B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild - type FICD . Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species . The elution profile of a monomeric FICDL258D - H363A mutant is also shown for reference , alongside the elution times of molecular weight standards .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild-type FICD. Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species. The elution profile of a monomeric FICDL258D-H363A mutant is also shown for reference, alongside the elution times of molecular weight standards.


C Representative immunoblot comparing the ability of wild - type and Arg371Ser FICD ( at a wide range of enzyme concentrations ) to catalyse the accumulation of BiP - AMP in the presence of physiological ATP concentrations ( 5 mM ) . At low enzyme concentrations ( and early time points ) the AMPylation defect imposed by Arg371Ser is manifest . At higher enzyme concentrations where the deAMPylation activity of the wild - type enzyme begins to dominate , the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP - AMP ( relative to the same concentration of wild - type enzyme ) . D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C ( mean values ± SEM , n = 4 , biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative immunoblot comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP in the presence of physiological ATP concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP (relative to the same concentration of wild-type enzyme). D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C (mean values ± SEM, n = 4, biological replicates).


F Representative immunoblot comparing the accumulation of BiP - AMP in reactions constituted with the indicated concentrations of wild - type and Arg371Ser FICD , performed as in A ( upper panel ) . The lower panel is a Coomassie - stained gel of the reaction constituents . An N - terminally extended version of the wild - type enzyme ( residues 45 - 458was used in lanes 2 and 5 - 7 , to distinguish its migration on the gel from the mutant ( residues 104 - 445 ) . The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F ( from n = 3 , biological replicates , mean values ± SEM ) .

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F Representative immunoblot comparing the accumulation of BiP-AMP in reactions constituted with the indicated concentrations of wild-type and Arg371Ser FICD, performed as in A (upper panel). The lower panel is a Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).


A Immunoblots of AMPylated proteins detected with a pan - AMP antibody , BiP , FICD and eIF2α ( a loading control ) in lysates of FICD ∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD . Signals corresponding to AMPylated BiP , auto - AMPylated FICDE234G , total BiP , FICD and eIF2α are indicated . The novel species reactive with the anti - FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S ( * ) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunoblots of AMPylated proteins detected with a pan-AMP antibody, BiP, FICD and eIF2α (a loading control) in lysates of FICD∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD and eIF2α are indicated. The novel species reactive with the anti-FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.


C Quantification of the data from repeats of the experiment as shown in B . The upper two graphs show the percent of cells with high CHOP : : GFP or high XBP : : Turq for each transfection . The lower panel provides quantitation of the median fluorescence intensity for each reporter . Mean ± SD , n = 4 , biological replicates .

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C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.


B On the left are immunoblots of AMPylated proteins detected with pan - AMP , BiP and eIF2α ( a loading control ) reactive antibodies in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) for the indicated time . In the panel on the right are immunoblots of samples immunoprecipitated with a BiP - specific antiserum from the lysates of the samples shown on the left .

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B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.


C Immunoblots as in B in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) and / or thapsigargin ( Tg ) for the indicated periods prior to harvest . D Quantification of ratio of the BiP - AMP to total BiP signal ( expressed in arbitrary units ) of repeats of the experiment shown in C . Mean ± SD of the BiP - AMP signal normalized to total BiP signal from the same samples are depicted , ( P values by two tailed t - test , n = 6 , biological replicates ) .

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C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).


A Representative immunoblot of AMPylated proteins in FICD ∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes ( replicated three times ) . Note that the hyperactive E234G mutant bearing the K124E - H131A mutations that compromise its ability to interact with BiP , is nonetheless consistently auto - AMPylated .

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A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.


C Bar diagram of the amount of alkaline phosphatase [ normalised to cytoplasmic luciferase ( a transfection marker ) ] secreted from FICD ∆ CHO cells co - expressing the indicated FICD enzymes . The K124E - H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [ Mean ± SD , P values by ANOVA with Šídák ' s multiple comparisons test , n = 8 - 16 ( biological replicates ) ] .

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C Bar diagram of the amount of alkaline phosphatase [normalised to cytoplasmic luciferase (a transfection marker)] secreted from FICD∆ CHO cells co-expressing the indicated FICD enzymes. The K124E-H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [Mean ± SD, P values by ANOVA with Šídák's multiple comparisons test, n = 8-16 (biological replicates)].


( A ) Oxygen consumption ratio , , in the indicated VSMC types .

(A) Oxygen consumption ratio, , in the indicated VSMC types.


( B ) mitochondrial ATP synthesis in the indicated VSMC types .

(B) mitochondrial ATP synthesis in the indicated VSMC types.


( C ) mitochondrial membrane potential ( MP ) , in the indicated VSMC types .

(C) mitochondrial membrane potential (MP), in the indicated VSMC types.


( A ) Cytosolic ATP synthesis in the indicated VSMC types , measured by incorporation of phosphate - 32 ( 32Pi ) into ADP . [ 32Pi ] - ATP was separated from 32Pi using the molybdate method , as explained in the Methods section .

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(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.


(B) Intracellular lactate concentration


( C ) external acidification in the indicated VSMC types .

(C) external acidification in the indicated VSMC types.


( D ) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi / mL calcium - 45 ( 45Ca2 + ) as a radiotracer .

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(D) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi/mL calcium-45 (45Ca2+) as a radiotracer.


( E ) Magnesium concentration in isolated mitochondria .

(E) Magnesium concentration in isolated mitochondria.


( B ) Representative time - course of 2 mmol / L phosphate on calcification of LmnaG609G / + VSMCs ( up ) . Calcification was visualized with Alizarin red . Representative microscopic images ( 10x ; scale bar : 100 µm ) showing calcification of treated and untreated LmnaG609G / + VSMCs ( down ) .

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(B) Representative time-course of 2 mmol/L phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 µm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down).


