The Schematic diagram shows that the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic MPP+-induced Cdk5 activation, and Cdk5 could phosphorylate the adjacent amino residue S1627 of R1628P mutation, thus activate LRRK2 kinase activity and cause neuronal death.

<p>The Schematic diagram shows that the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic MPP+-induced Cdk5 activation, and Cdk5 could phosphorylate the adjacent amino residue S1627 of R1628P mutation, thus activate LRRK2 kinase activity and cause neuronal death.</p

R1628P mutation do not alter the LRRK2 activity and cause neuronal toxicity directly.

<p><b>a.</b> Kinase activity of LRRK2 mutants tested by kinase assay using MBP as substrate. HEK293 cells were transfected with HA-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N as indicated. After 24 h, LRRK2 was immunoprecipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. The bottom panel shows equal expression of HA-LRRK2 and MBP used in the lysates. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 1A. The numbers are relative values, with WT set to 1. <b>c.</b> Kinase activity of LRRK2 mutants tested by kinase assay using LRRKtide as substrate. HA-tagged WT LRRK2 or mutants were immunoprecipitated as above, then <i>in vitro</i> LRRK2 kinase assay was performed using LRRKtide as substrate. <b>d.</b> Overexpression of R1628P mutation do not cause neuronal cell death. Primary-cultured cortical neurons were transfected with GFP-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N. After 48 h transfection, the dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. All the above results represent at least three independent experiments as the mean ± SD, *P<0.05, **P<0.01 (ANOVA).</p

Phosphorylation of R1628P mutant by Cdk5 increases the LRRK2 activity and causes cell death.

<p><b>a.</b> Plasmids of HA-tagged wild-type (WT) or R1628P mutant LRRK2 were cotransfected with Cdk5/p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. HA-LRRK2, Cdk5, and p35 levels were determined by Western blotting as a loading control. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3A. The numbers are relative values, with WT w/o Cdk5/p35 set to 1. <b>c.</b> Plasmids of HA-tagged wild-type (WT), R1628P, and S1627A:R1628P mutant LRRK2 were cotransfected with Cdk5 or dominant-negative form of Cdk5 (dnCdk5) and p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase activity was measured for 30min using LRRKtide as substrate. The results represent as the mean ± SD, **P<0.01 (compared with WT:control group) ##P<0.01 (compared with WT:Cdk5/p35 group) (ANOVA). <b>d.</b> Phosphorylation mimic of S1627 (S1627D) increased the LRRK2 kinase activity. HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P double mutant. LRRK2 kinase activities were measured as above. <b>e.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3D. The numbers are relative values, with WT set to 1. **P<0.01 (ANOVA) <b>f.</b> HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P. LRRK2 kinase activities were measured as above for indicated period of time using LRRKtide as substrate. **P<0.01 (compared with WT group, ANOVA)</p

Neurons ectopically expressing R1628P displayed a higher sensitivity to MPP+ in a Cdk5-dependent manner.

<p><b>a.</b> Neurons with the R1628P mutation display a higher sensitivity to MPP+. Primary-cultured cortical neurons from wild-type mice were transfected with GFP-tagged wild-type (WT) or R1628P mutant LRRK2 plasmids for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. <b>b.</b> Cdk5 deletion protects the neurons with the R1628P mutation from MPP+ toxicity. The above procedures were performed in primary-cultured cortical neurons from the neuronal Cdk5 conditional knockout mice. Single cell survival assay was conducted as above. The results represent at least three independent experiments as the mean ± SD, **P<0.01 (ANOVA). <b>c.</b> The higher sensitivity of R1628P to MPP+ requires the phosphorylation of S1627 on LRRK2. Primary-cultured cortical neurons from wild-type mice were transfected with GFP vector, GFP-tagged wild-type (WT) LRRK2 or R1628P, S1627A:R1628P, S1627D:R1628P mutant for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. The results represent at least three independent experiments as the mean ± SD, *P<0.05, (ANOVA)</p

Cdk5 could phosphorylate the adjacent S1627 in the LRRK2 R1628P mutant.

a. The R1628P mutation increase the binding affinity of LRRK2 with Cdk5. Primary-cultured cortical neurons were transfected with vehicle or LRRK2 (WT, R1628P). After 24 h, the exogenous LRRK2 was immunoprecipitated using anti-HA antibody, and the level of bound Cdk5 was measured by Western blotting. The bottom panel shows the loading control of HA-LRRK2 and Cdk5 used in the lysates. b. Cdk5 phosphorylates Serine 1627 (S1627) in R1628P mutant. Recombinant GST-tagged LRRK2 (COR domain, amino acids 1535~1878), including wild-type (WT), R1628P mutant and S1627A:R1628P double mutant, were purified. The GST-tagged LRRK2 recombinant protein were phosphorylated by active Cdk5/p35 and γ-32P-ATP in vitro, and the phosphorylation signals were analyzed by autoradiography. (top panel, about 60KD). The same membrane was probed with anti‑GST, Cdk5 or p35 antibody as a loading control (bottom panel). c. Cdk5 phosphorylates S1627 in cells. The HA-tagged LRRK2 (WT, R1628P and S1627A:R1628P) plasmids were cotransfected with Cdk5 and p35 in HEK293 cells. After 24 h of transfection, the LRRK2 were immuneprecipitated using an anti-HA antibody from lysates, and phosphorylation of LRRK2 were measured by Western blotting using a phospho-(Serine/Threonine)-Proline (pS/TP) antibody and anti-LRRK2 phospho S935 antibody. HA-LRRK2, Cdk5, p35, and actin levels were determined by Western blotting as a loading control.</p

