Subcellular Knockout of Importin β1 Perturbs Axonal Retrograde Signaling

Abstract

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.

Axonal transport of neural membrane protein 35 mRNA increases axon growth

“…For example, the microfilament protein b-actin allows for directional migration rather than overall motility of migrating fibroblasts when it is locally generated (Shestakova et al, 2001). Axonal translation of Importin b1 and RanBP1 is used to generate an Importin a/b heterodimer for transporting axonal signaling proteins to the cell body, with introduction of these proteins providing a precise temporal indicator of axon injury (Ben-Yaakov et al, 2012;Hanz et al, 2003;Perry et al, 2012;Yudin et al, 2008). Although NMP35 mRNA was initially cloned from developing sciatic nerve (Schweitzer et al, 1998) and NMP35 protein concentrates at synapses in post-mitotic neurons (Schweitzer et al, 2002), the possibility for localization of NMP35 mRNA was not considered in these early expression studies.…”

“…The myristoylation element in GFP coding sequence decreases its diffusion compared to non-modified GFP such that fluorescence recovery after photobleaching (FRAP) can be used to test for localized translation events by comparing recovery with and without active protein synthesis (Vuppalanchi et al, 2010;Yudin et al, 2008). Without a localizing UTR, the GFP myr mRNA is restricted to the neuronal cell body (Aakalu et al, 2001;Akten et al, 2011;Ben-Yaakov et al, 2012;Perry et al, 2012;Vuppalanchi et al, 2010;Willis et al, 2007;Yudin et al, 2008).…”

“…These included fluorescent intensities from FISH and immunofluorescence images calculated using Image J. One-way ANOVA with Bonferroni post-hoc test was used to test for significance the FRAP studies where more independent groups were compared as previously published (Akten et al, 2011;Ben-Yaakov et al, 2012;Perry et al, 2012;Vuppalanchi et al, 2010;Vuppalanchi et al, 2012;Yudin et al, 2008).…”

Sensing nerve injury at the axonal ER: Activated Luman/CREB3 serves as a novel axonally synthesized retrograde regeneration signal

“…Nuclear import of activated axonal TFs is mediated via their nuclear localization signal (NLS) binding to importin-α found throughout the axon. This interaction is heightened by the addition of importin-β, which is only axonally translated in response to axotomy, creating a high-affinity NLS-importin binding complex that retrogradely traffics target proteins to the cell body (11,12). The identification of multiple axonal TF transcripts in the injured nerve supports the view that many may serve as retrograde regeneration signals linking axonal to nuclear activities.…”

Targeting Axonal Protein Synthesis in Neuroregeneration and Degeneration

“…The formation of a new growth cone after axotomy of developing axons in vitro requires both local protein synthesis and degradation [30], and upon injury of mature axons, mRNAs and protein synthesis machinery are rapidly recruited into axons and intraaxonal translation is upregulated or re-activated within these mature axons [31][32][33][34]. Locally synthesized proteins are required for communication from the injured axons to their soma and likely participate in the formation of the growth bulb at the site of injury [35,36]. Several recent excellent reviews have been published covering the multifaceted role of local translation in injured axons [37][38][39]; here, we will focus on the question whether manipulation of the local translatome in injured axons might be of therapeutic value.…”