Journal: The Journal of Neuroscience

Article Title: Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease

doi: 10.1523/JNEUROSCI.3117-15.2016

Figure Lengend Snippet: Dopamine-specific knockdown of individual PHD isoforms in vivo. A, Representative in vivo analyses of PHD1, PHD2, and PHD3 ICC demonstrating expression in both DAergic and non-DAergic cell types in SN. SN sections were separately stained for PHD isoforms (green, ×40) and TH (upper right; red, ×40), and nuclei counterstained with DAPI (lower left; blue, ×40); merged, lower right. B, Gene targeting strategy for generating pTH-PHD knockdown (KD) lines and confirmation of PHD isoform-specific deletions. Top, Schematic of genetic location of floxed PHD alleles (PHD floxed) and expected deletion products (PHD cre), forward and reverse primers used for the PCR analyses (arrows), and expected sizes of PCR products before and after Cre-mediated deletion. Bottom, PCR confirming deletions in DAergic-enriched OB versus CB negative controls isolated from pTH-CRE-floxed PHD mice (KD) versus floxed PHD controls (CON). C, Representative Western blots demonstrating select PHD isoform reduction in striatal tissues from pTH-CRE-floxed PHD lines (left); densitometric quantitation (right); *p < 0.05 for PHD con versus KD; Tukey's multiple-comparison test.

Article Snippet: Human induced pluripotent stem cell (iPSC)-derived DAergic neurons were purchased from XCell Science.

Techniques: Knockdown, In Vivo, Expressing, Staining, Isolation, Western Blot, Quantitation Assay, Comparison

Journal: The Journal of Neuroscience

Article Title: Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease

doi: 10.1523/JNEUROSCI.3117-15.2016

Figure Lengend Snippet: Reductions in PHD2 activity results in neuroprotection against the mitochondrial neurotoxin MPTP in vivo and its metabolite MPP+ in vitro in a HIF1α-dependent fashion. A, Stereological TH+ SN cell counts of pTH-CRE-floxed PHD mice (PHD KD) versus floxed-only controls (PHD con) intraperitoneally injected with MPTP demonstrates a statically significant neuroprotective effect when PHD2 levels were reduced, but not in the case of reductions in the other two isoforms. Data are expressed as average SN TH+ cells per animal, n = 5 per condition. B, Locomotor activity was monitored via the cylinder test in PHD2 knockdown (KD) mice versus littermate controls before the animals were killed. Values are reported as number of rears (% control); *p < 0.05, saline versus MPTP-treated control; #p < 0.05 MPTP-treated PHD2 control versus MPTP-treated PHD2 KD. C, Representative in vivo analysis of dual PHD2 and TH ICC in PHD2 controls (top) versus KD SN (bottom), demonstrating reductions in PHD2 protein expression within DAergic SN neurons in PHD2 KD mice. SN sections were separately stained for PHD2 (right; green, ×40) and TH (middle; red, ×40); merged, yellow (left). Quantitation is shown as PHD2/TH ratio, *p > 0.05 PHD2 KD versus PHD2 control. D, Representative TH immunostaining of mesencephalic cultures from genetic PHD2 KD mice ± MPP+. E, TH+ cell quantification (right) of DAergic PHD2 KD under conditions of mitochondrial stress. Data are expressed as percentage of cell loss versus controls. F, G, MPP+-mediated losses in mitochondrial function as monitored by mitochondrial membrane potential (MMP) levels (F) and cell viability (G) in human iPSC-derived neurons are abrogated by pharmacological PHD2 inhibition. Data are expressed as percentage untreated control. *p < 0.001 versus control; **p < 0.01 versus MPP+. H, Representative TH immunostaining of mesencephalic cultures from PHD2 control mice in the presence of vehicle or MPP+ ± IOX2, ± HIF1α inhibition via 2-methoxyestradiol (MeOE2). I, TH+ cell quantitation demonstrates that neuroprotection afforded by PHD2 inhibition in these cells is prevented by reductions in HIF1α activity. Data are expressed as percentage untreated controls. *p < 0.01 versus control; #p < 0.01 versus MPP+, **p < 0.01 versus IOX2 MPP+; Tukey's multiple-comparison test.

Article Snippet: Human induced pluripotent stem cell (iPSC)-derived DAergic neurons were purchased from XCell Science.

Techniques: Activity Assay, In Vivo, In Vitro, Injection, Knockdown, Control, Saline, Expressing, Staining, Quantitation Assay, Immunostaining, Membrane, Derivative Assay, Inhibition, Comparison

Journal: The Journal of Neuroscience

Article Title: Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease

doi: 10.1523/JNEUROSCI.3117-15.2016

Figure Lengend Snippet: Reductions in PHD2 activity results in increases in expression of ATP13A2. A, RT-PCR analysis of ATP13A2 expression levels in mesencephalic cultures isolated from PHD2 control mice in the presence of pharmacological PHD2 inhibition via IOX2 demonstrates increased expression versus untreated controls. Data are reported as fold change. *p < 0.01 versus controls, unpaired t test. B, C, RT-PCR analyses of ATP13A2 levels in OB (B) and ST (C) tissues isolated from genetic PHD2 knock-out versus PHD2 control mice demonstrates upregulation gene expression in the context of in vivo reductions in DAergic PHD2 levels. The HIF1α target gene HO-1 was included as a positive control. Data are reported as fold change. *p < 0.01 versus controls, unpaired t test. D, Western blot analysis of ATP13A2 and HO-1 levels in striatal tissues isolated from PHD2 knockdowns (KDs) versus control mice demonstrates significant elevations in protein levels. Data are reported as percentage PHD2 control normalized to actin; *p < 0.01 versus PHD2, #p < 0.01 versus PHD2, unpaired t test. E, Western blot analysis of ATP13A2 levels in striatal tissues isolated from PHD1 and PHD3 KDs versus control mice demonstrates no significant change in protein levels. Data are reported as percentage PHD1 and PHD3 control normalized to actin, unpaired t test.

