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LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity

A Corrigendum to this article was published on 26 August 2014

A Corrigendum to this article was published on 26 August 2014

This article has been updated

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIβ (PKARIIβ). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2−/− mice after treatment with a Drd1 agonist. Notably, a Parkinson's disease–related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIβ also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinson's disease.

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Figure 1: Loss of Lrrk2 leads to a decreased number of mature spines and altered synaptic transmission.
Figure 2: Abnormal phosphorylation of cofilin in Lrrk2−/− neurons.
Figure 3: PKA signaling is involved in Lrrk2-dependent phosphorylation of cofilin.
Figure 4: LRRK2 interacts with PKARIIβ.
Figure 5: LRRK2 regulates the subcellular localization of PKARIIβ.
Figure 6: Loss of Lrrk2 causes alteration of PKA-dependent phosphorylation of GluR1 in young and aged mice.
Figure 7: The R1441C mutation impairs the regulatory function of LRRK2 on PKA.

Change history

  • 27 February 2014

    In the version of this article initially published, the G2385R mutation in Figure 7a,b was given as G2835R. The error has been corrected in the HTML and PDF versions of the article.

  • 18 June 2014

    In the version of this article initially published, the line graphs presented in Figure 1g,h were switched with ones from a different experiment. The error has been corrected in the HTML and PDF versions of the article.

  • 26 August 2014

    Nat. Neurosci. 17, 367–376 (2014); published online 26 January 2014; corrected after print 27 February 2014; corrected after print 18 June 2014 In the version of this article initially published, the line graphs presented in Figure 1g,h were switched with ones from a different experiment. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank H. Zhong (Vollum Institute) for providing PKA expression vectors, M. Cookson (National Institute on Aging) for providing human Lrrk2 expression vector, J. Shen and Y. Tong (Harvard University) for providing Lrrk2 R1441C knock-in mice, P. Lewis (University College London) for providing LRRK2 wild-type and R1441C recombinant proteins, and V. Alvarez (National Institute of Alcohol Abuse and Alcoholism), B. Ma (National Institute on Aging) and Z. Li, J.-M. Jia and S. Jiao (National Institute of Mental Health) for their advice and technical support. We also thank L. Donahue and N. Sastry for their assistance in editing the manuscript. This work was supported by the intramural research programs of National Institute on Aging (AG000944 and AG000928 to H.C.) and National Institute of Alcohol Abuse and Alcoholism (D.L.).

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Contributions

L.P., D.L. and H.C. conceived the project, designed the experiments and wrote the manuscript. L.P. generated and analyzed the biochemical and cell biology data presented in Figures 2,3,4,5,6,7 and Supplementary Figures 1,2,3,4,5,6,7,8,9,10. J.Y. generated and analyzed the data presented in Figures 4,5,6,7, Supplementary Figures 4,5,6,7,8,9,10 and performed the open-field tests shown in Figure 7. C.S. and D.L. performed the Golgi-Cox staining and electrophysiology experiments and data analysis presented in Figure 1. G.L. and J.Y. performed the immunofluorescence experiment of brain section shown in Supplementary Figure 2. C.X. conducted primary neuronal cultures, immunofluorescence staining and transfection experiments. L.S. generated the cofilin constructs shown in Figure 2. X.-L.G. performed the paired-pulse facilitation experiment presented in Figure 1. X.L. was actively involved in mouse generation. N.A.C. helped with electrophysiology experiments.

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Correspondence to Loukia Parisiadou or Huaibin Cai.

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Integrated supplementary information

Supplementary Figure 1 LRRK2 developmentally regulates dendritic spine maturation and synaptic transmission

(a) Western blot analysis of PSD95 expression in the striatal extract of P2, P7, P15, P21, and P30 LRRK2+/+ and LRRK2–/– mice. Actin was used as the loading control. (b) Bar graph depicts the quantification of PSD95 levels (n=3 mice per genotype and per time point). Data are represented as mean ± SEM. Unpaired t-test, t(4)=0.0643, 0.3217, 3.270, 3.411, and 0.4935, respectively. *P < 0.05. (c-e) Whiskers blots show average dendritic spine density (c), head diameter (d) and length (e) of SPNs in 1-month-old Lrrk2+/+ and Lrrk2–/– mice. NLrrk2+/+=761 spines and NLrrk2–/–=749 spines were from six neurons of three mice per genotype. Data represent mean ± SEM. Unpaired t-test, t(1508)=10.43, ****P<0.0001.

