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• W2912019839 abstract "•MLP is a regeneration-associated gene expressed in adult neurons of mammals•As an actin cross-linker, MLP facilitates filopodia formation in axonal growth cones•MLP overexpression facilitates CNS and PNS axon regeneration Muscle LIM protein (MLP) has long been regarded as a muscle-specific protein. Here, we report that MLP expression is induced in adult rat retinal ganglion cells (RGCs) upon axotomy, and its expression is correlated with their ability to regenerate injured axons. Specific knockdown of MLP in RGCs compromises axon regeneration, while overexpression in vivo facilitates optic nerve regeneration and regrowth of sensory neurons without affecting neuronal survival. MLP accumulates in the cell body, the nucleus, and in axonal growth cones, which are significantly enlarged by its overexpression. Only the MLP fraction in growth cones is relevant for promoting axon extension. Additional data suggest that MLP acts as an actin cross-linker, thereby facilitating filopodia formation and increasing growth cone motility. Thus, MLP-mediated effects on actin could become a therapeutic strategy for promoting nerve repair. Muscle LIM protein (MLP) has long been regarded as a muscle-specific protein. Here, we report that MLP expression is induced in adult rat retinal ganglion cells (RGCs) upon axotomy, and its expression is correlated with their ability to regenerate injured axons. Specific knockdown of MLP in RGCs compromises axon regeneration, while overexpression in vivo facilitates optic nerve regeneration and regrowth of sensory neurons without affecting neuronal survival. MLP accumulates in the cell body, the nucleus, and in axonal growth cones, which are significantly enlarged by its overexpression. Only the MLP fraction in growth cones is relevant for promoting axon extension. Additional data suggest that MLP acts as an actin cross-linker, thereby facilitating filopodia formation and increasing growth cone motility. Thus, MLP-mediated effects on actin could become a therapeutic strategy for promoting nerve repair. The visual system is frequently used as a versatile model for the investigation of CNS injuries and potential therapeutic approaches to promote axonal regeneration (Fischer and Leibinger, 2012Fischer D. Leibinger M. Promoting optic nerve regeneration.Prog. Retin. Eye Res. 2012; 31: 688-701Crossref PubMed Scopus (107) Google Scholar, Harvey, 2014Harvey A.R. Gene therapy and the regeneration of retinal ganglion cell axons.Neural Regen. Res. 2014; 9: 232-233Crossref PubMed Scopus (3) Google Scholar). Retinal ganglion cells (RGCs), the final output neurons of the vertebrate retina, are susceptible to genetic manipulations in vivo and in vitro, and their projecting axons within the optic nerve are readily accessible to minimally invasive surgery (Leibinger et al., 2013aLeibinger M. Andreadaki A. Diekmann H. Fischer D. Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration.Cell Death Dis. 2013; 4: e805Crossref PubMed Scopus (84) Google Scholar, Leibinger et al., 2016Leibinger M. Andreadaki A. Gobrecht P. Levin E. Diekmann H. Fischer D. Boosting central nervous system axon regeneration by circumventing limitations of natural cytokine signaling.Mol. Ther. 2016; 24: 1712-1725Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Like other CNS neurons, mature RGCs are normally unable to regenerate lesioned axons and die after optic nerve injury (Berkelaar et al., 1994Berkelaar M. Clarke D.B. Wang Y.C. Bray G.M. Aguayo A.J. Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats.J. Neurosci. 1994; 14: 4368-4374Crossref PubMed Google Scholar, Fischer et al., 2000Fischer D. Pavlidis M. Thanos S. Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.Invest. Ophthalmol. Vis. Sci. 2000; 41: 3943-3954PubMed Google Scholar), leading to irreversible visual loss. This regenerative failure has partly been attributed to an insufficient intrinsic regenerative capacity of CNS neurons as well as to axonal growth inhibitory molecules within CNS myelin and the glial scar (Fischer and Leibinger, 2012Fischer D. Leibinger M. Promoting optic nerve regeneration.Prog. Retin. Eye Res. 2012; 31: 688-701Crossref PubMed Scopus (107) Google Scholar, Silver and Miller, 2004Silver J. Miller J.H. Regeneration beyond the glial scar.Nat. Rev. Neurosci. 2004; 5: 146-156Crossref PubMed Scopus (2386) Google Scholar). However, various experimental approaches have been developed in recent years to promote axonal re-growth beyond the lesion site after optic nerve injury. For example, retrolental puncture of the lens capsule stimulates retinal astrocytes and Müller cells to secrete IL-6-type cytokines, thereby mediating neuroprotection and stimulating axon regeneration (Fischer et al., 2000Fischer D. Pavlidis M. Thanos S. Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.Invest. Ophthalmol. Vis. Sci. 2000; 41: 3943-3954PubMed Google Scholar, Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar, Leibinger et al., 2009Leibinger M. Müller A. Andreadaki A. Hauk T.G. Kirsch M. Fischer D. Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor.J. Neurosci. 2009; 29: 14334-14341Crossref PubMed Scopus (194) Google Scholar, Leon et al., 2000Leon S. Yin Y. Nguyen J. Irwin N. Benowitz L.I. Lens injury stimulates axon regeneration in the mature rat optic nerve.J. Neurosci. 2000; 20: 4615-4626Crossref PubMed Google Scholar, Müller et al., 2007Müller A. Hauk T.G. Fischer D. Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.Brain. 2007; 130: 3308-3320Crossref PubMed Scopus (195) Google Scholar). This so-called inflammatory stimulation (IS), which is associated with a change of gene expression (Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar), can thus be used to increase the regenerative capacity of mouse and rat RGCs (Leibinger et al., 2017Leibinger M. Andreadaki A. Golla R. Levin E. Hilla A.M. Diekmann H. Fischer D. Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.Proc. Natl. Acad. Sci. USA. 2017; 114: E5454-E5463Crossref PubMed Scopus (37) Google Scholar). To investigate the molecular mechanisms underlying IS-stimulated axon regeneration, we previously screened microarrays for genes that are differently expressed in the IS-induced regenerative state compared to naive and solely injured RGCs, respectively (Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar). Muscle LIM protein (MLP) was among the candidate genes found in this screen with an induction of mRNA expression after optic nerve injury alone and a significant stonger upregulation after additional IS, suggesting that this protein may be expressed in the adult CNS and potentially involved in regenerative processes (Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar). MLP is a cysteine-rich protein of the large LIM-domain-protein superfamily and has long been regarded as cardiac and skeletal muscle-specific before we found an expression in cholinergic amacrine cells, which was, however, restricted to the first few postnatal weeks (Levin et al., 2014Levin E. Leibinger M. Andreadaki A. Fischer D. Neuronal expression of muscle LIM protein in postnatal retinae of rodents.PLoS One. 2014; 9: e100756Crossref PubMed Scopus (7) Google Scholar). In myocytes, MLP is detectable from early stages of terminal differentiation to adulthood (Arber et al., 1994Arber S. Halder G. Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation.Cell. 1994; 79: 221-231Abstract Full Text PDF PubMed Scopus (394) Google Scholar) and has been implicated a key role in normal muscle physiology as well as pathogenesis. Mutations of MLP are directly associated with dilated and hypertrophic cardiomyopathies, and altered expression levels are observed in human failing hearts and various skeletal myopathies (Gehmlich et al., 2008Gehmlich K. Geier C. Milting H. Fürst D. Ehler E. Back to square one: what do we know about the functions of muscle LIM protein in the heart?.J. Muscle Res. Cell Motil. 