Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth <i>In Vitro</i> but Not Axonal Repair <i>In Vivo</i>

<div><p>Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes <i>Arg1</i> and <i>Gap43</i> and strongly promotes axon outgrowth <i>in vitro</i>. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion <i>in vivo</i>. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of <i>Nfil3</i>. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of <i>Nfil3</i> changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 <i>in vivo</i> does not recapitulate the regeneration-promoting effects that were previously observed <i>in vitro</i>, indicating that <i>in vivo</i> transcriptional control of regeneration is probably more complex and more robust against perturbation than <i>in vitro</i> data may suggest.</p></div

<i>Nfil3</i> deletion impairs functional recovery from peripheral nerve injury <i>in vivo</i>.

<p>(a) <i>Nfil3</i> KO mice show no differences in the total distance moved in the open field task (n = 12, <i>t</i>₍₂₂₎ = -0.27, <i>p</i> = 0.98). (b) The latency to fall off an accelerating rotarod was not affected in <i>Nfil3</i> KO mice (n = 12, F_(1,22) = 1.02, <i>p</i> = 0.32). (c) <i>Nfil3</i> KO mice have a significantly longer beam crossing latency than wildtype mice (main effect genotype, n = 11/10, F_(1,19) = 8.893, ^#<i>p</i> = 0.008). Post-hoc t-tests indicated indicate significant differences in performance at post-lesion days 5, 13, 15 and 17 (**<i>p</i> < 0.01, *<i>p</i> < 0.05). (d) <i>Nfil3</i> KO mice also make significantly more errors when crossing the beam (main effect genotype, n = 11/10, F_(1,19) = 7.145, ^#<i>p</i> = 0.015; interaction genotype*time, F_(12,228) = 2.131, ^$<i>p</i> = 0.016). Post-hoc t-tests indicate significant differences in performance at post-lesion days 9, 12, 13, 15 and 19 (**<i>p</i> < 0.01, *<i>p</i> < 0.05).</p

Gene ontology terms overrepresented within genes differentially regulated in <i>Nfil3</i> KO mice.

<p>Gene ontology terms overrepresented within genes differentially regulated in <i>Nfil3</i> KO mice.</p

<i>Nfil3</i> deletion enhances axon growth of DRG neurons in culture.

<p>(a) Example images of cultured embryonic DRG neurons from wildtype mice (top panels) and <i>Nfil3</i> KO mice (bottom panels) at 1, 5 and 8 days <i>in vitro</i> (DIV; scale bar: 500 <b>μ</b>m). (b) Quantification of axon lengths showed that the average axon length of <i>Nfil3</i> KO neurons was significantly higher compared to wildtype neurons at DIV1 (222±10 <b>μ</b>m vs. 178±9 <b>μ</b>m; n = 69/71), at DIV5 (931±101 <b>μ</b>m vs. 595±48 <b>μ</b>m; n = 26/31) and at DIV8 (1134±77 <b>μ</b>m vs. 806±57 <b>μ</b>m; n = 32/34) (Student’s <i>t</i> test; mean ± SEM; **<i>p</i> < 0.01).</p

Generation and validation of <i>Nfil3</i> KO mice.

<p>(a) A schematic representation of the knockout strategy is indicated. (b) Southern blot analysis confirming correct homologous recombination at the 5’ probe side using AvrII digestion yielding fragments of 11.4 kb (wildtype) and 7.6 kb (mutant), at the 3’ probe side using EcoRV digestion yielding fragments of 12.2 kb (wildtype) and 8.9 kb (mutant), and at the Neo cassette using NheI digestion yielding a band of 12 kb (mutant only). (c) <i>Nfil3</i> mRNA levels in <i>Nfil3</i> KO and wildtype brains as measured by quantitative real-time PCR. Gene expression was normalized against Gapdh expression.</p

NFIL3 deletion does not alter the expression of regeneration-associated genes.

<p>(a) Gene expression was profiled in <i>Nfil3</i> KO mice and wildtype controls at 2 days and at 5 days post-lesion, relative to non-injured control DRGs. Gene regulation values in wildtype animals at post-lesion day 5 show a highly significant correlation (r = 0.48, <i>p</i> < 2.2x10^-16) with previously published data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref031" target="_blank">31</a>] describing injury-induced gene expression changes in mouse DRGs at the same time point (GEO datasets GSM827127/8). (b) The expression of well-established regeneration-associated genes and/or NFIL3 target genes is not affected in <i>Nfil3</i> KO animals compared with wildtype controls. Of the 20 genes indicated here, 16 are in the core set of regeneration-associated genes identified in three or more independent microarray studies (bold print) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref032" target="_blank">32</a>], and 8 are experimentally validated NFIL3 target genes (underlined) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref011" target="_blank">11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127163#pone.0127163.ref012" target="_blank">12</a>]. No significant differences were observed between expression profiles of <i>Nfil3</i> KO animals and wildtype controls.</p

Gene ontology terms overrepresented within genes differentially regulated at 5 days post-lesion.

