Felicia Yu Hsuan Teng

Axonal regeneration in adult CNS neurons – signaling molecules and pathways

Failure of severed adult CNS axons to regenerate could be attributed to both a reduced intrinsic capacity to grow and an heightened susceptibility to inhibitory factors of the CNS extracellular environment. A particularly interesting and useful paradigm for investigating CNS axonal regeneration is its enhancement at the CNS branch of dorsal root ganglion (DRG) neurons after conditional lesioning of their peripheral branch. Recent reports have implicated the involvement of two well‐known signaling pathways utilizing separate transcription factors; the Cyclic AMP (cAMP) response element binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3), in conditional lesioning. The former appears to be the pathway activated by neurotrophic factors and Bcl‐2, while the latter is responsible for the neurogenic effect of cytokines [such as the leukemia inhibitory factor (LIF) and interleukin‐6 (IL‐6) elevated at lesion sites]. Recent findings also augmented earlier notions that modulations of the activity of another class of cellular signaling intermediate, the conventional protein kinase C (PKC), could result in a contrasting growth response by CNS neurons to myelin‐associated inhibitors. We discuss these signaling pathways and mechanisms, in conjunction with other recent reports of regeneration enhancement and also within the context of what is known about aiding regeneration of injured CNS axons.

Nogo‐A at CNS paranodes is a ligand of Caspr: possible regulation of K+ channel localization

We report Nogo‐A as an oligodendroglial component congregating and interacting with the Caspr–F3 complex at paranodes. However, its receptor Nogo‐66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo‐66 peptide and GST–Nogo‐66. Binding persisted even after phosphatidylinositol‐ specific phospholipase C (PI‐PLC) removal of GPI‐linked F3 from the cell surface, suggesting a direct interaction between Nogo‐66 and Caspr. Both Nogo‐A and Caspr co‐immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo‐A–Caspr and K+ channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT−/− mice), distances between the paired labeling of K+ channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo‐A congregation was markedly reduced. Our results demonstrate that Nogo‐A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon–glial junction architecture and possibly K+‐channel localization during development.

Why do Nogo/Nogo‐66 receptor gene knockouts result in inferior regeneration compared to treatment with neutralizing agents?

IN‐1, the monoclonal antibody against the exon 3‐encoded N‐terminal domain of Nogo‐A, and the Nogo‐66 receptor (NgR) antagonist NEP1‐40 have both shown efficacy in promoting regeneration in animal spinal cord injury models, the latter even when administered subcutaneously 1 week after injury. These results are supportive of the hypothesis that the Nogo–NgR axis is a major path for inhibition of spinal cord axonal regeneration and uphold the promises of these neutralizing agents in clinical applications. However, mice with targeted disruption of Nogo and NgR have, surprisingly, only modest regenerative capacity (if any) compared with treatment with IN‐1 or NEP1‐40. Disruption of the Nogo gene by various groups yielded results ranging from significant regenerative improvement in young mice to no improvement. Likewise, knockout of NgR produced some improvement in raphespinal and rubrospinal axonal regeneration, but not that of corticospinal neurons. Other than invoking possible differences in genetic background, we suggest here some possible and testable explanations for the above phenomena. These possibilities include effects of IN‐1 and NEP1‐40 on the CNS beyond neutralization of Nogo and NgR functions, and the latters possible role in the CNS beyond that of neuronal growth inhibition.

Cell Autonomous Function of Nogo and Reticulons : The Emerging Story at the Endoplasmic Reticulum

The myelin‐associated membrane protein reticulon‐4 (RTN4)/Nogo has been extensively studied with regards to its neurite outgrowth inhibitory function, both in limiting plasticity in the healthy adult brain and regeneration during central nervous system injury. These activities are presumably associated with Nogo splice isoforms expressed on the cell surface and function largely in trans, exerting an influence as an intercellular membrane‐bound ligand. Nogo, and other reticulon paralogues and orthologues, are however mainly localized to the endoplasmic reticulum (ER), and are likely to have cell autonomous functions that are not yet clear. Emerging evidence suggests that Nogo may have a role in modulating the morphology and functions of the ER. This role is apparently not essential for cell viability under normal growth conditions, but may be manifested under certain stress conditions. J. Cell. Physiol. 216: 303–308, 2008.