( C - F ) Measures of calcium in treated living LmnaG609G / + VSMCs ( C ) , treated fixed LmnaG609G / + VSMCs ( D ) , untreated living LmnaG609G / + VSMCs ( E ) , and untreated fixed LmnaG609G / + VSMCs ( F ) .

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(C-F) Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F).


( G ) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells ( ΔCa2 + ) .

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(G) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+).


( A ) Magnesium intake in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( B ) Plasma magnesium concentration in 34 - week - old untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( C ) Calcium content of aortas obtained from 34 - week - old wild - type mice and untreated and treated LmnaG609G / + mice ( n = 20 ) .

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(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).


( D ) Body masses of 34 - week - old untreated and treated LmnaG609G / + mice .

(D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice.


( E ) Kaplan - Meier graph for untreated and treated LmnaG609G / + mice ( n = 16 ) .

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(E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16).


( F ) Representative photographs of 40 - wk - old wild - type , untreated and treated LmnaG609G / + mice .

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(F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.


( A ) ATP concentration in liver homogenates obtained from 34 - week - old wild - type , untreated or treated LmnaG609G / + mice .

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(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.


( B ) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups .

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(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.


( C ) Magnesium concentration in isolated mitochondria .

(C) Magnesium concentration in isolated mitochondria.


( D ) Activities of the indicated mitochondrial complexes ( I , II , III , IV , and V ) in the absence or presence of rotenone ( 2 µmol / L ) , 2 - thenoyltrifluoroacetone ( TTFA ; 1 mmol / L ) , antimycin A ( 10 µmol / L ) , potassium cyanide ( KCN ; 1 mmol / L ) , or oligomycin ( 10 µmol / L ) .

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(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).


( B ) Light microscopy images of LGCs , FBGCs and osteoclasts stained with Giemsa staining ( upper panel ) . Representative Hoechst dye images acquired by ImageStream ( lower panel ) of sorted cells showing mononuclear ( mono ) and multinucleated ( multi ) populations for each cell type .

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(B) Light microscopy images of LGCs, FBGCs and osteoclasts stained with Giemsa staining (upper panel). Representative Hoechst dye images acquired by ImageStream (lower panel) of sorted cells showing mononuclear (mono) and multinucleated (multi) populations for each cell type.


( C ) RNA - seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type .

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(C) RNA-seq volcano plots highlighting the top 15 differentially expressed genes between mononuclear and multinucleated populations for each cell type.


( C ) MRC1 surface marker expression ( red ) acquired by ImageStream in mononuclear ( mono ) and multinucleated ( multi ) LGCs , FBGCs and osteoclasts stained for Hoechst . Bar graphs ( lower panel ) represent normalized MRC1 mean fluorescence intensity ( MFI ) , measured by ImageStream ; n = 4 donors .

(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.


( E ) CSF1R , MRC1 , TLR2 and FOS relative expression measured by qRT - PCR for LGCs ( upper ) , FBGCs ( middle ) and osteoclasts ( lower ) , following the cell membrane fusion regulator DC - STAMP knockdown . si - Ctrl , scrambled siRNA ; si - DC - STAMP , DC - STAMP siRNA ; n = 7 donors .

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(E) CSF1R, MRC1, TLR2 and FOS relative expression measured by qRT-PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC-STAMP knockdown. si-Ctrl, scrambled siRNA; si-DC-STAMP, DC-STAMP siRNA; n=7 donors.


( E ) The effect of lysosomotropic agents ( hydroxychloroquine , hydroxy ; ammonium chloride , NH4Cl ) and v - ATPase inhibitors ( Bafilomycin A1 , Baf A1 ; Concanamycin A , Con A ) on fusion and multinucleation in LGCs ( upper panel ) , FBGCs ( middle panel ) and osteoclasts ( lower panel ) . To test the effect of lysosome dysfunction on cellular iron , FeCl3 was supplemented . Error bars are median with interquartile range ; significance tested by one - way Anova followed by Sídák ' s multiple comparisons on log transformed data ; n = 4 donors ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 ; * * * * , P < 0 . 0001 .

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(E) The effect of lysosomotropic agents (hydroxychloroquine, hydroxy; ammonium chloride, NH4Cl) and v-ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl3 was supplemented. Error bars are median with interquartile range; significance tested by one-way Anova followed by Sídák's multiple comparisons on log transformed data; n=4 donors; *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001.


( E ) B7 - H3 surface marker expression ( red ) acquired by ImageStream in mononuclear and multinucleated LGCs , FBGCs and osteoclasts stained for Hoechst . Bar graphs ( lower panel ) show normalized B7 - H3 mean fluorescence intensity ( MFI ) , measured by ImageStream ; n = 4 donors . Error bars are mean ± SD ; significance tested by paired t - test ; * * , P < 0 . 01 ; ns , non - significant .

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(E) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) Phagocytosis quantified by ImageStream analysis in LGCs , FBGCs and osteoclasts . Representative images showing the uptake of S . Aureus bioparticles ( yellow ) in mononuclear ( mono ) and multinucleated ( multi ) cells . Bar graphs ( lower panel ) show normalized S . Aureus bioparticles mean fluorescence intensity ( MFI ) measured by ImageStream ; n = 5 donors . Error bars are mean ± SD ; significance tested by paired t - test ; * * , P < 0 . 01 ; ns , non - significant .

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(C) Phagocytosis quantified by ImageStream analysis in LGCs, FBGCs and osteoclasts. Representative images showing the uptake of S. Aureus bioparticles (yellow) in mononuclear (mono) and multinucleated (multi) cells. Bar graphs (lower panel) show normalized S. Aureus bioparticles mean fluorescence intensity (MFI) measured by ImageStream; n=5 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.