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

<div><p>Background</p><p>Recent studies have linked certain single nucleotide polymorphisms in the <i>leucine-rich repeat kinase 2 (LRRK2)</i> gene with Parkinson’s disease (PD). Among the mutations, <i>LRRK2</i> c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.</p><p>Principle Findings</p><p>Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.</p><p>Conclusion</p><p>Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.</p></div

Image_1_Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation.TIF

Ubiquitin-proteasome pathway (UPS) and autophagy-lysosome pathway (ALP) are the two major protein degradation pathways, which are critical for proteostasis. Growing evidence indicates that proteasome inhibition-induced ALP activation is an adaptive response. Transcription Factor EB (TFEB) is a master regulator of ALP. However, the characteristics of TFEB and its role in proteasome inhibition-induced ALP activation are not fully investigated. Here we reported that the half-life of TFEB is around 13.5 h in neuronal-like cells, and TFEB is degraded through proteasome pathway in both neuronal-like and non-neuronal cells. Moreover, proteasome impairment not only promotes TFEB accumulation but also facilitates its dephosphorylation and nuclear translocation. In addition, proteasome inhibition-induced TFEB accumulation, dephosphorylation and nuclear translocation significantly increases the expression of a number of TFEB downstream genes involved in ALP activation, including microtubule-associated protein 1B light chain-3 (LC3), particularly LC3-II, cathepsin D and lysosomal-associated membrane protein 1 (LAMP1). Furthermore, we demonstrated that proteasome inhibition increases autophagosome biogenesis but not impairs autophagic flux. Our study advances the understanding of features of TFEB and indicates that TFEB might be a key mediator of proteasome impairment-induced ALP activation.</p

Data_Sheet_1_Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation.ZIP

Ubiquitin-proteasome pathway (UPS) and autophagy-lysosome pathway (ALP) are the two major protein degradation pathways, which are critical for proteostasis. Growing evidence indicates that proteasome inhibition-induced ALP activation is an adaptive response. Transcription Factor EB (TFEB) is a master regulator of ALP. However, the characteristics of TFEB and its role in proteasome inhibition-induced ALP activation are not fully investigated. Here we reported that the half-life of TFEB is around 13.5 h in neuronal-like cells, and TFEB is degraded through proteasome pathway in both neuronal-like and non-neuronal cells. Moreover, proteasome impairment not only promotes TFEB accumulation but also facilitates its dephosphorylation and nuclear translocation. In addition, proteasome inhibition-induced TFEB accumulation, dephosphorylation and nuclear translocation significantly increases the expression of a number of TFEB downstream genes involved in ALP activation, including microtubule-associated protein 1B light chain-3 (LC3), particularly LC3-II, cathepsin D and lysosomal-associated membrane protein 1 (LAMP1). Furthermore, we demonstrated that proteasome inhibition increases autophagosome biogenesis but not impairs autophagic flux. Our study advances the understanding of features of TFEB and indicates that TFEB might be a key mediator of proteasome impairment-induced ALP activation.</p

Image_3_Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation.tif

Ubiquitin-proteasome pathway (UPS) and autophagy-lysosome pathway (ALP) are the two major protein degradation pathways, which are critical for proteostasis. Growing evidence indicates that proteasome inhibition-induced ALP activation is an adaptive response. Transcription Factor EB (TFEB) is a master regulator of ALP. However, the characteristics of TFEB and its role in proteasome inhibition-induced ALP activation are not fully investigated. Here we reported that the half-life of TFEB is around 13.5 h in neuronal-like cells, and TFEB is degraded through proteasome pathway in both neuronal-like and non-neuronal cells. Moreover, proteasome impairment not only promotes TFEB accumulation but also facilitates its dephosphorylation and nuclear translocation. In addition, proteasome inhibition-induced TFEB accumulation, dephosphorylation and nuclear translocation significantly increases the expression of a number of TFEB downstream genes involved in ALP activation, including microtubule-associated protein 1B light chain-3 (LC3), particularly LC3-II, cathepsin D and lysosomal-associated membrane protein 1 (LAMP1). Furthermore, we demonstrated that proteasome inhibition increases autophagosome biogenesis but not impairs autophagic flux. Our study advances the understanding of features of TFEB and indicates that TFEB might be a key mediator of proteasome impairment-induced ALP activation.</p

Image_2_Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation.TIF

Ubiquitin-proteasome pathway (UPS) and autophagy-lysosome pathway (ALP) are the two major protein degradation pathways, which are critical for proteostasis. Growing evidence indicates that proteasome inhibition-induced ALP activation is an adaptive response. Transcription Factor EB (TFEB) is a master regulator of ALP. However, the characteristics of TFEB and its role in proteasome inhibition-induced ALP activation are not fully investigated. Here we reported that the half-life of TFEB is around 13.5 h in neuronal-like cells, and TFEB is degraded through proteasome pathway in both neuronal-like and non-neuronal cells. Moreover, proteasome impairment not only promotes TFEB accumulation but also facilitates its dephosphorylation and nuclear translocation. In addition, proteasome inhibition-induced TFEB accumulation, dephosphorylation and nuclear translocation significantly increases the expression of a number of TFEB downstream genes involved in ALP activation, including microtubule-associated protein 1B light chain-3 (LC3), particularly LC3-II, cathepsin D and lysosomal-associated membrane protein 1 (LAMP1). Furthermore, we demonstrated that proteasome inhibition increases autophagosome biogenesis but not impairs autophagic flux. Our study advances the understanding of features of TFEB and indicates that TFEB might be a key mediator of proteasome impairment-induced ALP activation.</p