Supplementary Figure 2 Actin-based morphological changes in Lrrk2–/– neurons involve cofilin phosphorylation

(a) Western blot analysis of Lrrk2+/+ and Lrrk2–/– striatal extracts of indicated actin binding protein during development. The full-length images are shown in Supplementary Fig. 3a. (b) Immuno-staining of pS3 cofilin (green) in the CA1 hippocampal of P15 Lrrk2+/+ and Lrrk2–/– mice and Topro 3 (blue). Scale bar: 10μm. (c) Western blot analysis of indicated proteins in the whole brain homogenate of P2, P5, and P15 non-transgenic (nTg) and BAC wild-type Lrrk2 transgenic (Tg) mice. The full-length images are shown in Supplementary Fig. 3b. (d) Co-immunoprecipitation of HA-tagged LRRK2 with endogenous cofilin and HSP90 from forebrain homogenates of P15 CaMKII-tTA single and CaMKII-tTA/tetO-Lrrk2 double transgenic mice. The full-length images are shown in Supplementary Fig. 3c. (e) The full-length images of Fig. 2a.

Supplementary Figure 3 The full-length images of Supplementary Fig. 2.

(a-c) The full-length images of Supplementary Fig. 2a (a), 2c (b), and 2d (c).

Supplementary Figure 4 PKA pathway is involved in LRRK2-dependent phosphorylation of cofilin.

(a) Western blot analysis of phosphorylated LIMK1/2 (p-LIMK1/2), total LIMK2, pSSH1L (S978), total SSH1L, PP1, and 14-3-3ζ proteins in the forebrain of P15 LRRK2+/+ and LRRK2–/– pups. The full-length images are shown in Supplementary Fig. 5a. (b) Quantification analysis of p-LIMK1 levels normalized to total LIMK1/2 levels derived of three independent experiments. Data represent min to max. Unpaired t-test, t(4)=1.087. (c) The level of phosphorylated SSH1L (p-SSH1L) normalized against the total SSH1L level in respective samples. Data represent min to max. Unpaired t-test, t(4)=1.087. N=3 per genotype and per time point. Data represent mean ± SEM. Unpaired t-test, t(4)=1.278. (d,e) Western blot analysis of cortical extracts derived of Lrrk2+/+ (d) and Lrrk2–/– (e) pups treated with indicated pharmacological compounds. The full-length images are shown in Supplementary Fig. 5b,c. (f, g) Bar graphs show quantification analysis of pS3/total cofilin levels. Bars represent mean± SEM (n=3). Unpaired t-test, *P<0.05, **P<0.01, ***P<0.001. (h) Western blot analysis of phosphorylated (p) and total CREB in cultured Lrrk2+/+ and Lrrk2–/– cortical neurons at 15DIV treated with DMSO or FSK. The full-length images are shown in Supplementary Fig. 5d. (i, j) Whiskers plots represent the quantification of pCREB levels normalized to total CREB levels (n=3 independent experiments per genotype). Data are represented as min to max. Unpaired t-test, Lrrk2+/+: t(4)=6.404, *p<0.05. Lrrk2–/–: t(4)=0.8727. (k) Western blot and quantification analysis of pCREB in the striatal nuclear extracts of Lrrk2+/+ and Lrrk2–/– mice at P15 (n=3 per genotype). The full-length images are shown in Supplementary Fig. 5e. (l) Whiskers plot shows min to max. Unpaired t-test, t(4)=4.310, *p<0.05. (m,n) The full-length images of Fig. 3a, 3c.

Supplementary Figure 5 The full-length images of Supplementary Fig. 4.

(a-e) The full-length images of Supplementary Fig. 3a (a), 3d (b), 3e (c), 3h (d), and 3k (e).

Supplementary Figure 6 LRRK2 regulation lies downstream of cAMP production.

(a) cAMP levels in cultured Lrrk2+/+ and Lrrk2–/– cortical neuronal extracts (n=3 independent experiments per genotype with duplication of each sample). Data are represented as mean ± SEM. Unpaired t-test, t(4)=1.771, p=0.1020. (b) Cultured cortical neurons treated with vehicle (DMSO) or FSK were subject to cAMP production ELISA assay. Bar shows the response to FSK as a percentage changes relative to the average value of DMSO treated neurons from three independent experiments. (c) Western blot illustrating PSD95, LRRK2 and PKARIIβ protein levels in different subcellular fractions (described in detail in Materials and Methods section) of both Lrrk2+/+ and Lrrk2–/– brains. The full-length images are shown in Supplementary Fig. 7a. (d) Striatal extracts at different ages during development were tested for the presence of pPKARIIβ, PKARIIβ, and PKACα in both genotypes. The full-length images are shown in Supplementary Fig. 7b. (e) Co-IP of MBP-tagged PKARIIβ, GST-tagged LRRK2 and PKACα recombinant proteins using a PKACα antibody. LRRK2 does not affect the interaction between PKACα and PKARIIβ. The full-length images are shown in Supplementary Fig. 7c. Four independent experiments were performed. Unpaired t-test, t(6)=1.5685, p=0.1329. (f) Co-staining of endogenous PKACα (green) and PKARIIβ (red) in cultured Lrrk2+/+ and Lrrk2–/– hippocampal neurons at 15DIV. Scale bar: 10μm. (g-m) The full-length images of Fig. 4a-e, 4h, and 4i.

