Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway

Protein kinase A (PKA) controls major aspects of neurite outgrowth and morphogenesis and plays an essential role in synaptic plasticity and memory. However, the molecular mechanism(s) of PKA action on neurite sprouting and activity are still unknown. Here, we report that in response to neurotrophin or cAMP stimulation the RING ligase praja2 ubiquitinates and degrades NOGO-A, a major inhibitor of neurite outgrowth in mammalian brain. Genetic silencing of praja2 severely inhibited neurite extension of differentiating neuroblastoma cells and mesencephalic neurons and axon outgrowth and sprouting of striatal terminals in developing rat brain. This phenotype was rescued when both praja2 and NOGO-A were depleted, suggesting that NOGO-A is, indeed, a biologically relevant target of praja2 in neuronal cells. Our findings unveil a novel mechanism that functionally couples cAMP signaling with the proteolytic turnover of NOGO-A, positively impacting on neurite outgrowth in mammalian brain.Neurite outgrowth plays an essential role in embryonic development, neuronal differentiation, and central nervous system (CNS) plasticity. Outgrowth can be also altered in several neurological disorders, as well as by neuronal injury and degeneration (1, 2). Extracellular signals, such as neurotrophins (NTFs) and neurotransmitters, regulate neurite outgrowth, dendritic arborization, and synaptic activity, establishing a dynamic neuronal network in developing and adult CNS. NTFs and neurotransmitters act at the cell membrane by generating intracellular second messengers that, in turn, reversibly modulate the activity of signaling proteins and effector enzymes (3, 4).cAMP is an ancient second messenger that controls a variety of biological cues. In neurons, essential functions such as neurite outgrowth and morphogenesis, synaptic transmission, and plasticity require tightly regulated responses to cAMP/protein kinase A (PKA) stimulation (5). PKA holoenzyme localizes in subcellular microdomains through interactions with A-Kinase-Anchor-Proteins (AKAPs). AKAP forms a local transduction unit, which includes signaling/metabolic enzymes, receptors, ion channels, adaptor molecules, and mRNAs (6, 7). Space-restricted activation of PKA provides a control mechanism to direct, integrate, and locally attenuate the cAMP cascade (8). praja2 belongs to a growing family of mammalian RING ligases abundantly expressed in the brain that finely tune the stability of intracellular substrates and play an essential role in critical aspects of cell signaling. In response to cAMP stimulation, praja2 couples ubiquitination and proteolysis of the inhibitory PKA regulatory (R) subunits by the proteasome to a sustained cAMP/PKA signaling, significantly impacting on synaptic plasticity and long-term memory (9). In addition to enhancing cAMP signaling, the role of praja2 in neuronal differentiation and dendritic network in the CNS and the molecular targets involved are unknown.NOGO-A is a member of the reticulon (RTN) family of integral membrane proteins with a conserved C terminus reticulon homology domain (RHD) and abundantly expressed in oligodendrocytes and in distinct neuronal subpopulations (10, 11). NOGO-A was originally identified as a potent inhibitor of neurite outgrowth (1, 10). In the adult CNS and in injured neurons, NOGO-A restricts the capacity of an axon to grow and regenerate. Genetic ablation of NOGO-A promotes neuritogenesis and fasciculation of oligodendrocytes and culture of dorsal root ganglion neurons, functionally improving neuronal plasticity and recovery of postischemic adult rat brain (12).Although the role of NOGO-A in neurite outgrowth is well established, regulation of NOGO-A levels in differentiating neurons and the mechanism(s) involved have been, to date, unknown. Here, we report a novel mechanism of neuritogenesis based on proteolytic turnover of NOGO-A (13). In response to cAMP or neurothrophin stimulation, RING ligase praja2 ubiquitinates and degrades NOGO-A. Proteolysis of NOGO-A by praja2 is functionally linked to neurite outgrowth in both differentiating neurons and developing rat brain.