( C ) TRAP immunohistochemistry ( upper panel ) and hydroxyapatite resorption ( lower panel ) in LGCs , FBGCs and osteoclasts . Note the presence of TRAP + MGCs in all cell types and hydroxyapatite resorption only in osteoclasts .

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(C) TRAP immunohistochemistry (upper panel) and hydroxyapatite resorption (lower panel) in LGCs, FBGCs and osteoclasts. Note the presence of TRAP+ MGCs in all cell types and hydroxyapatite resorption only in osteoclasts.


( D ) Oxygen consumption rate ( OCR , upper panel ) , its related readout ( middle panel ) and extracellular acidification rate ( ECAR , lower panel ) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts ; n = 4 donors . ( E ) Extracellular acidification rate ( ECAR , left ) and glycolysis ( right ) in mononuclear and multinucleated osteoclasts ; n = 4 donors .

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(D) Oxygen consumption rate (OCR, upper panel), its related readout (middle panel) and extracellular acidification rate (ECAR, lower panel) measured by extracellular flux analysis in sorted mononuclear and multinucleated osteoclasts; n=4 donors. (E) Extracellular acidification rate (ECAR, left) and glycolysis (right) in mononuclear and multinucleated osteoclasts; n=4 donors.


( F ) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts ; n = 3 donors .

(F) Mitochondria copy number measured by qPCR in mononuclear and multinucleated osteoclasts; n=3 donors.


( G ) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number ; n = 6 donors .

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(G) Relative expression of mitochondrial transcripts in sorted mononuclear and multinucleated osteoclasts after normalisation by mitochondrial copy number; n=6 donors.


B Latency to enter dark compartment for Gadd45a - WT ( white bars , n = 11 ) and Gadd45a - KO ( blue bars , n = 11 ) . A significant reduction was found in the Gadd45a - KO at 24 hours and 7 days after the training . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * = p < 0 . 05 .

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B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


C In situ hybridization to detect Gadd45a mRNA in adult wild - type hippocampus ( n = 8 animals ; antisense , left panel ; sense , middle panel ) and in Gadd45a - KO sections ( n = 6 animals ; right panel ) . Scale bars : 500 µm ( black bar ) .

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C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar).


D Experience - dependent changes in mRNA levels were evaluated under control conditions ( home cage ) and exposed to the PA ( context , and context + shock groups ; n = 6 for all groups ) , respectively . Hippocampal Gadd45a mRNA levels remained unchanged , while Gadd45b was induced ( context ; t10 = 2 . 427 , context + shock t10 = 2 . 319 ) . Concomitantly , mRNA levels of activity - regulated cytoskeleton protein ( Arc ) , as a proxy of neuronal activation , were increased in mice exposed to the PA context ( t10 = 3 . 406 ) and context + shock ( t10 = 3 . 141 ) . Values shown are mean ± SEM ; 1 - Way ANOVA and unpaired t - test : * = p < 0 . 05 , * * * = p < 0 . 001 .

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D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.


Gadd45a mRNA level in the hippocampus of Gadd45a - overexpressing mice ( red , n = 7 ) was significantly increased ( t10 = 4 . 189 ) as compared to Gadd45a - WT ( n = 5 ) . Values shown are mean ± SEM ; unpaired t - test : * * = p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Gadd45a mRNA level in the hippocampus of Gadd45a-overexpressing mice (red, n=7) was significantly increased (t10=4.189) as compared to Gadd45a-WT (n=5). Values shown are mean ± SEM; unpaired t-test: ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( Nex - Cre ( - / - ) , n = 5 ) and Gadd45a - overexpressing hippocampi ( Nex - Cre ( + / - ) , n = 5 ) 8 weeks after AAV injection . Note that the Gadd45α - HA signal in Gadd45a - overexpressing mice is predominantly found in the stratum pyramidale ( sp ) but also in the stratum oriens and stratum radiatum ( so and sr , respectively ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (Nex-Cre(-/-), n=5) and Gadd45a-overexpressing hippocampi (Nex-Cre(+/-), n=5) 8 weeks after AAV injection. Note that the Gadd45α-HA signal in Gadd45a-overexpressing mice is predominantly found in the stratum pyramidale (sp) but also in the stratum oriens and stratum radiatum (so and sr, respectively). Scale bar: 250 µm.


D Cellular distribution of Gadd45α was analyzed by HA - western blot on nuclear and cytoplasmic protein fractions of Gadd45a - WT and Gadd45a - overexpressing mice ( n = 2 for both groups ) . HA was found predominantly in the nuclear fraction of Gadd45a - overexpressing hippocampi , with a smaller but recognizable expression also in the cytoplasm .

D Cellular distribution of Gadd45α was analyzed by HA-western blot on nuclear and cytoplasmic protein fractions of Gadd45a-WT and Gadd45a-overexpressing mice (n=2 for both groups). HA was found predominantly in the nuclear fraction of Gadd45a-overexpressing hippocampi, with a smaller but recognizable expression also in the cytoplasm.


E Latency to enter the dark compartment for Gadd45a - WT ( white bars , n = 10 ) and Gadd45a - overexpressing mice ( red bars , n = 12 ) in the PA test . A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a - overexpresion group . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=10) and Gadd45a-overexpressing mice (red bars, n=12) in the PA test. A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a-overexpresion group. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05.


