension, and adhesion. However, little is known about the expression of intrinsic chemorepulsive proteins and their receptors, after peripheral nerve injury and during nerve regeneration. Here we report the effect of peripheral nerve injury on the expression of the genes encoding sema III/coll-1 and its receptor neuropilin-1, which are known to be expressed in adult sensory and/or motor neurons. We have shown that peripheral nerve crush or transection results in a decline in sema III/coll-1 mRNA expression in injured spinal and facial motor neurons. This decline was paralleled by an induction in the expression of the growth-associated protein B-50/GAP-43. As sema III/coll-1 returned to normal levels following nerve crush, B-50/GAP-43 returned to precrush levels. Thus, the decline in sema III/coll-1 mRNA coincided with sensory and motor neuron regeneration. A sustained decline in sema III/coll-1 mRNA expression was found when regeneration was blocked by nerve transection and ligation. No changes were observed in neuropilin-1 mRNA levels after injury to sensory and motor neurons, suggesting that regenerating peripheral neurons continue to be sensitive to sema III/coll-1. Therefore we propose that a decreased expression of sema III/coll-1, one of the major ligands for neuropilin-1, during peripheral nerve regeneration is an important molecular event that is part of the adaptive response related to the success of regenerative neurite outgrowth occurring following peripheral nerve injury.

The Nogo-66 Receptor NgR1 Is Required Only for the Acute Growth Cone-Collapsing But Not the Chronic Growth-Inhibitory Actions of Myelin Inhibitors

Neuronal Nogo-66 receptor 1 (NgR1) has been proposed to function as an obligatory coreceptor for the myelin-derived ligands Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) to mediate neurite outgrowth inhibition by these ligands. To examine the contribution of neuronal NgR1 to outgrowth inhibition, we used two different strategies, genetic ablation of NgR1 through the germline and transient short hairpin RNA interference (shRNAi)-mediated knock-down. To monitor growth inhibition, two different paradigms were used, chronic presentation of substrate-bound inhibitor to measure neurite extension and acute application of soluble inhibitor to assay growth cone collapse. We find that regardless of the NgR1 genotype, membrane-bound MAG strongly inhibits neurite outgrowth of primary cerebellar, sensory, and cortical neurons. Similarly, substrate-bound OMgp strongly inhibits neurite outgrowth of NgR1 wild-type and mutant sensory neurons. Consistent with these results, shRNAi-mediated knock-down of neuronal NgR1 does not result in a substantial release of L-MAG (large MAG) inhibition. When applied acutely, however, MAG-Fc and OMgp-Fc induce a modest degree of growth cone collapse that is significantly attenuated in NgR1-null neurons compared with wild-type controls. Based on our findings and previous studies with Nogo-66, we propose that neuronal NgR1 has a circumscribed role in regulating cytoskeletal dynamics after acute exposure to soluble MAG, OMgp, or Nogo-66, but is not required for these ligands to mediate their growth-inhibitory properties in chronic outgrowth experiments. Our results thus provide unexpected evidence that the growth cone-collapsing activities and substrate growth-inhibitory activities of inhibitory ligands can be dissociated. We also conclude that chronic axon growth inhibition by myelin is mediated by NgR1-independent mechanisms.