2008; 29: 155-158Crossref PubMed Scopus (23) Google Scholar, Knöll et al., 2002Knöll R. Hoshijima M. Hoffman H.M. Person V. Lorenzen-Schmidt I. Bang M.L. Hayashi T. Shiga N. Yasukawa H. Schaper W. et al.The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy.Cell. 2002; 111: 943-955Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, Sanoudou et al., 2006Sanoudou D. Corbett M.A. Han M. Ghoddusi M. Nguyen M.A. Vlahovich N. Hardeman E.C. Beggs A.H. Skeletal muscle repair in a mouse model of nemaline myopathy.Hum. Mol. Genet. 2006; 15: 2603-2612Crossref PubMed Scopus (39) Google Scholar, Winokur et al., 2003Winokur S.T. Chen Y.W. Masny P.S. Martin J.H. Ehmsen J.T. Tapscott S.J. van der Maarel S.M. Hayashi Y. Flanigan K.M. Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation.Hum. Mol. Genet. 2003; 12: 2895-2907Crossref PubMed Scopus (172) Google Scholar). The two LIM domains of MLP independently mediate interactions with multiple proteins at different subcellular locations, thus determining its complex and diverse functional roles in muscle development and cytoarchitecture (Vafiadaki et al., 2015Vafiadaki E. Arvanitis D.A. Sanoudou D. Muscle LIM protein: master regulator of cardiac and skeletal muscle functions.Gene. 2015; 566: 1-7Crossref PubMed Scopus (53) Google Scholar). For example, cytoplasmic MLP interacts either directly with actin to confer cross-linking and to promote actin bundling, or indirectly with actin-binding proteins such as cofilin or α-actinin to increase actin dynamics in myocytes (Arber and Caroni, 1996Arber S. Caroni P. Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ.Genes Dev. 1996; 10: 289-300Crossref PubMed Scopus (202) Google Scholar, Flick and Konieczny, 2000Flick M.J. Konieczny S.F. The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin.J. Cell Sci. 2000; 113: 1553-1564PubMed Google Scholar, Hoffmann et al., 2014Hoffmann C. Moreau F. Moes M. Luthold C. Dieterle M. Goretti E. Neumann K. Steinmetz A. Thomas C. Human muscle LIM protein dimerizes along the actin cytoskeleton and cross-links actin filaments.Mol. Cell. Biol. 2014; 34: 3053-3065Crossref PubMed Scopus (37) Google Scholar, Louis et al., 1997Louis H.A. Pino J.D. Schmeichel K.L. Pomiès P. Beckerle M.C. Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression.J. Biol. Chem. 1997; 272: 27484-27491Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, Papalouka et al., 2009Papalouka V. Arvanitis D.A. Vafiadaki E. Mavroidis M. Papadodima S.A. Spiliopoulou C.A. Kremastinos D.T. Kranias E.G. Sanoudou D. Muscle LIM protein interacts with cofilin 2 and regulates F-actin dynamics in cardiac and skeletal muscle.Mol. Cell. Biol. 2009; 29: 6046-6058Crossref PubMed Scopus (43) Google Scholar). MLP may also act as a mechanical stress sensor, entering and accumulating in nuclei of cardiomyocytes, where it modulates gene expression (Boateng et al., 2007Boateng S.Y. Belin R.J. Geenen D.L. Margulies K.B. Martin J.L. Hoshijima M. de Tombe P.P. Russell B. Cardiac dysfunction and heart failure are associated with abnormalities in the subcellular distribution and amounts of oligomeric muscle LIM protein.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H259-H269Crossref PubMed Scopus (78) Google Scholar, Buyandelger et al., 2011Buyandelger B. Ng K.E. Miocic S. Piotrowska I. Gunkel S. Ku C.H. Knöll R. MLP (muscle LIM protein) as a stress sensor in the heart.Pflugers Arch. 2011; 462: 135-142Crossref PubMed Scopus (44) Google Scholar, Knöll et al., 2002Knöll R. Hoshijima M. Hoffman H.M. Person V. Lorenzen-Schmidt I. Bang M.L. Hayashi T. Shiga N. Yasukawa H. Schaper W. et al.The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy.Cell. 2002; 111: 943-955Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, Kong et al., 1997Kong Y. Flick M.J. Kudla A.J. Konieczny S.F. Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD.Mol. Cell. Biol. 1997; 17: 4750-4760Crossref PubMed Scopus (239) Google Scholar). The present study investigated whether MLP protein is expressed in the adult CNS and whether it is involved in regenerative processes of injured axons. Indeed, we found a correlation of MLP expression with the regenerative state of rat RGCs, making it a regeneration-associated protein. MLP co-localized with the actin cytoskeleton in axonal growth cones, and its overexpression markedly promoted axonal regeneration in culture and in vivo. Contrary to that, knockdown or expression of a dominant-negative variant showed the opposite effect. Although MLP was absent in murine RGCs or peripheral sensory neurons, exogenous expression in these neurons significantly increased axon growth and optic nerve regeneration. Thus, MLP also has physiological functions outside of muscle tissue and might be a potential target for the development of therapeutic strategies to facilitate nerve repair, either alone or in combination with other approaches. We previously detected MLP protein in a subpopulation of adult rat sensory neurons upon sciatic nerve crush (Levin et al., 2017Levin E. Andreadaki A. Gobrecht P. Bosse F. Fischer D. Nociceptive DRG neurons express muscle lim protein upon axonal injury.Sci. Rep. 2017; 7: 643Crossref PubMed Scopus (6) Google Scholar). To investigate whether similar induction might also occur in adult CNS tissue, we initially performed quantitative real-time PCR and then western blot analyses on retinal lysates from uninjured control rats or 5 d after optic nerve crush (ONC). Additional experimental groups received lens injury-induced IS either alone or in combination with ONC to increase the regenerative capacity of RGCs (Fischer and Leibinger, 2012Fischer D. Leibinger M. Promoting optic nerve regeneration.Prog. Retin. Eye Res. 2012; 31: 688-701Crossref PubMed Scopus (107) Google Scholar, Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar). As expected, very low mRNA expression, but no MLP protein was detected in naive control retinae or after IS only (Figures 1A–1C). However, transcript (Figure 1A) and protein levels (Figures 1B and 1C) were induced upon ONC and further increased upon ONC+IS. These results were verified by immunohistochemistry on flat-mounted retinae (Figure 1D), showing no signal in naive and IS-treated retinae, but a few MLP-positive RGCs with axons (identified by βIII-tubulin labeling) 5 d after ONC. Additional IS further increased their number and staining intensity (Figure 1D) and retinal cross-sections revealed MLP expression in injured RGCs only (Figure 1D). Next, we determined the time course of retinal expression (Figures 1E–1I). Induction of Mlp transcripts were already detected 3 d after ONC+IS and progressively increased at 5 and 7 d (Figure 1E). Western blots, revealed steady MLP levels from 5 d to at least 14 d after surgery (Figures 1F and 1G). As RGCs continuously die within this time period (Fischer et al., 2000Fischer D. Pavlidis M. Thanos S. Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.Invest. Ophthalmol. Vis. Sci. 2000; 41: 3943-3954PubMed Google Scholar, Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar), these results indicated preferential survival of MLP-expressing RGCs and/or continued rising expression in surviving neurons over time. Quantification in retinal flat-mounts revealed similar percentages of MLP-positive neurons in the surviving population of RGCs at 7 and 14 d after ONC+IS (Figures 1H and 1I). However, the percentage of strongly stained RGCs significantly increased from 7 to 14 d (Figure 1I), indicating further accumulation of MLP in the surviving RGCs. We then investigated whether MLP expression is similarly induced in cultured adult rat RGCs upon axon and dendritic loss by the dissociation process. Quantitative real-time PCR revealed significantly increased amounts of Mlp transcripts after 3 d compared to 2 hr in culture (Figure 2A). Consistently, protein expression was detected in ∼3% of βIII-tubulin-positive RGCs at 2 d and ∼10% at 3 d in culture (Figures 