Supplementary Figure 7 LRRK2 regulates PKARIIβ dendritic spine localization.

(a-c) The full-length images of Supplementary Fig. 6c (a), 6d (b), and 6e (c). (d) Fluorescent images of GFP-PKACα and mCherry in dendrites of transfected Lrrk2+/+ and Lrrk2–/– hippocampal neurons at 15DIV. Scale bars: 5μm. (e) Representative image of cultured Lrrk2–/– hippocampal neurons co-transfected with GFP-PKARIIβ (green), mCherry (red) and myc-LRRK2 (blue). The e1 and e2 are enlarged images of the boxed areas in (e). Scale bars: 20μm (e), 20μm (e1), and 2μm (e2). (f) Representative images showing spines and their parental dendrites from cultured Lrrk2+/+ hippocampal neurons co-transfected with mCherry and either GFP-PKARIIb, GFP-PKARIIβΔ2-5 or GFP-PKARIIβΔ2-5MTBD. Scale bars: 2μm. (g) Images of cultured Lrrk2–/– hippocampal neuron co-transfected with mCherry and PKARIIβΔ2-5MTBD. Scale bars: 2μm. (h) The full-length images of Fig. 5d.

Supplementary Figure 8 Abnormal phosphorylation of GluR1 in Lrrk2–/– mice.

(a,b) Western Blot analysis of cultured LRRK2+/+ and LRRK2–/– cortical neurons treated with DMSO or FSK (50μM for 1hr) for the expression of phosphorylated (pS845) and total GluR1. The full-length images are shown in Supplementary Fig. 9a,b. (c) Western blot analyses show the effect of LRRK2 kinase inhibitor LRRK2-IN-1 on FSK-induced phosphorylation of GluR1 S845 and cofilin S3 in cultured cerebral cortical neurons. Culture cerebral cortical neurons (8DIV) derived from P0 Lrrk2+/+ mouse pups with or without 5μM LRRK2-IN-1 for 30min prior to DMSO or 50μM FSK treatment for additional 1h. The phosphorylation of LRRK2 S935 serves as a positive control for LRRK2-IN-1 inhibitory activity. The top band in the pS935 blot is a non-specific signal reactive to LRRK2 S935 antibody. The full-length images are shown in Supplementary Fig. 9c. (d,e) Bar graphs show alteration of GluR1 pS845 (d) and cofilin pS3 (e) in cultured cortical neurons treated with PKA activator FSK and LRRK2 inhibitor LRRK2-IN-1. Three independent cultures were used under each experimental condition. Data represent mean ± SEM. One-way ANOVA plus Tukey's Multiple Comparison Test, ****P<0.0001, ns: not significant. (f) Western blot analysis of pS831 and total GluR1 in the striatum of P21 Lrrk2+/+ and Lrrk2–/– mice after treated with saline (0) or Drd1 agonist SKF81297 at 2 and 10mg per kg bodyweight. The full-length images are shown in Supplementary Fig. 9d. (g) Quantification analysis of pS831 GluR1 ratio in the striatal tissues of P21 mice treated with SKF81297. N=4 per genotype. Data represent mean±SEM. Unpaired t-test, t(6)=0.2216, 1.413, and 1.223, respectively. (h-j) The full-length images of Fig. 6a,c, and f, respectively.

Supplementary Figure 9 The full-length images of Supplementary Fig. 8.

(a-d) The full-length images of Supplementary Fig. 8a (a), 8b (b), 8c (c), and 8f (d).

Supplementary Figure 10 The full-length images of Fig. 7.

(a-e) The full-length images of Fig. 7a (a), 7c (b), 7g (c), 7i (d), and 7k (e). (f) Western blot shows the specificity of pS3 cofilin antibody. HEK293 cells were transfected with GFP-tagged wild-type (WT), S3A, and S3D mutant cofilin constructs. The pS3 cofilin antibody only recognized the GFP-WT cofilin but not the ones with mutations at the S3 residue. (g) Western blot shows the specificity of PKARIIb antibody. Mouse N2a cell lines were transfected with control and four independent PKARIIβ siRNA. A significant reduction of PKARIIb protein levels was found in all PKARIIβ siRNA-transfected cells. (h) Western blot shows the specificity of PKACα antibody. N2a cells were transfected with control and four independent PKACα siRNA. A significant reduction of PKACα protein levels was found in all PKACα siRNA-transfected cells.

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Parisiadou, L., Yu, J., Sgobio, C. et al. LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity. Nat Neurosci 17, 367–376 (2014). https://doi.org/10.1038/nn.3636

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