B LTP protocol was applied to 16 slices of Gadd45a - WT mice ( white dots , n = 5 animals ) and 11 slices of Gadd45a - KO mice ( blue dots , n = 5 animals ) , resulting in a significantly ( p < 0 . 05 , Student t - test ) lowered induction of LTP in Gadd45a - KO mice as compared with Gadd45a - WT . Values shown are mean ± SEM . C Gadd45a - overexpressing mice ( red dots , n = 10 slices from 6 animals ) presented a slightly increased LTP as compared with Gadd45a - WT ( white dots , n = 8 slices from 6 animals ) , which became more pronounced during the late phase of the experiment . Values shown are mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B LTP protocol was applied to 16 slices of Gadd45a-WT mice (white dots, n=5 animals) and 11 slices of Gadd45a-KO mice (blue dots, n=5 animals), resulting in a significantly (p<0.05, Student t-test) lowered induction of LTP in Gadd45a-KO mice as compared with Gadd45a-WT. Values shown are mean ± SEM. C Gadd45a-overexpressing mice (red dots, n=10 slices from 6 animals) presented a slightly increased LTP as compared with Gadd45a-WT (white dots, n=8 slices from 6 animals), which became more pronounced during the late phase of the experiment. Values shown are mean ± SEM.


A MA plot of DESeq differential expression analysis with the 1 % false discovery rate ( FDR ) hits in red ( 5 / 402 ; and with > 2 - fold change 1 / 65 , up - / down - regulated respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A MA plot of DESeq differential expression analysis with the 1% false discovery rate (FDR) hits in red (5/402; and with >2-fold change 1/65, up-/down-regulated respectively).


D UCSC browser view of reads - per - million - normalised RNA - seq coverage for Grin2a in Gadd45a - WT ( black ) and Gadd45a - KO ( blue ) representative samples . Gene structure of Grin2a ( colored in purple ) , and the direction of transcription ( red arrow ) . Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4 - 8 .

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D UCSC browser view of reads-per-million-normalised RNA-seq coverage for Grin2a in Gadd45a-WT (black) and Gadd45a-KO (blue) representative samples. Gene structure of Grin2a (colored in purple), and the direction of transcription (red arrow). Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4-8.


E Detailed view of the extended 3 ´ UTR of Grin2a showing a significant 5 ´ < 3 ´ gradient in RNA - seq coverage . Note that the normalised read coverage is similar between genotypes at the actual 3 ´ UTR end but differs significantly upstream including the annotated exons . For subsequent experiments , position of the designed primers covering proximal and distal parts of the 3 ´ UTR are indicated below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Detailed view of the extended 3´UTR of Grin2a showing a significant 5´<3´ gradient in RNA-seq coverage. Note that the normalised read coverage is similar between genotypes at the actual 3´UTR end but differs significantly upstream including the annotated exons. For subsequent experiments, position of the designed primers covering proximal and distal parts of the 3´UTR are indicated below.


F , G Validation of Gadd45α - regulated levels of Grin2a mRNA by qPCR with primers covering exons 3 - 4 . Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions ( white bars ; Gadd45a - WT = 7 and Gadd45a - KO = 7 ) and 1 hour after PA ( Gadd45a - WT = 5 , black bars ; Gadd45a - KO = 6 , blue bars ) . Random primed cDNA levels ( F ) , were very similar in all the experimental groups . Oligo ( dT ) - primed cDNA ( G ) revealed a significant increase of Grin2a 1h after PA , which was only observed in Gadd45a - WT mice . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * * = p < 0 . 01 , * * * = p < 0 . 001 . H , I mRNA levels of proximal vs . distal parts of the 3 ´ UTR analyzed in the same set of oligo ( dT ) - primed cDNA . ( H ) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a - WT . ( I ) Distal parts of the 3 ´ UTR remained unchanged ( n = 6 for all groups ) . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G Validation of Gadd45α-regulated levels of Grin2a mRNA by qPCR with primers covering exons 3-4. Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT=7 and Gadd45a-KO=7) and 1 hour after PA (Gadd45a-WT=5, black bars; Gadd45a-KO=6, blue bars). Random primed cDNA levels (F), were very similar in all the experimental groups. Oligo(dT)-primed cDNA (G) revealed a significant increase of Grin2a 1h after PA, which was only observed in Gadd45a-WT mice. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01, *** = p<0.001. H, I mRNA levels of proximal vs. distal parts of the 3´UTR analyzed in the same set of oligo(dT)-primed cDNA. (H) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a-WT. (I) Distal parts of the 3´UTR remained unchanged (n=6 for all groups). Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


A , B NR2A protein levels were analyzed in the hippocampus of mice under control conditions ( white bars ; Gadd45a - WT , n = 13 and Gadd45a - KO , n = 14 ) and 1 hour after PA ( black bars ; Gadd45a - WT = 14 , Gadd45a - KO = 14 ) . NR2A signal ( 160 kDa ) was normalized to the loading control ( tubulin ( 55 kDa ) for the total fraction , and synaptotagmin ( 68 kDa ) for the synaptosomal fraction ) and calculated as percentage of control . In Gadd45a - WT samples ( A ) , NR2A protein levels were unchanged in the total fraction during memory formation , while in the synaptosomal fraction , a significant increase was detected ( t20 = 3 . 199 ) . In Gadd45a - KO hippocampi ( B ) , levels of NR2A protein in both fractions remained unaltered during memory consolidation . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * * = p < 0 . 01 .

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A, B NR2A protein levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT, n=13 and Gadd45a-KO, n=14) and 1 hour after PA (black bars; Gadd45a-WT=14, Gadd45a-KO=14). NR2A signal (160 kDa) was normalized to the loading control (tubulin (55 kDa) for the total fraction, and synaptotagmin (68 kDa) for the synaptosomal fraction) and calculated as percentage of control. In Gadd45a-WT samples (A), NR2A protein levels were unchanged in the total fraction during memory formation, while in the synaptosomal fraction, a significant increase was detected (t20=3.199). In Gadd45a-KO hippocampi (B), levels of NR2A protein in both fractions remained unaltered during memory consolidation. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01.