2B and 2C). Thus, similar to ONC in vivo, MLP-protein induction in culture was low, but clearly found in the cytoplasm and in the nuclei of RGCs (Figure 2D). Within the axonal compartment, MLP was particularly detected in growth cones and co-localized with F-actin (Figure 2E). We also subjected rats to ONC+IS and anterogradely labeled regenerating axons in the optic nerve by intravitreal injection of fluorescent cholera toxin subunit B (CTB). These nerves were isolated 14 d after injury and used for tissue clearing after MLP immunostaining to visualize the protein throughout the whole optic nerve (Figure 2F). Image analysis revealed that 74% ± 4.5% of axonal growth cones in the distal part of the nerve were MLP-positive. Hence, as in cell culture, endogenous MLP was also localized in axonal growth cones in vivo. Activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway have been shown to contribute to axon regeneration (Fischer and Leibinger, 2012Fischer D. Leibinger M. Promoting optic nerve regeneration.Prog. Retin. Eye Res. 2012; 31: 688-701Crossref PubMed Scopus (107) Google Scholar). To test whether MLP expression depends on the activity of one of these pathways, we cultured adult rat RGCs in the presence of the JAK/STAT3 pathway inhibitor AG490, the PI3K inhibitor LY294002, or the mTOR inhibitor rapamycin. After either 2 hr or 3 days, RGCs were fixed for immunocytochemical staining. At concentrations that blocked neurite growth without affecting survival (Figures S1D and S1F), AG490 did not significantly affect MLP, but GAP43 expression (Figures S1A–S1C and S1E). However, LY294002 and rapamycin markedly suppressed MLP expression and neurite growth, while inhibition of phosphatase and tensin homolog (PTEN) by bisperoxovanadium (BPV) had the opposite effects (Figures S1A–S1D). In contrast to MLP, GAP43 expression was not affected by LY294002, rapamycin, or BPV (Figures S1B and S1E). We next tested whether inhibition of mTOR may also suppress injury-induced MLP expression in RGCs in vivo. To this end, rats were treated either with rapamycin or vehicle and received ONC + IS. Retinal flat-mounts were prepared 5 days afterwards. Rapamycin treatment reduced MLP expression compared to similar treated controls (Figures S1I and S1J), as well as the number of pS6-positive RGCs (Figures S1G and S1H), indicating effective mTOR inhibition. Thus, MLP protein expression is PI3K/AKT/mTOR activity dependent, but JAK/STAT3 independent. The rather small proportion of MLP-positive RGCs (<10% at 3 d) in cultures from naive (in vivo untreated) retinae (Figures 2C and S2H) and the occurring extensive axotomy-induced cell death at later time points (Grozdanov et al., 2010Grozdanov V. Muller A. Sengottuvel V. Leibinger M. Fischer D. A method for preparing primary retinal cell cultures for evaluating the neuroprotective and neuritogenic effect of factors on axotomized mature CNS neurons.Curr. Protoc. Neurosci. 2010; Chapter 3 (Unit 3.22)PubMed Google Scholar) precluded reliable knockdown experiments to functionally assess the role of endogenous MLP in these cultures. For this reason, we prepared retinal cultures 7 d after ONC+IS in vivo, when the protein was already detectable in >50% of all RGCs (Figures 1 and S2H) and the RGCs were already in a regenerative state (Fischer et al., 2004Fischer D. Petkova V. Thanos S. Benowitz L.I. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation.J. Neurosci. 