Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin a5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

The extensive lengths of neuronal processes necessitate efficient mechanisms for communication with the cell body. Neuronal regeneration after nerve injury requires new transcription; thus, long-distance retrograde signalling from axonal lesion sites to the soma and nucleus is required. In recent years, considerable progress has been made in elucidating the mechanistic basis of this system. This has included the discovery of a priming role for early calcium waves; confirmation of central roles for mitogen-activated protein kinase signalling effectors, the importin family of nucleocytoplasmic transport factors and molecular motors such as dynein; and demonstration of the importance of local translation as a key regulatory mechanism. These recent findings provide a coherent mechanistic framework for axon-soma communication in the injured nerve and shed light on the integration of cytoplasmic and nuclear transport in all eukaryotic cells.

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.

Summary Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells, however so far evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of importin β1 in lesioned axons coordinates a retrograde injury signaling complex transported to the neuronal cell body. Here we show that a long 3′ untranslated region (3′UTR) directs axonal localization of importin β1. Conditional targeting of this 3′UTR region in mice causes subcellular loss of importin β1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of importin β1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of importin β1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins, and is required for efficient retrograde signaling in injured axons.

Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate ~900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and ~4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified ~400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.

G protein-activated K؉ channels (GIRKs; Kir3) are activated by direct binding of G␤␥ subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G i/o family, preferably G␣ i3 and G␣ i2 , are donors of G␤␥ for GIRK. How this specificity is achieved is not known. Here, using a pulldown method, we confirmed the presence of G␣ i3-GDP binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5-3-O-(thio)triphosphate, reduced the binding of G␣ i3 by a factor of 2-4. G␣ i1-GDP bound to GIRK1 and GIRK2 much weaker than G␣ i3-GDP . Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G i3 activates GIRK more efficiently than G i1 , as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified G␤␥ in excised membrane patches was strongly augmented by coexpression of G␣ i3 and less by G␣ i1 . Differences in physical interactions of GIRK with GDP-bound G␣ subunits, or G␣␤␥ heterotrimers, may dictate different extents of G␣␤␥ anchoring, influence the efficiency of GIRK activation by G␤␥, and play a role in determining signaling specificity.

SummarySize homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing. Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development. Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1. Thus, molecular motors critically influence cell-length sensing and growth control.

Cardiac and neuronal G protein-activated K؉ channels (GIRK; Kir3) open following the binding of G␤␥ subunits, released from G i/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free G␤␥, but a G␤␥-independent component has also been envisaged. We investigated G␤␥ dependence of the basal GIRK activity (A GIRK,basal ) quantitatively, by titrated expression of G␤␥ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used G␤␥ scavenger, myristoylated C terminus of ␤-adrenergic kinase (m-c␤ARK), reduced A GIRK,basal by 70 -80% and eliminated the acetylcholine-evoked current (I ACh ). However, we found that m-c␤ARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed G␤␥ scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A GIRK,basal by up to 90%. Expression of GIRK was accompanied by an increase in the level of G␤␥ and G␣ in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of G␤␥ and its constitutive association with GIRK may underlie the excessively high A GIRK,basal observed at high expression levels of GIRK. Only 10 -15% of A GIRK,basal persisted upon expression of both m-phosducin and c␤ARK. These results demonstrate that a major part of I basal is G␤␥-dependent at all levels of channel expression, and only a small fraction (<10%) may be G␤␥-independent.G protein-activated, inwardly rectifying K ϩ channels (GIRK, Kir3) 1 mediate postsynaptic inhibitory effects of various neurotransmitters in the brain and atrium via seven-helix, G protein-coupled receptors (GPCRs) linked to pertussis toxin-sensitive G proteins of the G i/o family. Opening of the channels is the result of a direct binding of G␤␥ subunits released from the G␣ i/o ␤␥ heterotrimers (1-4). The channel can also be activated by cytosolic Na ϩ and membranal phosphatidylinositol 4,5-bisphosphate (PIP 2 ); the latter is essential for proper GIRK gating by both Na ϩ and G␤␥ (3, 5, 6). Whereas the physiological role of neurotransmitter-induced GIRK activity is well established, the basal activity of these channels (A GIRK,basal ) is often regarded as negligible and physiologically unimportant. This feature distinguishes GIRK from many other K ϩ channels of the Kir family, such as Kir1 and Kir2, which show high intrinsic activity under physiological conditions and are often referred to as "constitutively active." Low A GIRK,basal is supposed to ensure high signal-to-noise ratio for GIRK-related neurotransmitter signaling and to minimize participation of GIRK in resting membrane K ϩ conductance (see Ref. 2). However, some classical and many recent studies challenge this concept. In sinoa...