C Gadd45α pulldown experiments were carried out in wild - type hippocampal lysates ( n = 12 for all groups ) . Percentage of input recovery was unchanged between GFP - FLAG ( green bars ) and Gadd45α - FLAG ( black bars ) for genes that are not regulated by Gadd45α , such as succinate dehydrogenase complex flavoprotein subunit A ( Sdha , no 3 ´ UTR extension ) , and mitogen - activated protein kinase 6 ( Map2K6 , containing 3 ´ UTR extension of 10 kb ) . On the contrary , Gadd45α - FLAG bound fraction revealed a significant increase in Grin2a ( t22 = 4 . 992 ) and Grm5 mRNA ( t22 = 4 . 939 ) as compared to GFP - FLAG fraction . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * * * = p < 0 . 001 .

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C Gadd45α pulldown experiments were carried out in wild-type hippocampal lysates (n=12 for all groups). Percentage of input recovery was unchanged between GFP-FLAG (green bars) and Gadd45α-FLAG (black bars) for genes that are not regulated by Gadd45α, such as succinate dehydrogenase complex flavoprotein subunit A (Sdha, no 3´UTR extension), and mitogen-activated protein kinase 6 (Map2K6, containing 3´UTR extension of 10 kb). On the contrary, Gadd45α-FLAG bound fraction revealed a significant increase in Grin2a (t22=4.992) and Grm5 mRNA (t22=4.939) as compared to GFP-FLAG fraction. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: *** = p<0.001.


B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a - overexpression ( q24 = 4 . 094 ) and Gadd45aK45E - overexpression groups ( q24 = 4 . 726 ) ( n = 9 for all groups ) . Values shown are mean ± SEM ; Tukey ´ s multiple comparison test : * = p < 0 . 05 , * * = p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a-overexpression (q24 = 4.094) and Gadd45aK45E-overexpression groups (q24 = 4.726) (n=9 for all groups). Values shown are mean ± SEM; Tukey´s multiple comparison test: * = p<0.05, ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( left ) , Gadd45a - overexpression ( middle ) and Gadd45aK45E - overexpression ( right ) samples 8 weeks after AAV injection ( n = 3 for all groups ) . Upper row depicts the entire hippocampus , while center and lower rows show magnifications of the CA3 area in which the HA signal ( red ) is not restricted to the nuclei ( blue and white ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (left), Gadd45a-overexpression (middle) and Gadd45aK45E-overexpression (right) samples 8 weeks after AAV injection (n=3 for all groups). Upper row depicts the entire hippocampus, while center and lower rows show magnifications of the CA3 area in which the HA signal (red) is not restricted to the nuclei (blue and white). Scale bar: 250 µm.


D Gadd45α - overexpression was confirmed at the protein level by HA - western blot on hippocampal samples from Gadd45a - WT , Gadd45a - overexpression and Gadd45aK45E - overexpression mice ( n = 3 for all groups ) . Hippocampus scale bars : 250 µm ; CA3 scale bars : 50µm .

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D Gadd45α-overexpression was confirmed at the protein level by HA-western blot on hippocampal samples from Gadd45a-WT, Gadd45a-overexpression and Gadd45aK45E-overexpression mice (n=3 for all groups). Hippocampus scale bars: 250 µm; CA3 scale bars: 50µm.


E Latency to enter the dark compartment for Gadd45a - WT ( white bars , n = 12 ) , Gadd45a - overexpression ( red bars , n = 11 ) and Gadd45aK45E - overexpression mice ( grey bars , n = 11 ) in the PA test . Note that , unlike Gadd45a - overexpression mice , those expressing the mutated form of Gadd45α ( no RNA - binding capacity ) did not show an increase in memory retention . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * = p < 0 . 05 , * * * = p < 0 . 001 .

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E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=12), Gadd45a-overexpression (red bars, n=11) and Gadd45aK45E-overexpression mice (grey bars, n=11) in the PA test. Note that, unlike Gadd45a-overexpression mice, those expressing the mutated form of Gadd45α (no RNA-binding capacity) did not show an increase in memory retention. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05, *** = p<0.001.


( A ) Electron micrographs of MEFs . Scale bar ; 1 μm . High - magnification images depicting representative electron - dense materials are shown . Scale bar ; 0 . 2 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Electron micrographs of MEFs . Scale bar; 1 μm. High-magnification images depicting representative electron-dense materials are shown. Scale bar; 0.2 μm.


( B ) Citrate synthase activity ( absorbance / min ) was measured in MEFs . Error bars represent ± SD from eight independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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(B) Citrate synthase activity (absorbance/min) was measured in MEFs . Error bars represent ±SD from eight independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( C , D ) Oxygen consumption rates ( OCRs ) of MEFs were measured on an XF24 Analyzer . ( C ) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin ( 2 μg / mL ) ( Oligo ) , FCCP ( 2 μM ) , and rotenone ( 1 μM ) plus actinomycin ( 2 μM ) ( R & A ) . ( D ) OCR ( pmol O2 / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( E ) ATP production ( pmol / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( F ) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype . MEFs were treated with tunicamycin ( 5 μg / ml ) or vehicle control in the presence or absence of bafilomycin A1 ( 400 nM ) , and endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin in the absence of bafilomycin , which were normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( G ) Survival rate of MEFs treated with tunicamycin ( 5 μg / ml ) , thapsigargin ( 1 μM ) , or hydrogen peroxide ( 100 μM ) . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( A ) Absolute [ Ca2 + ] m changes in MEFs of indicated genotype in response to bradykinin ( 2 . 5 μM ) . ( B ) Basal and peak values of Ca2 + transients ( μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). Error bars represent ±SD from six independent experiments. Data information: For graph P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( C ) Peak values of Ca2 + transients in MEFs transfected with IP3R or shRNA against IP3R , or treated with 2 - AP ( 20 mM ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( D ) Peak values of Ca2 + transients in MEFs transfected with VDAC1 or shRNA against VDAC1 . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( E ) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm ) . Scale bar : 100 nm . Data represent percentage of ERmitochondrial contacts per mitochondria perimeter . Error bars represent ± SD from 40 images MEFs Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(E) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm). Scale bar: 100 nm. Data represent percentage of ERmitochondrial contacts per mitochondria perimeter. Error bars represent ±SD from 40 images MEFs Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( F ) In situ PLA images using anti - IP3R and anti - VDAC1 antibodies . Scale bar : 20 μm . Data represent fluorescent in MEFs normalized LRRK2 + / + MEFs . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(F) In situ PLA images using anti-IP3R and anti-VDAC1 antibodies. Scale bar: 20 μm. Data represent fluorescent in MEFs normalized LRRK2+/+ MEFs. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( G ) Peak values of Ca2 + transients in MEFs transfected with synthetic tethering protein ( TOM - mRFP - ER ) to induce artificial tethering of the ER and mitochondria . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(G) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Representative immunoblots of MAM components in MEFs of indicated genotype . MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right .

