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. 2016 Mar 22;113(12):3365-70.
doi: 10.1073/pnas.1516697113. Epub 2016 Mar 7.

Neuronal profilins in health and disease: Relevance for spine plasticity and Fragile X syndrome

Kristin Michaelsen-Preusse  1 , Sabine Zessin  1 , Gayane Grigoryan  1 , Franziska Scharkowski  1 , Jonas Feuge  1 , Anita Remus  2 , Martin Korte  3
Affiliations

Neuronal profilins in health and disease: Relevance for spine plasticity and Fragile X syndrome

Kristin Michaelsen-Preusse et al. Proc Natl Acad Sci U S A. .

Abstract

Learning and memory, to a large extent, depend on functional changes at synapses. Actin dynamics orchestrate the formation of synapses, as well as their stabilization, and the ability to undergo plastic changes. Hence, profilins are of key interest as they bind to G-actin and enhance actin polymerization. However, profilins also compete with actin nucleators, thereby restricting filament formation. Here, we provide evidence that the two brain isoforms, profilin1 (PFN1) and PFN2a, regulate spine actin dynamics in an opposing fashion, and that whereas both profilins are needed during synaptogenesis, only PFN2a is crucial for adult spine plasticity. This finding suggests that PFN1 is the juvenile isoform important during development, whereas PFN2a is mandatory for spine stability and plasticity in mature neurons. In line with this finding, only PFN1 levels are altered in the mouse model of the developmental neurological disorder Fragile X syndrome. This finding is of high relevance because Fragile X syndrome is the most common monogenetic cause for autism spectrum disorder. Indeed, the expression of recombinant profilins rescued the impairment in spinogenesis, a hallmark in Fragile X syndrome, thereby linking the regulation of actin dynamics to synapse development and possible dysfunction.

