Red Emissive Two-Photon Probe for Real-Time Imaging of Mitochondria Trafficking

Mitochondria trafficking plays an essential role for supplying energy in the neuronal system. We report here a red emissive two-photon probe for mitochondria (CMT-red) that showed high selectivity and robust staining ability for mitochondria, high photostability under a two-photon microscopy imaging condition, and low cytotoxicity. This probe can be easily loaded into live cells and tissue and used for real-time, high resolution imaging of the mitochondria trafficking in primary cortical neurons as well as in rat hippocampal tissue

LRRK2 interacts with p62.

<p><b>A</b>. HEK 293T cells were transiently co-transfected with FLAG-tagged p62 (FLAG-p62), c-myc-tagged LRRK2 (myc-LRRK2), or both. Twenty-four hours after transfection, total cell lysates were subjected to immunoprecipitation with anti-c-myc monoclonal antibody 9E10. Binding of p62 to LRRK2 was analyzed with western blotting. <b>B</b>. Binding of endogenous LRRK2 to p62 was examined in HEK 293T cells via immunoprecipitation with the indicated quantities of anti-p62 antibody (0.25~1.5 μg). <b>C</b>. The P2 fraction of total rat brain lysate was subjected to immunoprecipitation with the indicated quantities of anti-p62 antibody (0.5~1.5 μg). Immune complexes were resolved by SDS-PAGE, followed by western blotting against the indicated antibodies. One microgram of rabbit anti-GFP antibody was used as a negative control (IgG). <b>D</b>. HEK 293T cells were transiently transfected with myc-tagged LRRK2 WT or mutant expression constructs (G2019S, R1441C, D1994A, or G2385R) and FLAG-tagged p62. Co-immunoprecipitation was performed as shown in panel A. <b>E</b>. Bar graph shows the relative binding of p62 to mutant LRRK2, normalized to LRRK2 WT. The data were obtained from four independent experiments; n.s. indicates p > 0.05 versus WT binding, analyzed with one-way ANOVA.</p

LRRK2 is degraded by both the UPS and the ALP.

<p><b>A</b>. Primary rat cortical neurons at 14 days <i>in vitro</i> were incubated DMSO, 5 μM MG132, 10 mM 3-methyladenine (3-MA), 50 μM chloroquine, or 50 nM bafilomycin A1 for 2 h. Representative immunoblots for the indicated proteins from four to five independent experiments are shown. <b>B</b>. LRRK2 expression level relative to that in the DMSO-treated sample was quantified by using ImageJ software. Bar graph data represent the means of LRRK2 expression ± SEM (DMSO, 1.00 ± 0.08; MG132, 2.46 ± 0.52; 3-MA, 2.22 ± 0.51; chloroquine, 3.32 ± 0.70; bafilomycin A1, 3.73 ± 0.93; n = 4–5; *p < 0.05, paired <i>t</i>-test).</p

Mapping of the binding domains between LRRK2 and p62.

<p><b>A</b>. Schematic representation showing LRRK2 domains and the LRRK2 fragments (F1-F8) used in panel A. F1: amino acids (aa) 1–480, N-terminus segment 1; F2: aa 480–895, N-terminus segment 2; F3: aa 895–1329, N-terminus segment 3; F4: aa 981–1503, LRR + GTPase; F5: aa 981–1298, LRR; F6: aa 1534–1857, COR; F7: aa 1866–2139, kinase; F8: aa 2125–2528, WD40 domain. <b>B</b>. p62 interacts the N-terminal region of LRRK2. GFP-tagged p62 was co-transfected with the indicated FLAG-LRRK2 fragment constructs F1–F8 in HEK 293T cells as shown in panel B. Immunoprecipitation was carried out by using rabbit anti-GFP antibody, and the binding domains were detected with mouse anti-FLAG antibody. <b>C</b>. Schematic representation showing p62 domains and p62 fragments (G1-G6) used in panel A. G1: GFP alone; G2: aa 1–103, p62 Phox and Bem1 (PB1); G3: aa 1–163, p62 PB1 and zinc finger (ZZ); G4: aa 1–251, p62 ΔLIR/ΔUBA; G5: aa 1–386, p62 ΔUBA; G6: aa 1–440, p62 full length. ΔTB, ΔSMIR, ΔSMIR/TB indicate the deletion mutants of p62 aa 225–251, aa 166–224, aa 166–251, respectively. All mutant constructs were sequence-proved with complete fidelity. LIR, microtubule-associated protein 1A/1B-light chain 3-interaction region; UBA, ubiquitin-associated domain. <b>D</b>. LRRK2 interacts with p62 through the SMIR of p62. HEK 293T cells were co-transfected with GFP-p62 fragment constructs (G1-G6) and myc-tagged LRRK2. Co-immunoprecipitation assays were performed using anti-myc (left panel) or anti-GFP antibody (right panel). Arrowhead indicates immunoglobulin heavy chain (IgH).</p

LRRK2 regulates Ser351 phosphorylation of p62.