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(A) Representative immunoblots of MAM components in MEFs of indicated genotype. MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right.


( C ) Peak values of Ca2 + transients in MEFs transfected with deletion constructs of LRRK2 ( G2019S ) . Error bars represent ± SD from six independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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(C) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( D ) Binding assays to detect interactions between deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases . HEK293 cells were transfected with deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases , and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the V5 or HA epitope , as indicated the right of each panel .

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(D) Binding assays to detect interactions between deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases. HEK293 cells were transfected with deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases, and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the V5 or HA epitope, as indicated the right of each panel.


Peak values of Ca2 + transients in MEFs transfected with ligase - active MARCH5 ( WT ) , MULAN ( WT ) , and Parkin ( W403A ) [ Parkin ( WA ) ] or dominant - negative forms of MARCH5 ( H43W ) [ MARCH ( HW ) ] , MULAN ( C339A ) [ MULAN ( CA ) ] , Parkin ( C431A ) [ Parkin ( CA ) ] ( E ) Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


Peak values of Ca2 + transients in MEFs transfected with active USP30 or inactive USP30 ( C77S ) [ USP ( CS ) ] ( F ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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Peak values of Ca2+ transients in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( A ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2 + / + and LRRK2 ( G2019S ) - expressing MEFs treated with vehicle ( Control ) or tunicamycin ( 1 μg / ml ) . Endogenous PERK , MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Data represent the ratio of phosphorylated to total protein levels . Error bars represent ± SD from independent experiments . Data information : For graph A the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) Immunoprecipitation / immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin ( 5 μg / ml ) . LRRK2 ( G2019S ) MEFs were also pretreated with LRRK2 - IN - 1 ( 1 μM ) . MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the Myc epitope , phosphoserine or mitofin2 as indicated to the right of each panel . Mfn2 : mitofusin 2 , Ub : ubiquitin . Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin . Error bars represent ± SD from four independent experiments . Data information : For graph B , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Peak values of Ca2 + transients in MEFs treated with LRRK2 - IN - 1 ( 1 μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) ( Upper panel ) Diagram showing full - length PERK Myc at the N - terminus and FLAG at the C - terminus ( M - PERK - F ) . S1P recognition sequence R33SLL is mutated to A33SLL ( M - PERK ( R33A ) - F ) . ( Lower panel ) Immunoblot of PERK and PERK ( R33A ) from transfected MEFs under tunicamycin . MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method . Lysates were immunoprecipitated with antibody against FLAG . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody as indicated to the right .

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(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.


( C ) In vitro kinase assay using isolated PERK , isolated E3 ubiquitin ligase and [ γ - 32P ] ATP . Reaction mixture was subjected to SDS / PAGE . Blots were with antibody against Myc , FLAG or phosphoserine . [ γ - 32P ] ATP - incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ) .

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(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).


( D ) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases , HA - tagged Ubl , and His - tagged mitofusin2 in the presence of E1 enzyme , UbcH7 and ATP . E3 ubiquitin ligases were initially phosphorylated PERK or PERK ( K618R ) in vitro . Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination . itofusin 2 with Ni - NTA subjected to SDS / PAGE . Blots were with antibody against HA or mitofusin2 . Mfn2 : mitofusin 2 , Ub : ubiquitin .

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(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.


( E ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2 ( G2019S ) - expressing MEFs transfected with the increasing amounts of LRRK2 - d1 - V5 ( 1 , 5 , 25 μg / 106 cells ) treated with tunicamycin ( 1 μg / ml ) . Endogenous MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Endogenous mitofusin 2 was immunoblotted with anti - mitofusin 2 antibody . Data represent the ratio of phosphorylated to total protein levels of MARCH5 , MULAN and Parkin , and the ratio of mitofusin 2 to actin . Error bars represent ± SD from independent experiments . Data information : For graph E , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2 . HEK293 cells were transfected with PERK and either MARCH5 , MULAN or Parkin , LRRK2 , LRRK2 ( D1994A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc or V5 , and precipitates were immunoblotted with antibody indicated the right .

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(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.


( B ) In vitro kinase assay for auto - phosphorylated LRRK2 using isolated LRRK2 mutants and [ γ - 32P ] ATP with or without LRRK2 - IN - 1 ( 1 μM ) . Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS / PAGE . Blots were with antibody against V5 or phosphoserine . [ γ - 32P ] ATP - incorporated LRRK2 visualized by autoradiography .

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(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.


( C ) Binding assays to detect interactions between phosphomimetic LRRK2 ( S1292D ) or phosphorylation - defective LRRK2 ( S1292A ) and E3 ubiquitin ligases . HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope . Precipitated proteins were subjected to SDS / PAGE , for immunoblotwith antibody against V5 or Myc .