2004; 24: 8726-8740Crossref PubMed Scopus (246) Google Scholar). Expectedly, these cultures contained many more MLP-positive RGCs than “naive” preparations (68% ± 4 at 1 d). Interestingly, the proportion of neurite-bearing RGCs that had spontaneously regenerated after 24 hr was significantly higher in the MLP-expressing RGC population (59% for RGCs with strong [++] and 49% for RGCs with moderate [+] MLP expression) compared to MLP-negative (−) RGCs (30%) (Figures S2A and S2B), implying that neurons’ regenerative state was correlated with endogenous MLP expression. To study a functional involvement of MLP in neurite growth, we first blocked its induction using efficient and previously characterized short hairpin RNA (shRNA) (Levin et al., 2017Levin E. Andreadaki A. Gobrecht P. Bosse F. Fischer D. Nociceptive DRG neurons express muscle lim protein upon axonal injury.Sci. Rep. 2017; 7: 643Crossref PubMed Scopus (6) Google Scholar). Recombinant adeno-associated viruses (AAVs) were used to deliver either Mlp-specific shRNA (shMlp) or scrambled control shRNA (shScr) into RGCs in vivo, both achieving comparable transduction rates of ∼50% as indicated by GFP co-expression (Figure S2C). Accordingly, immunostained retinal flat-mounts showed no MLP in GFP-positive/shMlp-transduced RGCs 7 days after ONC+IS (Figure S2D). Consistent with the transduction rate of the shMLP-AAV, quantitative real-time PCR (Figure S2E) and western blot analysis (Figures S2F and S2G) revealed about 50% reduced MLP expression in the entire retinae, verifying efficient knockdown in transduced RGCs. We then prepared retinal cultures to determine spontaneous neurite growth within 24 hr (Figures S2H–S2J). Comparable neurite lengths were quantified for GFP-positive/shScr transduced RGCs and non-transduced RGCs, indicating that AAV-mediated expression of GFP or non-targeting control shRNA per se had no influence on neurite growth (Figure S2J). In contrast, shMlp-mediated knockdown of the protein in GFP-positive RGCs reduced neurite length by 54% compared to non-transduced cells in the same cultures (Figures S2I and S2J). The specificity of this effect and its dependence on endogenous MLP expression were verified with a second experiment using retinal cultures from naive/uninjured rats, that showed only very few MLP-positive RGCs after 2 d (see Figures 2C and S2H). In these cultures, transduction with shMlp had no discernable effect on neurite outgrowth compared to non-transduced controls at 3 d in cell culture (Figure S2K). In all these experiments (Figures S2I–S2K), the number of RGCs per well was not significantly affected by any treatment. As induction of endogenous MLP upon axonal injury seemed to facilitate neurite growth of RGCs, we investigated whether exogenous MLP expression might further promote this effect. To this end, recombinant AAV encoding MLP and GFP (MLP-AAV) were intravitreally injected into adult rat eyes, transducing ∼50% of all RGCs (Figure S3A). Accordingly, RGCs of these animals expressed MLP even without prior ONC- or ONC+IS-mediated induction (Figure S3B), and retinal cultures prepared 3 weeks after viral application contained transduced, MLP/GFP-expressing next to non-transduced, MLP/GFP-negative RGCs (Figure S3C). Because viral transduction per se did not affect neurite growth (Figures S2J), non-transduced RGCs in the same cultures were used as an internal control. Compared to these, MLP overexpression induced a 2.7-fold increase in RGC neurite length (Figures S3C and S3D). Addition of ciliary neurotrophic factor (CNTF) to the medium similarly promoted neurite growth of MLP-expressing as well as non-transduced RGCs, demonstrating that the effects of the cytokine and MLP were independent from each other (Figure S3D). As disinhibition toward inhibitory molecules is also relevant for successful CNS regeneration, we grew MLP-expressing and non-transduced RGCs on inhibitory myelin extract. However, in contrast to the disinhibitory ROCK inhibitor Y27632, neurite growth on myelin was similarly reduced for transduced and non-transduced RGCs compared to cultures on laminin (Figure S3E). In all culture experiments (Figures S3C–S3E), the numbers of RGCs per well were similar and not significantly affected by any treatment. Thus, MLP expression correlated with the regenerative state and facilitated neurite growth without conferring disinhibition. We next assessed the role of MLP in optic nerve regeneration in vivo using shRNA-mediated knockdown or RGC-specific overexpression, respectively. Adult