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1.
Figure 2.

Figure 2. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Localization of exogenous ATP13A2 to intracellular acidic vesicles in cortical neurons. (A) Confocal fluorescence microscopy reveals the co-localization of exogenous V5-tagged human ATP13A2 with GFP-LC3 (autophagosomes), RFP-Rab5A (early endosomes), GFP-Rab7A (late endosomes) and LAMP1-RFP (lysosomes) in neuronal soma and processes. Exogenous GFP-tagged mouse ATP13A2 is specifically labeled by the ATP13A2 antibody (LMNR1). (B) ATP13A2-V5 localizes to punctate structures located upon βIII-tubulin-positive neuronal processes, whereas ATP13A2-GFP fails to co-localize with endogenous synaptophysin-1, a marker of synaptic vesicles. Cytofluorograms and correlation coefficients (Rcoloc) indicate the extent of co-localization between exogenous ATP13A2 and each marker. Confocal images are representative of at least two independent cultures. Scale bar: 10 µm.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
2.
Figure 1.

Figure 1. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Development of an ATP13A2-specific antibody. (A) HEK-293T cells transiently expressing V5-tagged human ATP13A2 (left panel) or full-length untagged mouse ATP13A2 (right panel) were probed with an antibody to ATP13A2 (LMNR1) to reveal a specific protein band of 130–150 kDa (arrows) in transfected cells (+) that is absent from mock transfected cells (−). (B) Soluble protein extract from whole mouse brain was probed with ATP13A2-specific antibody, LMNR1 (left panel). The LMNR1 antibody detects a major protein species of ∼130 kDa (indicated by arrows), corresponding to the expected mass of endogenous ATP13A2. This protein species was not detected following pre-absorption of the LMNR1 antibody with an excess of peptide antigen (right panels). (C) Subcellular fractionation of whole mouse brain tissue. ATP13A2 (LMNR1 antibody) is enriched in the light membrane (P3, microsomal) fraction, but also detected in heavy (P2), synaptosomal (LP1), synaptic vesicle-enriched (LP2) membrane fractions. The distribution of marker proteins demonstrates the enrichment of lysosomes (LAMP1; P3), mitochondria/heavy membranes (TIM23; P2 and LP1), ER (PDI; P2, P3, LP1 and LP2), Golgi (Giantin; P2 and P3) and synaptosomes/synaptic vesicles (synaptophysin 1; P2, P3, LP1 and LP2). Molecular mass markers are indicated in kilodaltons. Blots are representative of at least two independent experiments.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
3.
Figure 5.

Figure 5. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

ATP13A2 modulates the size and number of LC3-positive autophagosomes in neurons. (A) Soluble extracts from rat primary cortical neurons co-transfected with control or ATP13A2-specific shRNAs or V5-tagged human ATP13A2 and GFP-LC3 plasmids at a 10:1 molar ratio were probed with antibodies to GFP to reveal LC3-I and LC3-II isoforms or β-tubulin as a control for protein loading. (B) Quantitative analysis of western blots revealing a normal ratio of LC3-II to LC3-I (left), but reduced steady-state levels of total GFP-LC3 (right) due to ATP13A2 silencing or overexpression in cortical neurons. Bars represent the mean ± SEM (n = 4 experiments). (C and E) Representative confocal fluorescent images indicating GFP-LC3-positive puncta in cortical neurons following (C) overexpression of human ATP13A2 or (E) shRNA-mediated silencing of ATP13A2, compared with control plasmids (empty vector or control shRNA). Scale bar: 5 µm. (D and F) Quantitative analysis of GFP-LC3-positive puncta indicates (D) the increased length and number of puncta due to ATP13A2 overexpression or (F) the reduced length of puncta due to ATP13A2 silencing. Bars represent the mean ± SEM length of puncta (micrometers) or percent particles/cell compared with the control condition (n = 9 neurons/condition from three experiments). Histograms indicate the mean frequency distribution of GFP-LC3-positive puncta length in micrometers for each condition (n = 9 neurons/condition). *P < 0.05, **P < 0.01 or ***P < 0.005 compared with control assessed by unpaired Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
4.
Figure 3.