Keywords: FMRP; FXS; actin; profilin; spinogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PFN1 expression is developmentally regulated in the hippocampus and important for spine formation. (A) Western blot analysis of hippocampal profilin levels revealed a significant reduction of PFN1 levels throughout development, whereas PFN2a levels are only altered mildly. (B) Examples for Western blots of PFN1 and PFN2a levels, three samples for each time point. (C) Spine numbers in primary embryonic cultures were significantly reduced following RNAi-mediated knockdown of PFN1 at 17 DIV but not at 34 DIV, the overexpression of PFN1 only affected 34 DIV neurons. (D and E) Spine subtype composition of neurons analyzed in C. Abbreviations: m, mushroom; s, stubby; t, thin; f, filopodia. (D) Neurons at 17 DIV; (E) neurons at 34 DIV, PFN ↑: overexpression of PFN1. (F–I) PFN1 knockdown led to a significant reduction in spine density in CA1 neurons (F and G) and CA3 neurons (H and I); example image of CA1 (G) or CA3 (I) apical dendrites of control or shPFN1-expressing cells. (Scale bars, 5 µm.) a1 proximal apical (50–200 µm from soma), a2 distal apical tufts, b basal dendrites. Means, SEM, and P values, as well as statistic tests used are depicted in Table S1. *P < 0.05; **P < 0.01; ***P < 0.001 (all compared to ctrl/P0); #P < 0.05 (compared to P14).
Fig. S1.
Fig. S1.
(A) primary embryonic neuron transfected with shPFN1 + eGFP-F, immunostaining using anti-PFN1 reveals loss of PFN1 in the transfected neurons compared with neighboring cells. (Scale bar, 20 µm.) (B) Quantification of knockdown by intensity measurement of the immunostaining seen in A; 20 shPFN1-transfected neurons were compared with neighboring control cells. (C) Cofilin and phospho-cofilin levels were analyzed via immunohistochemistry in primary embryonic hippocampal cultures transfected with the control vector shLuciferase, shPFN1, or shPFN2a (all n = 10); fluorescence intensity was compared with neighboring untransfected neurons. The quantification revealed a mild increase in cofilin expression for PFN2a transfected cells compared with shLuciferase expressing neurons, whereas the phospho-cofilin levels were unaltered upon knockdown of both profilin isoforms compared with control transfected neurons. (D) Spine-type quantification for the organotypic slice cultures described in Fig. 1 F–I. Mushroom (m), stubby (s), thin (t) showed a decrease in the relative number of mushroom spines accompanied by an increase in the number of stubby spines. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
PFN2a but not PFN1 is important for spine stability, synapse function, and spine plasticity. (A) Motility was increased in the absence of PFN2a; example spines for each imaging time point of a control cell and a PFN2a-deficient neuron. (Scale bar, 1 µm.) White arrows indicate an example spine. Absolute changes in spine length (B) or head width (C) were significantly increased when averaged for 5-min imaging intervals in PFN2a-deficient pyramidal neurons. (D) Example traces of mEPSCs in control cells or PFN isoform deficient pyramidal cells. (E) The frequency of mEPSCs was significantly reduced upon knockdown of profilin isoforms; however, a significant decrease in amplitude (F) could only be found in PFN2a-deficient neurons. (G) Changes in spine head width 60 min after cLTP induction normalized to before stimulation; activity-dependent structural plasticity was virtually absent in PFN2a-deficient pyramidal neurons. (H) Rescue experiments overexpressing recombinant profilin isoforms in shPFN2a mApple-transfected cells. Only PFN2a was able to rescue the defect in spine plasticity; only recombinant PFN2a was significantly enriched in spines upon stimulation. ns, not significant. (I) Example images of a cell transfected with shPFN2a mApple + YFP-PFN2a before and after stimulation. (Scale bar, 1 µm.) Means, SEM, and P values, as well as statistic tests used are depicted in Table S1. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S2.
Fig. S2.
(A) Absolute changes in spine length or head diameter for a 5-min imaging interval in shPFN2a- (n = 7) or control transfected cells (n = 6), dashed line indicates the optical resolution limit. Note that only PFN2a-deficient cells show changes above the resolution limit of 200 nm. (B) Example images of PFN2a-deficient cells showing the increase in spine length motility. (Scale bar, 2 µm.) (C) Electrophysiological recording of the Schaffer collaterals in four organotypic slice cultures using the cLTP induction protocol; after 10 min of baseline recording, 10 mM glycine was applied for 10 min. Recording during the stimulation was stopped and resumed afterward with recordings every 2 min. (D) Changes in the diameter of spine heads of control transfected CA1 neurons with (n = 9) or without stimulation (n = 4); analyzed were the time point directly before stimulation compared with 60 min after cLTP induction or the changes within a 60-min time window without stimulation. Dashed lines indicate the optical resolution limit. Note that only stimulated spines display changes above the resolution limit of 200 nm. (E) Depicted is the fraction of spines of control transfected neurons showing changes in head diameter above the resolution limit and the relative amount of growth per single spine compared with before the stimulation. (F) Example confocal microscopic images of a spine before and 60 min after stimulation. (Scale bar, 2 µm.) (G) Comparison of activity-dependent spine head growth in CA1 and CA3 cells revealed no significant difference between pyramidal cell types. In both neuronal cell types PFN2a deficiency let to an almost complete impairment of activity-dependent spine growth. ***P < 0.001.
Fig. 3.
Fig. 3.
Profilin isoforms regulate spine actin dynamics in an opposing manner. (A) Actin dynamics were analyzed using FRAP expressing eGFP-actin in pyramidal neurons; example spine (dashed circle, diameter: 1 µm) before and at different time points after the 405-nm laser bleaching impulse. (B–D) CA3 control cells were compared with neurons deficient in PFN isoforms, depicted are the recovery curve (B), turnover time (C), and stable actin fraction (D). (E) Quantification of the turnover time and (F) stable actin fraction in control cells (gray) or PFN2a-deficient CA3 neurons (orange) before, at 15–30 min (30′), and at 60–75 min (75′) after cLTP induction. Means, SEM, and P values, as well as statistic tests used are depicted in Table S1. *P < 0.05; **P < 0.01.
Fig. S3.
Fig. S3.
(A) Fluorescence recovery curve for spines of control (mApple + GFP-actin) expressing CA1 and CA3 pyramidal neurons. (B) The turnover time significantly differed between CA1 and CA3 neurons (CA1 15 ± 0.6 s, CA3 23.7 ± 1.8 s, P < 0.001). (C) The stable fraction indicated by the fluorescence, which did not recover after 112 s, was not different between pyramidal neuron subtypes (CA1 23 ± 1.5%, CA3 26 ± 1.8%). (D) Fluorescence recovery curve for spines of control (mApple + GFP-actin) expressing CA3 neurons and CA3 cells expressing the shRNA control vector directed against firefly luciferase (shLuciferase); no side effects of shRNA transfection could be observed regarding turnover time (E) and stable actin fraction (F). (G–I) CA1 control cells were compared with neurons deficient in PFN isoforms. Depicted are the recovery curve (G), turnover time (H), and stable actin fraction (I). (J and K) Recovery curves of spines before and after induction of cLTP. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
PFN1 but not PFN2a expression is dysregulated in a mouse model of the FXS. (A) Example Western blots for profilin isoforms in whole-brain lysates derived from adult WT or fmr1 KO mice. (B) Quantification of protein levels revealed a significant reduction for PFN1 in fmr1 KO animals. (C) Hippocampal PFN1 protein levels were reduced as well at P0 in fmr1 KO mice. (D) Representative gel of a RIP using an antibody against FMRP followed by RT-PCR specific for either PFN1 or PFN2a mRNA identified only the mRNA of PFN1 as a target of FMRP (three animals, 2–4 mo of age were analyzed). Arc was used as a positive control; analysis of spine length (E) or head width (F) in WT, fmr1 KO and fmr1 KO neurons expressing recombinant PFN1 or PFN2a revealed an increase in long, thin spines in fmr1 KO neurons that could be only fully rescued by overexpression of PFN1. (G) Example images of WT, fmr1 KO, and fmr1 KO cells expressing recombinant profilin isoforms. (Scale bar, 2 µm.) Means, SEM, and P values as well as statistic tests used are depicted in Table S1. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S4.
Fig. S4.
The developmental regulation of PFN levels was quantified in fmr1 KO animals (three to six animals per time point), whereas the level of PFN1 significantly decreased compared with P0 PFN2a levels remained unaltered, ANOVA with post hoc Bonferroni correction. ***P < 0.001.
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References

    1. Fischer M, Kaech S, Knutti D, Matus A. Rapid actin-based plasticity in dendritic spines. Neuron. 1998;20(5):847–854. - PubMed
    1. Chen LY, Rex CS, Casale MS, Gall CM, Lynch G. Changes in synaptic morphology accompany actin signaling during LTP. J Neurosci. 2007;27(20):5363–5372. - PMC - PubMed
    1. Fukazawa Y, et al. Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo. Neuron. 2003;38(3):447–460. - PubMed
    1. Honkura N, Matsuzaki M, Noguchi J, Ellis-Davies GC, Kasai H. The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines. Neuron. 2008;57(5):719–729. - PubMed
    1. Hotulainen P, et al. Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J Cell Biol. 2009;185(2):323–339. - PMC - PubMed

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