(C) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.


( D ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK - ΔN in the presence of LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . HEK293 cells were transfected with MARCH5 , MULAN , Parkin , and PERK - ΔN LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc , and precipitates were immunoblotted with antibody indicated the right .

(D) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.


( A ) Immunoblot of LC3 and p62 . MEFs were transfected with PERK - ΔN or PERK shRNA . Endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 .

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(A) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.


( B ) Peak values of Ca2 + transients in MEFs transfected with PERK - ΔN or PERK shRNA . Error bars represent ± SD from six independent experiments .

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(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.


D ) In the 6 months old p32cKO heart , p62 and multi - ubiquitin staining patterns were co - localized . Scale bar , 5 µm . E ) In the 6 months old p32cKO heart , the staining patterns of p62 and the mitochondrial marker protein , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm .

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D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.


( F ) Western blot analysis of ubiquitinated proteins using an anti - multi - ubiquitin antibody in the 6 months old WT and p32cKO heart ( n = 4 mice per group ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

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(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( B ) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart . Tissues were excited at a wavelength of 540 ( upper panel ) or 470 ( lower panel ) and emission spectra were collected with a confocal microscope at wavelengths ( band path ) of 580 - 630 nm ( upper panel ) or 510 - 560 nm ( lower panel ) . Enlarged view of the white squares is shown right . Scale bars , 20 µm . The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM ( three months old mice , n = 11 ; 6 months old mice , n = 9 - 14 , 9 months old mice , n = 9 - 15 , 12 months old mice , n = 11 - 21 ) . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 005 .

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(B) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.


( D ) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts . Quantification is shown in the lower panel ( n = 4 mice per group ) . GAPDH was used as an internal control . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

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(D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( A ) LC - MS / MS metabolomic analysis of NAD + , NADH , NADP + , nicotinamide and NAAD in the 6 months old WT and p32cKO hearts . NAD + and NADP + showed significantly different levels between WT and p32cKO hearts ( n = 12 ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * p < 0 . 001 , * * * p < 0 . 0002 .

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(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.


( B ) Real - time PCR analysis of RNA expression of NAD - synthesizing enzymes in the 6 months old WT and p32cKO hearts . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT mice vs p32cKO mice ( n = 6 ) , * * * p < 0 . 005 , * p < 0 . 05 . The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows . NAM : nicotinamide , NA : nicotinic acid , QA : quinolic acid , NMN : nicotinamide mononucleotide .

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(B) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.


( G ) Nmnat3 mRNA expression in 3T3 - L1 cells after 1 mM CAP treatment for 72 h . The HIF1α inhibitor ( 20 or 50 µM ) was added 2 h before the CAP treatment . Error bars are presented as mean ± SD of three independent experiments . Student ' s t - test was performed on WT cells vs WT cells treated CAP and ( or ) HIF1α inhibitor , * * p < 0 . 01 , * p < 0 . 05 .

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(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.


( I ) Nmnat3 mRNA expression in 3T3 - L1 cells after 150 µM CoCl2 treatment for 72 h ( n = 3 ) . Error bars are presented as mean ± SD of three independent experiments . Statistical significance was assessed by Student ' s t - test , * * p < 0 . 01 .

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(I) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.


( A ) Lysosomal acidification is impaired in p32KO MEFs . Representative images of WT and p32KO MEFs , stained with dextran - Oregon Green ( 488 ) and dextran - TMRM . Scale bar , 5 µm . Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification , and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification . Dextran - Oregon Green is quenched under acidic pH , and thus an increased green : red ratio denotes impaired lysosomal acidification . The average ± SEM of the green : red ratio in at least 30 cells in two independent experiments is presented in the plot ( normalized to WT green / red = 1 ) . Statistical significance was assessed by Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 .

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(A) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.


( E ) Nmnat3 ( v1 ) overexpression in p32KO MEFs rescued lysosomal function . LysoTracker Red - stained p32KO MEFs were quantified by flow cytometry . Error bars are presented as mean ± SEM of 4 independent experiments . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 001 .

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(E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.


( A ) WT MEFs were stained with dextran - Oregon Green and dextran - TMRM to examine lysosomal acidification after 10 nM FK866 treatment . FK866 decreased lysosomal acidification , which was rescued by 1 mM NMN pretreatment . The stained MEFs were quantified using a microscope ( BZ - X800 , KEYENCE ) . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

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(A) WT MEFs were stained with dextran-Oregon Green and dextran-TMRM to examine lysosomal acidification after 10 nM FK866 treatment. FK866 decreased lysosomal acidification, which was rescued by 1 mM NMN pretreatment. The stained MEFs were quantified using a microscope (BZ-X800, KEYENCE). Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( F ) To examine autophagosome and autolysosome formation , we used DAPRed , which indicates autophagosomes , and DALGreen , which indicates autolysosomes . After starvation , co - localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells . Scale bars , 10 µm . Quantification is shown on the right . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

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(F) To examine autophagosome and autolysosome formation, we used DAPRed, which indicates autophagosomes, and DALGreen, which indicates autolysosomes. After starvation, co-localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells. Scale bars, 10 µm. Quantification is shown on the right. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( D ) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs , the membrane pellet ( bound ) and supernatant ( free ) were subjected to immunoblotting with GAPDH and PGK1 antibodies .

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(D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies.


( I ) After addition of GAP , NAD and Pi , ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin / luciferase kit . Iodoacetate ( IA ; 4 µM ) was used to inhibit GAPDH activity . Three independent experiments were performed . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * p < 0 . 002 .

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(I) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002.