<p>Gene ontology terms overrepresented within genes differentially regulated at 5 days post-lesion.</p

Dominant-negative inhibition of NFIL3 impairs regenerative axon growth <i>in vivo</i>.

<p>(a) Overview of the experimental design. At day 0 L4/L5 DRGs were injected with AAV5 virus expressing either DN-NFIL3 and GFP, or GFP only. At day 14 the animals received a unilateral crush of the sciatic nerve. At day 21 we transsected the sciatic nerve 1 cm distal from the crush and treated the proximal stump with the retrograde tracer FastBlue. The distal stump was removed for histological analysis. At day 27 animals were sacrificed, and the DRGs were removed for histological analysis. (b) Examples of control and DN-NFIL3 treated DRG sections stained with anti-βIII-tubulin in red, anti-GFP in green, and showing FastBlue labeling in blue (scale bar: 100 <b>μ</b>m). (c) The total fraction of FastBlue-positive βIII-tubulin expressing neurons was slightly lower in DN-NFIL3-treated animals compared with controls (n = 8, t₍₁₄₎ = 1.180, <i>p</i> = 0.25). (d) When the quantification of FastBlue-positive cells was limited to GFP-positive (i.e. virally transduced) neurons, a significant reduction was observed in DN-NFIL3-treated animals compared with controls (n = 8, t_(9.214) = 2.390, <i>p</i> = 0.040). (e) Examples of control and DN-NFIL3 treated sciatic nerve sections stained with anti-βIII-tubulin (scale bar: 100 <b>μ</b>m). (f) No significant difference was observed in fiber densities in the sciatic nerve at 1 cm distal of the crush (n = 8, t₍₁₄₎ = 0.095, <i>p</i> = 0.925).</p

TRIM3 Regulates the Motility of the Kinesin Motor Protein KIF21B

<div><p>Kinesin superfamily proteins (KIFs) are molecular motors that transport cellular cargo along the microtubule cytoskeleton. KIF21B is a neuronal kinesin that is highly enriched in dendrites. The regulation and specificity of microtubule transport involves the binding of motors to individual cargo adapters and accessory proteins. Moreover, posttranslational modifications of either the motor protein, their cargos or tubulin regulate motility, cargo recognition and the binding or unloading of cargos. Here we show that the ubiquitin E3 ligase TRIM3, also known as BERP, interacts with KIF21B via its RBCC domain. TRIM3 is found at intracellular and Golgi-derived vesicles and co-localizes with the KIF21B motor in neurons. <i>Trim3</i> gene deletion in mice and TRIM3 overexpression in cultured neurons both suggested that the E3-ligase function of TRIM3 is not involved in KIF21B degradation, however TRIM3 depletion reduces the motility of the motor. Together, our data suggest that TRIM3 is a regulator in the modulation of KIF21B motor function.</p> </div

The E3 ligase TRIM3 is not involved in the degradation of the motor protein KIF21B.

<p>(A, B) KIF21B half life analysis using cycloheximide (CHX) chase experiments. More than 50% of KIF21B degrades within 48 hours in cultured hippocampal neurons (DIV16) derived from wildtype (+/+) mice. TRIM3 genetic depletion does not alter the half life of KIF21B, as assessed through evaluation of relative KIF21B signal intensities. Optineurin and actin served as controls. Relative signal intensity of KIF21B/actin ratios in %. n=3, each. 4h: wildtype (+/+) 100%, knockout (-/-) 100%; 8h: wildtype (+/+) 93.9±9.2, knockout (-/-) 110.4±12.9; 24h: wildtype (+/+) 57.8±11.5, knockout (-/-) 61.5±12.0; 48h: wildtype (+/+) 39.4±10.3, knockout (-/-) 35.4±9.6. (C, D) Relative signal intensities of KIF21B and KIF5 in hippocampal lysates remain equal across the genotypes (wildtype (+/+) versus <i>Trim3</i> knockout (-/-)). Relative signal intensity of KIF/NSE ratios in %. n=4 each. KIF21B: wildtype (+/+) 0.55±0.08, knockout (-/-) 0.69±0.03; KIF5: wildtype (+/+) 0.70±0.07, knockout (-/-): 0.72±0.03. ns: not significant. (E, F) Overexpression of TRIM3 does not alter endogenous KIF21B protein levels. Cultured hippocampal neurons (DIV10) were transfected with vectors encoding HA-TRIM3 or HA, respectively. Coexpression of GFP served as transfection control and volume marker. Cells were fixed and stained for endogenous KIF21B at DIV14. Somatic KIF21B signal intensity: HA-control: set to 100%, n= 40; HA-TRIM3: 104±17%, n=38. (Scale bars in E: 20 µm.) Data: means±SEM.</p