Open Brain Gene Product Rab23: Expression Pattern in the Adult Mouse Brain and Functional Characterization

The gene mutated in the mouse open brain (opb) phenotype antagonizes sonic hedgehog‐mediated signaling and encodes a small GTPase of the Rab family, Rab23. To date, the brain expression profile and exact mechanism of function of the Rab23 protein has remained unknown. Specific antibodies generated against Rab23 showed that the protein is highly enriched in the adult rodent brain and present in low levels in multiple tissues of the adult rodent. Rab23 is found in the cytosol as well as being associated with the plasma and endosomal membranes. In the adult mouse brain, Rab23 is found in βIII tubulin (TuJ) positive neuronal cell bodies and are most prominent in the cortex, hypothalamus and the cerebellum. It is, however, absent from glial fibrillary acidic protein (GFAP) positive astrocytes or CNPase positive oligodendrocytes. Despite the plasma membrane/endosomal membrane localization of Rab23, neither overexpression of the GTP‐restricted nor the GDP‐bound mutant forms affect internalization of transferrin or epidermal growth factor. Exogenous overexpression of Rab23 or its mutants also did not affect the morphological differentiation of thalamic neurons in culture. Expression of Rab23 in the adult brain is suggestive, however, of having a postnatal function beyond its role in embryonic development.

Nogo‐66 and myelin‐associated glycoprotein (MAG) inhibit the adhesion and migration of Nogo‐66 receptor expressing human glioma cells

Malignant gliomas are common and aggressive brain tumours associated with significant morbidity and mortality. We showed in this report that substratum adherence and migration by human U87MG glioma cells in culture were significantly attenuated by the extracellular domains of Nogo‐A (Nogo‐66) and the myelin‐associated glycoprotein (MAG). U87MG cells contained significant amounts of endogenous Nogo‐66 receptor (NgR), and treatment of the cells with phosphatidylinositol‐specific phospholipase C (PI‐PLC) or NgR antibodies resulted in an increase in their ability to adhere to, or migrate through, Nogo‐66‐ and MAG‐coated substrates. Nogo‐66 and MAG may therefore modulate glioma growth and migration by acting through the NgR, a phenomenon that has potential therapeutic implications.

Nogo signaling and non-physical injury-induced nervous system pathology.

The Nogo gene products were described first as myelin‐associated inhibitors that prevent neuronal regeneration upon injury. Recent findings have also implicated Nogo in several neuronal pathologies that are not induced by physical injury. Nogo‐A may be an important determinant of autoimmune demyelinating diseases, as active immunization with Nogo‐A fragments attenuates the symptoms of experimental autoimmune encephalomyelitis (EAE). Nogo‐A levels are elevated markedly in hippocampal neurons of patients with temporal lobe epilepsy (TLE), in brain and muscle of patients with amyotrophic lateral sclerosis (ALS), and in schizophrenic patients. Concrete evidence for a direct role of Nogo‐A in the latter neuropathies is not yet available, but such a role is logically in line with new findings associated with localization of Nogo‐A and Nogo–Nogo‐66 receptor (NgR)‐mediated signaling. We speculate on possible linkages between the effect of aberrant elevation of Nogo levels and the signaling consequences that could lead to nervous system pathology.

Nogo-A and Nogo-66 receptor in amyotrophic lateral sclerosis.

Nogo/reticulon (RTN)‐4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo‐A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo‐A in ALS aetiology is supported by the findings that Nogo‐A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo‐A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo‐66 receptor (NgR) by either agonistic or antagonistic Nogo‐66‐derived peptides protects against p75 neurotrophin receptor (p75NTR)‐dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75NTR, and this association could preclude pro‐apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75NTR in ALS disease progression may provide important, therapeutically exploitable information.

Emerging cues mediating astroglia lineage restriction of progenitor cells in the injured/diseased adult CNS.

Other than specific neurogenic regions, the adult central nervous system (CNS) is not conducive for neuronal regeneration and neurogenesis, particularly at sites of injury or neurodegeneration. Engraftment of neural stem/progenitor cells into non-neurogenic regions or sites of injury/disease invariably results mainly in astroglia differentiation. The reasons for such a lineage restriction have not been well defined. Recent findings have brought to light some underlying novel mechanistic basis for this preferential differentiation into astroglia. The more oxidized state of pathological brain tissue leads to upregulation of the protein deacetylase sirtuin 1 (Sirt1). Sirt1 appears to stabilize a co-repressor complex of Hairy/enhancer of split (Hes)1, thereby suppressing expression of the proneuronal transcription factor Mash1, and directs progenitor cell differentiation towards the glia lineage. Sirt1 upregulated by CNS inflammation may also inhibit neuronal differentiation. Myelin-associated inhibitors such as Nogo, acting through the Nogo-66 receptor (NgR), also appear to promote neural stem/progenitor cell differentiation into astrocytes. Understanding the molecular basis of glia lineage restriction of neural progenitors in the injured or diseased CNS would provide handles to improving the success of stem cell-based transplantation therapy.

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H2O2) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H2O2-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.