Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Transfection, Isolation, Fractionation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Derivative Assay, Membrane, BrdU Incorporation Assay, Synthesized

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques:

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition, Disruption, Control, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition, Cell Culture

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Isolation, Fractionation, SDS Page, Control, Pulse Chase, Transfection

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

doi: 10.64898/2026.03.19.712868

Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

Techniques: Control, Infection, Comparison

Journal: bioRxiv

Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

doi: 10.64898/2026.03.19.712868

Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

Techniques: Control, Infection, Comparison

Journal: Frontiers in Systems Neuroscience

Article Title: Is there a correlation between functional recovery of manual dexterity after motor cortex lesion and initial motor learning slope in the intact state?

doi: 10.3389/fnsys.2026.1754760

Figure Lengend Snippet: Two hypotheses to be tested. (A) Cartoon illustrating the typical time course of manual dexterity score for an adult “untreated” macaque monkey (“monkey a”), based on the modified Brinkman Board task executed with the dominant hand, including the initial motor learning phase, with a steep learning curve slope (“Ln. Sl. a”). The end of the learning phase is characterized by a plateau. Then, a unilateral experimental M1 lesion contralateral to the tested hand took place (purple vertical line), which provoked a total loss of manual dexterity during a few weeks. It was followed by a spontaneous, progressive functional recovery, until reaching a single plateau of recovery. The dashed line represents the corresponding recovery curve slope (“Rc. Sl. a”). In absence of treatment, the plateau remained stable for months to years. The vertical arrow represents the recovered post-lesion score. (B) Same as in panel (A) , but for a gentle learning curve slope represented by an untreated “monkey b.” Note that both slopes ( learning curve slope and recovery curve slope ) are clearly less steep than for “monkey a.” Seven monkeys included in the present study corresponded to such a time course, as depicted in panels (A,B) , with various learning and recovery curve slopes . The hypothesis 1 tested in the present report argues that the initial motor learning slope is correlated with the slope of functional recovery from a M1 lesion, possibly following the relationship between learning curve slope and recovery curve slope as depicted in panel (D) . As a consequence, the hypothesis 2 argues that in case of “steep initial motor learning” a higher recovered score of manual dexterity after M1 lesion will be reached than in case of “gentle initial motor learning.” (C) Same as in panels (A,B) , but for a M1 lesioned monkey subjected to a treatment (either anti-Nogo-A antibody or ANCE autologous cellular therapy; n = 6 monkeys). The M1 lesion is indicated by the vertical purple line, followed by the treatment onset (red arrow pointing down). Following the total loss of manual dexterity, a first plateau of functional recovery took place, reflecting an initial spontaneous recovery. This first plateau of recovery was used to derive six additional data points to the seven data points illustrated in panels (A,B) . Overall, 13 data points were representative of the spontaneous recovery post-M1 lesion [ ; see also for more detail]. As a result of treatment, these six monkeys exhibited a second plateau of functional recovery, representing the effect of the treatment (red curve). As for the first plateau, a recovery curve slope (“Rc. Sl.”) and a recovery score (red arrow pointing up) can be derived specifically for the second plateau , corresponding overall to six “treatment” data points. (D) Expected correlation between the recovery curve slope and the initial learning curve slope , for both plateau 1 and plateau 2 (see above).

Article Snippet: The author declares that the anti-Nogo-A antibody used in this study was provided by Novartis AG.

Techniques: Modification, Functional Assay, Gentle, Derivative Assay