rats received intravitreal injections of either control GFP-AAV, shMlp-AAV, or MLP-AAV (see Figures S2C–S2G, S3A, and S3B) and were subjected to ONC+IS 3 weeks thereafter. The enhanced regenerative capacity of RGCs upon IS treatment enabled regeneration of GAP43-positive axons into the distal part of the crushed optic nerve in GFP-AAV-injected control animals (Figures 3A, 3B, and S4A–S4C). In comparison, the number and length of regenerating axons was significantly reduced upon neuronal Mlp knockdown, while MLP overexpression, on the other hand, increased axon regeneration (Figures 3A, 3B, and S4A–S4C). These effects on optic nerve regeneration were independent of neuronal survival as the numbers of RGCs in retinal flat-mounts were comparable for all 3 experimental groups both after sole ONC or ONC+IS (Figures 3C and 3D). Hence, the role of MLP was restricted to axon growth promotion. We next investigated whether MLP might be also expressed in axotomized RGCs or sensory dorsal root ganglion (DRG) neurons of mice. As in rats, very few Mlp transcripts and no MLP protein were detected in naive adult BL6-mouse retinae (Figures S5A and S5B) or DRG neurons of the same animals (Figures S5L–S5N). However, MLP expression was not induced upon ONC or ONC+IS in the retina or in sensory neurons after sciatic nerve crush (SNC) (Figures S5L–S5O) either, revealing a difference between rats and mice. This result was not caused by a selective specificity of the antibody for rat MLP as the respective mouse protein was readily detected in murine heart lysate (Figures S5B and S5M). To investigate whether the lack of MLP induction was due to the mouse strain (inbred versus outbred), we also measured retinal MLP expression in C57BL/6,129/Ola(B6CF1) (inbred) and jOrl:SWISS mice (outbred). However, both strains showed very similar results. This rather unexpected finding hence presented us the opportunity to determine the effect of ectopic MLP expression on axon regeneration without endogenous expression/induction in the background. Intravitreal injection of MLP-AAV in adult mice transduced ∼60% of RGCs (Figure S5C) and RGC-specific MLP expression was detectable in retinal whole mounts as well as cell cultures (Figures S5D and S5E). As in rat cultures, neurite growth of naive mouse RGCs was significantly increased upon MLP expression compared to non-transduced cells (Figures S5F and S5G). Again, CNTF equally promoted neurite growth of MLP-expressing and non-transduced RGCs further, indicating additive effects (Figures S5F and S5G). To exclude the possibility that this outcome might have been influenced by MLP expression prior to cell culture preparation, as retinae were routinely isolated 3 weeks after intravitreal AAV transduction in vivo, we took advantage of a recently described baculoviral approach to efficiently and swiftly transduce adult RGCs after culture preparation (Levin et al., 2016Levin E. Diekmann H. Fischer D. Highly efficient transduction of primary adult CNS and PNS neurons.Sci. Rep. 2016; 6: 38928Crossref PubMed Scopus (9) Google Scholar). To this end, mouse retinal cell cultures were treated with baculoviruses encoding either HA-tagged MLP (MLP-HA-bv) or Cre-recombinase (Cre-HA-bv) as control (Figure S5H), achieving ∼50% transduction of RGCs and recombinant expression in less than 24 hr (Levin et al., 2016Levin E. Diekmann H. Fischer D. Highly efficient transduction of primary adult CNS and PNS neurons.Sci. Rep. 2016; 6: 38928Crossref PubMed Scopus (9) Google Scholar). As before, MLP expression markedly increased RGC neurite lengths in comparison to control t" @default.
• W2912019839 created "2019-02-21" @default.
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• W2912019839 date "2019-01-01" @default.
• W2912019839 modified "2023-09-29" @default.
• W2912019839 title "Muscle LIM Protein Is Expressed in the Injured Adult CNS and Promotes Axon Regeneration" @default.
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