Figure 3. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Increased levels of ATP13A2 in PD/DLB brains. (A) Soluble extracts derived from the striatum of normal control (nos 1–4) or PD/DLB (nos 1–5) subjects probed with ATP13A2 antibody (LMNR1), and β-actin antibody as a control for protein loading. (B) Densitometric analysis of western blots revealing increased levels of ATP13A2 in soluble extracts from striatum or medial frontal gyrus of PD/DLB subjects compared with normal control subjects. Data represent the mean ± SEM levels of ATP13A2 normalized to β-actin and are expressed as a percent of control levels (n = 4 for control or n = 5 for PD/DLB subjects). (C) Representative DAB immunostaining with ATP13A2 antibody (LMNR1) in the substantia nigra pars compacta from normal control or PD subjects. Staining indicates ATP13A2 localized to characteristic neuromelanin-positive dopaminergic neurons. Scale bar: 100 µm (upper panel) or 20 µm (lower panel). (D) Semi-quantitative analysis of DAB intensity corresponding to ATP13A2 levels in individual substantia nigra dopaminergic neurons from control or PD subjects. Data represent the mean ± SEM intensity of ATP13A2 staining in individual dopaminergic neurons (n = 1449/control or 1257/PD neurons) taken from at least three subjects per group. (E) Representative confocal fluorescent image of an intraneuronal LB from the cingulate cortex of a DLB subject (no. 8) immunolabeled with phospho-S129-α-synuclein (pαSyn, green) and ATP13A2 (LMNR1, red) antibodies. (F) Semi-quantitative analysis of ATP13A2 fluorescence intensity in cortical pyramidal neurons with (LB+, n = 86 neurons) or without (LB−, n = 2990 neurons) LBs. Data represent the mean ± SEM fluorescence intensity of ATP13A2 in cortical pyramidal neurons from the cingulate cortex of a single DLB subject. **P < 0.001 analyzed by Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
5.
Figure 8.

Figure 8. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. (A) Rat primary cortical neurons were co-transfected with V5-tagged human ATP13A2 (or control empty vector) and mito-DsRed2 plasmids at a 10:1 molar ratio at DIV 9 to label transfected neurons. At DIV 12, mito-DsRed2-labeled mitochondria in individual neurons were monitored by time-lapse, live-cell confocal microscopy. Shown are representative time-lapse image series of mitochondria at baseline (over 120 s) and following acute cadmium exposure (over 240 s) in neurons. (B) Time course of average mitochondrial length induced by acute cadmium exposure (at time 0 s) indicating delayed cadmium-induced fragmentation in neurons overexpressing human ATP13A2. Data were fitted to a second-order kinetic model. (C) ATP13A2 overexpression does not alter mean basal or cadmium-induced (peak) mitochondrial length (left graph), but increases the half-life (t1/2) of cadmium-induced mitochondrial fragmentation (right graph). (DF) Similar experiments were conducted on primary cortical neurons co-transfected with non-silencing control shRNA or ATP13A2-specific shRNA and mito-DsRed2 plasmids at a 10:1 molar ratio. (D) Representative time-lapse image series of mitochondria at baseline and following cadmium treatment. (E) Time course of average mitochondrial length induced by cadmium exposure (at time 0 s) and determination of (F) mean basal and cadmium-induced (peak) length or the rate of mitochondrial fragmentation. ATP13A2 silencing reduces basal and cadmium-induced mitochondrial length (left graph) and reduces the half-life of cadmium-induced fragmentation (right graph). Data represent n = 15 neurons/condition sampled from five independent experiments. *P < 0.05 or ***P < 0.005 compared with the appropriate control condition as indicated by lines, or by comparing basal versus cadmium conditions, assessed by unpaired Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
6.
Figure 4.

Figure 4. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Modulation of ATP13A2 expression impairs neurite outgrowth of midbrain dopaminergic neurons. (A) Soluble extracts from HEK-293T cells transiently co-expressing untagged mouse ATP13A2 and rodent-specific ATP13A2 (sh-ATP13A2) or non-silencing control (sh-control) shRNA plasmids were probed with an antibody to ATP13A2 (LMNR1) to demonstrate shRNA-mediated knockdown of ATP13A2. β-tubulin indicates equivalent protein loading. (B) Soluble extracts from rat primary cortical neurons transiently expressing control or ATP13A2-specific shRNAs, or untagged mouse ATP13A2, were probed with antibodies to ATP13A2 (LMNR1) or β-tubulin as a control for protein loading. ATP13A2-specific shRNA reduces the levels of endogenous ATP13A2 compared with control shRNA, whereas overexpression of mouse ATP13A2 is also detected. (C) Rat primary midbrain dopaminergic neurons co-transfected at DIV 3 with shRNA (control or ATP13A2-specific) or V5-tagged human ATP13A2 (or control empty vector) and GFP plasmids at a 10:1 molar ratio. Cultures were fixed at DIV 7. Representative fluorescent micrographs reveal the co-labeling of dopaminergic neurons with GFP and TH for each condition. Axonal processes are indicated by arrows. Scale bar: 100 µm. (D and E) Quantitative analysis of the length of GFP+/TH+ dopaminergic neurites reveals a significant shortening of axonal processes due to (D) the shRNA-mediated silencing of ATP13A2 or (E) the overexpression of human ATP13A2, compared with control neurons (sh-control or empty vector). Bars represent the mean ± SEM length of TH+ neurites in micrometers (n = 39–42 neurons for shRNAs or n = 28–39 neurons for hATP13A2) sampled across four independent cultures. Histograms indicate the mean frequency distribution of neurite length for each condition (n = 4 experiments). *P < 0.05 or **P < 0.01 compared with control plasmids assessed by unpaired Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
7.
Figure 6.