<p><b>A</b>. Primary cortical neurons were infected with lentivirus that contain LRRK2 shRNA or a non-target shRNA sequence (NT shRNA) for 7 days, and endogenous protein expression levels were analyzed via western blotting with the indicated antibodies. <b>B</b>. Relative phosphorylation levels of p62 after LRRK2 knockdown normalized to NT shRNA were quantified. Summary data are presented as means ± SEM from three independent experiments (NT shRNA, 1.00 ± 0.00; pSer351, 2.92 ± 0.74; pSer403, 3.10 ± 1.09; n = 3; *p < 0.05, paired <i>t</i>-test). <b>C</b>. GFP-p62 WT or Ser351Glu (S351E) was co-transfected with vector control, WT, or G2019S myc-LRRK2 in HEK 293T cells. Endogenous Keap1 bindings to p62 were analyzed via immunoprecipitation with anti-GFP (p62) antibody. <b>D</b>. Quantification data from panel C represent means of relative Keap1 binding to p62 ± SEM (WT LRRK2, 1.05 ± 0.19; GS LRRK2, 0.67 ± 0.11; vector, 1.51 ± 0.08; n = 3; *p < 0.05, paired <i>t</i>-test).</p

Stability of LRRK2 is regulated by p62.

<p><b>A</b>. HEK 293T cells were transfected with 0.25, 0.5, or 1.0 μg FLAG-p62 expression plasmid or control vector for 24 h. Bafilomycin A1 was treated for 2 h. Endogenous LRRK2 expression was detected with western blotting by using LRRK2 antibody from total cell lysates. Quantification data from panel A represent means of LRRK2 expression ± SEM (Vec, 1.00 ± 0.00; 0.25 μg p62, 0.59 ± 0.12; 0.5 μg p62, 0.34 ± 0.15; 1.0 μg p62, 0.46 ± 0.15; n = 3; *p < 0.05, paired <i>t</i>-test). <b>B</b>. Primary cortical neurons were treated with bafilomycin A1 for 2 h and endogenous protein levels were detected with western blotting using the indicated antibodies. <b>C</b>. Primary cortical neurons were infected with lentivirus that overexpress GFP (control) or p62 (p62 OE) under a ubiquitin promoter. Neurons were also infected with lentivirus that harbor p62 small hairpin RNA (shRNA) under an H1 promoter to knock down the expression of p62 (p62 KD). Five to seven days after infection, the neurons were treated with DMSO or 5 μM MG132 for 2 h. Endogenous LRRK2 expression was examined with western blotting. Arrowhead indicates the exogenous expression of 3 X FLAG-tagged p62, in which bands were shifted owing to the increased molecular weight of p62 fused with three tandem FLAG epitopes. <b>D</b>. Quantification of band intensity of LRRK2 relative to that of the control is shown. Means ± SEM (control, 1.00 ± 0.00; P62 OE, 0.60 ± 0.11; p62 KD, 1.12 ± 0.49; n = 4 for p62 OE, n = 13 for p62 KD; *p < 0.05, n.s. indicates p > 0.05, paired <i>t</i>-test). <b>E</b>. A FLAG-p62 expression plasmid was transfected in HEK 293T cells. Twenty-four hours after transfection, the cells were treated with 100 nM rapamycin for 2 h, and protein expression levels were analyzed with western blotting with the indicated antibodies. <b>F</b>. Bar graph data represent the means of normalized LRRK2 expression ± SEM (Vec, 1.00 ± 0.00; Vec with rapamycin, 0.39 ± 0.08; p62, 0.35 ± 0.05; p62 with rapamycin, 0.51 ± 0.04; n = 3, *p < 0.01, n.s. indicates p > 0.05, paired <i>t</i>-test). <b>G</b>. HEK 293T cells were co-transfected with WT or G2019S myc-LRRK2 and phosphomimetic mutant FLAG-p62 Ser403Glu (S403E) or dephosphorylated mutant Ser403Ala (S403A). LRRK2 expression was detected with western blotting. <b>H</b>. Bar graph data represent the means of normalized LRRK2 expression ± SEM from panel G (Vec, 1.00 ± 0.00; S403E, 0.50 ± 0.01; S403A, 1.28 ± 0.66; S403E in G2019S, 0.47 ± 0.11; n = 3–4, *p < 0.05, paired <i>t</i>-test).</p

Image_2_N-Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors.TIF

<p>N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.</p

Data_Sheet_1_N-Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors.pdf

<p>N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.</p

<i>Snap23</i>^Δ/Δ blastocysts die prior to uterine implantation.

<p>(<b>A</b>) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated <i>Snap23</i>^Δ/wt females mated with <i>Snap23</i>^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018444#pone-0018444-t002" target="_blank">Table 2</a>). (<b>B</b>) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the <i>Snap23</i> deleted allele (<i>Snap23</i>^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the <i>Snap23</i>^wt allele (266 bp) and for the <i>Snap23</i>^Δ allele (492 bp) are indicated.</p

Oligonucleotide primers used in this study and estimated size of PCR products.

<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Snap23</i>, and to generate probes for Southern blot analysis. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p