B Image sequence from confocal time lapse shows the early steps in neuronal differentiation . Two transient basal protrusions are extended along the basal surface of the neural tube ( 6h 26 to 14h 01 ) and then retracted ( 15h 31 to 17h 31 ) . The apical attachment is also retracted ( 17h 31 to 19h 53 ; e . g . - 1h 30 , ) before the axon is extended ( 20h 38 to 21h 50 ) . Main images are maximum projections and insets are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

B Image sequence from confocal time lapse shows the early steps in neuronal differentiation. Two transient basal protrusions are extended along the basal surface of the neural tube (6h 26 to 14h 01) and then retracted (15h 31 to 17h 31). The apical attachment is also retracted (17h 31 to 19h 53; e.g. -1h 30,) before the axon is extended (20h 38 to 21h 50). Main images are maximum projections and insets are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


C Image sequence from confocal time lapse shows a neuron before , during and after axon initiation . Prior to axon initiation , the neuron extends multiple small , transient pre - axonal protrusions ( - 2h to - 20m ) . The nascent axon is extended ( 0h ) and maintained for a short period ( 0h to 30m ) before axon growth begins ( 45m to 2h 20 ) . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

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C Image sequence from confocal time lapse shows a neuron before, during and after axon initiation. Prior to axon initiation, the neuron extends multiple small, transient pre-axonal protrusions (-2h to -20m). The nascent axon is extended (0h) and maintained for a short period (0h to 30m) before axon growth begins (45m to 2h 20). Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


D Plots showing axon position on the soma ( e . g . asterisk in Figure 1C 0h ) relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 86 cells from 8 experiments ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 95 . 3o ; transverse view P < 0 . 001 , mean = 148 . 9o ) . Data analysed using Moore ' s modification of the Rayleigh ' s test .

D Plots showing axon position on the soma (e.g. asterisk in Figure 1C 0h) relative to the cell centroid at 0,0 for dorsal and transverse view (n = 86 cells from 8 experiments). Axon position is not random (dorsal view P < 0.001, mean = 95.3o; transverse view P < 0.001, mean = 148.9o). Data analysed using Moore's modification of the Rayleigh's test.


F Lateral and transverse reconstructions of DoLA neuron at the time of axon initiation ( 0h ) and during axon growth ( 4h ) . Data information : All scale bars = 10 µm .

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F Lateral and transverse reconstructions of DoLA neuron at the time of axon initiation (0h) and during axon growth (4h). Data information: All scale bars = 10 µm.


G - M Plots showing axon position on the soma relative to cell centroid at 0 , 0 in dorsal and transverse view for DoLA ( G ; n = 7 cells ) , CiA ( H ; n = 10 cells ) , CiD ( I ; n = 3 cells ) , VeLD ( J ; n = 5 cells ) , CoSA ( K ; n = 11 cells ) , CoB ( L ; n = 6 cells ) and UCoD ( M ; 15 cells ) neuronal subtypes .

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G-M Plots showing axon position on the soma relative to cell centroid at 0,0 in dorsal and transverse view for DoLA (G; n = 7 cells), CiA (H; n = 10 cells), CiD (I; n = 3 cells), VeLD (J; n = 5 cells), CoSA (K; n = 11 cells), CoB (L; n = 6 cells) and UCoD (M; 15 cells) neuronal subtypes.


A Image sequence showing centrosome position of a neuron labelled with membrane and centrosome labels during apical process retraction . The centrosome is retracted close to the tip of the apical process . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

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A Image sequence showing centrosome position of a neuron labelled with membrane and centrosome labels during apical process retraction. The centrosome is retracted close to the tip of the apical process. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


B ( i ) Image sequence showing a neuron labelled with a membrane marker during apical retraction without any observable abscission event ( n = 64 / 72 cells from 6 experiments ) . ( ii ) High resolution images of boxed section in ( i ) . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

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B (i) Image sequence showing a neuron labelled with a membrane marker during apical retraction without any observable abscission event (n = 64/72 cells from 6 experiments). (ii) High resolution images of boxed section in (i). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


C ( i ) Low magnification overview of the spinal cord of a cilium reporter line , Tg ( actb2 : arl13b - GFP ) from a single confocal slice . ( ii ) High resolution image sequence of boxed section in ( i ) . One GFP - labelled cilium moves from apical surface towards the basal surface of the spinal cord ( n = 13 cilia from two experiments ) . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

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C (i) Low magnification overview of the spinal cord of a cilium reporter line, Tg(actb2:arl13b-GFP) from a single confocal slice. (ii) High resolution image sequence of boxed section in (i). One GFP-labelled cilium moves from apical surface towards the basal surface of the spinal cord (n = 13 cilia from two experiments). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


D ( i ) Low magnification overview of the spinal cord of a Tg ( actb2 : arl13b - GFP ) embryo labelled with centrin - RFP from a single confocal slice . ( ii ) Image sequence of boxed section in ( i ) . A cilium and centrosome move together from apical surface towards the basal surface of the spinal cord ( n = 2 cells from one experiment ) . Insets show high magnification of cilium - centrosome pair . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

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D (i) Low magnification overview of the spinal cord of a Tg(actb2:arl13b-GFP) embryo labelled with centrin-RFP from a single confocal slice. (ii) Image sequence of boxed section in (i). A cilium and centrosome move together from apical surface towards the basal surface of the spinal cord (n = 2 cells from one experiment). Insets show high magnification of cilium-centrosome pair. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


E Graph showing length of cilium as it moves from apical surface ( 0 % ) to close to basal surface ( 100 % . Cilium length did not change ( P > 0 . 05 for n = 6 / 7 cilia from two experiments ; non - linear regression ) .

E Graph showing length of cilium as it moves from apical surface (0%) to close to basal surface (100%. Cilium length did not change (P > 0.05 for n = 6/7 cilia from two experiments; non-linear regression).