Article Snippet: Human induced pluripotent stem cell (iPSC)-derived DAergic neurons were purchased from XCell Science.

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Inhibition, Knock-Out, Gene Expression, In Vivo, Positive Control, Western Blot

Journal: The Journal of Neuroscience

Article Title: Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease

doi: 10.1523/JNEUROSCI.3117-15.2016

Figure Lengend Snippet: PHD2 inhibition in vivo abrogates basal and MPTP-mediated increases in cytosolic ferrous iron levels via increases in lysosomal iron storage. A, Cytosolic ferrous iron levels as measured by the calcein dequenching assay in striatal DAergic synaptosomes isolated from PHD2 knockdowns (KDs) versus PHD2 controls ± MPTP or saline, demonstrating that PHD2 KD results in reductions both basally and following MPTP treatment. Values are reported as percentage PHD2 control; *p < 0.05 versus PHD2 controls; #p < 0.05 versus PHD2 KD; &p < 0.05 versus MPTP-treated PHD2 controls; Tukey's multiple-comparison test. B, Cytosolic ferrous iron levels following treatment with bafilomycin reported as fold increases versus MPTP, demonstrating increases in lysosomal iron stores in PHD2 KDs; p̂ > 0.05 versus MPTP-treated PHD2 control; ^p̂ > 0.05 versus MPTP-treated PHD2 KD.

Article Snippet: Human induced pluripotent stem cell (iPSC)-derived DAergic neurons were purchased from XCell Science.

Techniques: Inhibition, In Vivo, Isolation, Saline, Control, Comparison

Journal: The Journal of Neuroscience

Article Title: Regulation of ATP13A2 via PHD2-HIF1α Signaling Is Critical for Cellular Iron Homeostasis: Implications for Parkinson's Disease

doi: 10.1523/JNEUROSCI.3117-15.2016

Figure Lengend Snippet: Knockdown (KD) of ATP13A2 expression in cultured human DAergic cells results in reduced ability to maintain lysosomal iron storage and abrogates PHD2 inhibition-mediated neuroprotection under conditions of stress. A, RT-PCR analysis of ATP13A2 levels in SH-SY5Y cells stably transfected with lentiviral ATP13A2 shRNA shows a significant reduction in ATP13A2 expression. Data are expressed as fold change; *p < 0.01 versus scrambled shRNA controls; unpaired t test. B, Representative Western blot of ATP13A2 levels in ATP13A2 shRNA transfected cells versus controls. C, Quantitation following normalization to actin (right) demonstrates corresponding reductions in ATP13A2 protein levels. Data are reported as percentage scrambled shRNA; *p < 0.01 versus scrambled; unpaired t test. D, Cytosolic ferrous iron measurements via calcein dequenching assay in stable differentiated ATP13A2 shRNA versus scrambled control SY5Y cells demonstrate that levels are increased following ATP13A2 KD both basally and following treatment with MPP+; bafilomycin results in significantly higher increases in cytosolic ferrous iron in MPP+ controls versus MPP+-treated ATP13A2 KD cells. Values are presented as percentage control; *p < 0.05 versus scrambled controls; #p < 0.05 versus MPP+, &p < 0.05 versus MPP+-treated controls; p̂ < 0.05 versus MPP+-treated KDs; Tukey's multiple-comparison test. E, RT-PCR analysis of ATP13A2 expression levels in the absence and presence of IOX2 demonstrates increased expression in scrambled controls, which is reduced by ATP13A2 shRNA. Data are reported as fold change; *p < 0.01 versus scrambled control; #p < 0.05 versus IOX2 alone; Tukey's multiple-comparison test. F, Cytosolic ferrous iron levels in MPP+-treated versus control cells in the absence and presence of IOX2 and ATP13A2 KD; values are presented as percentage controls. IOX2 prevents MPP+-mediated increases in cytosolic ferrous iron and this is reversed by knockdown of ATP13A2; *p < 0.05 versus controls; #p < 0.05 versus MPP+ alone; &p < 0.05 versus IOX2 alone; p̂ < 0.05 versus IOX2 KD; Tukey's multiple-comparison test. G, Cell viability as assessed by CyQuant fluorescence assay in differentiated control versus ATP13A2 KD cells in the absence or presence of IOX2 and MPP+; values are presented as percentage control. MPP+-mediated losses in cell viability are exacerbated by ATP13A2 KD both basally and in the presence of IOX2; *p < 0.05 versus controls, #p < 0.05 versus MPP+-control, &p < 0.05 versus MPP+, IOX2-treated control; Tukey's multiple-comparison test.

Article Snippet: Human induced pluripotent stem cell (iPSC)-derived DAergic neurons were purchased from XCell Science.

Techniques: Knockdown, Expressing, Cell Culture, Inhibition, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, shRNA, Western Blot, Quantitation Assay, Control, Comparison, CyQUANT Assay, Fluorescence