Figure 6. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Silencing of ATP13A2 expression regulates the kinetics of intracellular pH in neurons. (A) Rat primary cortical neurons were co-transfected with V5-tagged human ATP13A2 (or control empty vector) and EGFP plasmids at a 10:1 molar ratio at DIV 9 to label transfected neurons. At DIV 12, neurons were loaded with SNARF-1 fluorescent dye to monitor the intracellular pH of individual EGFP-positive neurons by time-lapse, live-cell confocal microscopy. Following time-lapse imaging of baseline pH over a 120 s period, neurons were exposed to 1 mm cadmium and EGFP-positive neurons were imaged over a 360 s period. Representative images of EGFP-positive neurons loaded with SNARF-1 dye are shown in the left image, and pseudo-color encoded mosaics indicating basal (top row) and progressive cadmium-induced acidification (bottom rows) with time are shown in the right image. Warmer tones indicate acidic pH as indicated by lower color scale bar. Scale bar: 50 µm. (B) Average time course of intracellular pH induced by acute cadmium exposure fitted to a first-order kinetic model (AR3). Cadmium was added at time point 0 s. (C) Fitted parameters of basal and peak intracellular pH after cadmium exposure (left graph) and the rate of pH change following cadmium exposure (t1/2 in seconds; right graph) are indicated. (DF) Similar experiments were conducted on primary cortical neurons co-transfected with non-silencing control shRNA or ATP13A2-specific shRNA and EGFP plasmids at a 10:1 molar ratio. (D) Representative confocal images of EGFP-positive cortical neurons loaded with SNARF-1 under basal conditions or induced by acute cadmium exposure. (E) Average time course of intracellular pH following cadmium treatment (fitted to AR3 model) and (F) basal and peak pH (left) or rate (right) induced by cadmium exposure. Data represent n = 15 neurons/condition sampled from five independent experiments. **P < 0.01 compared with control condition assessed by unpaired Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.
8.
Figure 7.

Figure 7. From: PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Modulation of ATP13A2 expression reduces basal intracellular calcium concentration. (A) Rat primary cortical neurons were co-transfected with V5-tagged human ATP13A2 (or control empty vector) and DsRed plasmids at a 10:1 molar ratio at DIV 9 to label transfected neurons. At DIV 12, neurons were loaded with Fluo-4 AM fluorescent dye to monitor the intracellular calcium levels of individual DsRed-positive neurons by time-lapse, live-cell confocal microscopy. Representative time-lapse series of baseline intracellular calcium levels (over 120 s) and following acute cadmium exposure (over 240 s) in DsRed-positive neurons. Warmer tones indicate higher intracellular calcium concentrations as indicated by lower color scale bar. (B) ATP13A2 overexpression reduces the levels of basal and cadmium-induced intracellular calcium in neurons. (C) Time course of average intracellular calcium levels induced by cadmium exposure (at time 0 s) expressed as increased calcium levels over initial basal levels for each neuron. Data were fitted to a first-order kinetic model, and the mean (D) rate (half-life, t1/2) and (E) maximum peak calcium levels were determined. (FJ) Similar experiments were conducted on primary cortical neurons co-transfected with non-silencing control shRNA or ATP13A2-specific shRNA and DsRed plasmids at a 10:1 molar ratio. (F) Representative time-lapse series of baseline and cadmium-induced intracellular calcium levels. (G) ATP13A2 silencing reduces the levels of basal and cadmium-induced intracellular calcium in neurons. (H) Time course of intracellular calcium levels induced by cadmium exposure and determination of mean (I) rate and (J) maximum calcium levels. Data represent n = 15 neurons/condition sampled from five independent experiments. **P < 0.01 or ***P < 0.005 compared with the appropriate control condition as indicated by lines, or by comparing basal versus cadmium conditions, assessed by unpaired Student's t-test.

David Ramonet, et al. Hum Mol Genet. 2012 April 15;21(8):1725-1743.

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