A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus

Abstract

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.

A Neuronal Isoform of Protein Kinase G Couples Mitogen-Activated Protein Kinase Nuclear Import to Axotomy-Induced Long-Term Hyperexcitability inAplysiaSensory Neurons

“…8 A). Similar patches have been seen after the import of other proteins into Aplysia nuclei (Ambron et al, 1992;Schmied et al, 1993;Gunstream et al, 1995).…”

“…3A). In addition, because proteins are retrogradely transported along Aplysia axons at a rate of 1.5 mm/hr (Ambron et al, 1992;Schmied et al, 1993;Gunstream et al, 1995), the delay is consistent with the transport of apPKG from the crush site, which was located 2 cm from the ganglion. We then placed a ligation proximal to the crush site and found that activated apPKG and total apPKG protein accumulated on the distal side of the ligation, relative to the crush site (Fig.…”

“…8 A). Alexalabeled BSA also remained in the cytoplasm after injection, as expected (Ambron et al, 1992;Schmied et al, 1993;Gunstream et al, 1995). In contrast, injected Alexa-labeled active recombinant vertebrate ERK1, which is imported into the nucleus in a variety of cell types (Karin, 1994) rapidly entered the nucleus where it was distributed in discrete patches (Fig.…”

“…Nevertheless, type-I PKGs have a putative NLS (Gudi et al, 1997), and apPKG has a short stretch of positively charged amino acids ( 453 KCLKKKHI) in the ATP-binding domain that could function as an NLS. The import of proteins into the nucleus of Aplysia neurons is readily assessed by injecting their fluorescently labeled cognates directly into the cell body (Ambron et al, 1992;Schmied et al, 1993;Gunstream et al, 1995). We therefore injected Alexalabeled recombinant apPKG into the soma of SNs after 2 d in vitro, severed the neurites to initiate an injury response, and, 30 min later, examined the cells by fluorescence microscopy.…”

Memory-Like Alterations inAplysiaAxons after Nerve Injury or Localized Depolarization

“…The observation of depolarization-induced LTH in intact axons shows that axonal LTH can be produced by mechanisms other than damming of Na ϩ channels at the proximal stump of a severed axon (Devor and Govrin-Lippmann, 1983;Devor et al, 1993). The distance of the treated nerve segment from the neuronal soma (2-4 cm) and the blockade of synaptic transmission in the ganglion during induction and testing of LTH, coupled with reported rates of axonal transport in Aplysia (ϳ1.5 mm/hr) (Ambron et al, 1992;Gunstream et al, 1995;Sung et al, 2004), suggests that the axonal LTH seen 24 hr after treatment does not depend on transport of retrograde signals that produce transcriptional or translational effects in the soma Sung et al, 2004) and subsequent anterograde transport of plasticity-mediating molecules to the axonal test site.…”