Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Dusp26 is targeted to the OMM via its NH2-terminal amino acid residues. A HEK293 cells were transiently transfected using control vector alone (lanes 1 and 5) or plasmids encoding Dusp26-Flag and Myc-Pink1 (lanes 2–4 and 6–8). Cytoplasm and mitochondrial fractions were analyzed by immunoblotting using antibody to Dusp26-Flag or Pink1. Hsp60 and β-actin were used to indicate mitochondria and cytoplasmic purity. B Cytoplasmic and mitochondrial fractions were isolated and immunoblotting analysis was performed to detect endogenous Dusp26 in HEK293 and SH-SY5Y cell lines. UQCRE2 and β-actin show purity of mitochondria and cytoplasmic fractions, respectively. Lanes 1–2, indicate cytoplasmic and lanes 3–4 indicate mitochondrial fractions. C, D Dusp26 domain structure and gene fragments that were fused to EGFP (C). HEK293 cells were transiently transfected using Mito-ds-Red and Dusp26 (aa1–34)-EGFP, Dusp26 (aa29–211)-EGFP or Dusp26 (1–211)-EGFP constructs. Cells were cultured on a coverslip, fixed and examined by fluorescent microscope. Scale bar 5 µm. E HEK293 cells transiently expressing Dusp26-Flag were untreated, or treated with proteinase K for 20 min (see “Materials and methods”) [32]. Following treatment, mitochondrial fractions were analyzed by immunoblotting using antibodies to the indicated proteins. F HEK293 cells were transiently transfected using Dusp26-Flag plasmids. Immunofluorescent analyses were performed with cells that were cultured on coverslips and were either untreated, treated with 0.01% digitonin or 1% triton. Cells were then stained with antibody to the indicated proteins. Scale bar 2 µm. All experiments were performed three times and results were consistent

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Isolation, Construct, Cell Culture, Microscopy, Expressing, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: SH-SY5Y cells exhibit mitochondrial impairment following loss of Dusp26. A Dusp26 expression levels in parental and two Dusp26 deficient SH-SY5Y clones (#1 and #2) were determined by immunoblotting. The Dusp26-deficient cell lines were generated by targeting the exon 2 region of Dusp26 DNA using CRISPR/CAS9 system. β-actin is loading control. B, C Phase contrast images of parental and Dusp26-deficient SH-SY5Y cell lines #1 and #2. Loss of Dusp26 causes apparent cell morphological alteration. Quantification of the cells with neurites (C). 10–15 microscopic fields from independent cultures for each cell line were analyzed for the presence of neurites. D, E Quantification of cell proliferation and cell death. 105 parental and Dusp26-deficient cells (#2) were plated and cell number was determined at the indicated times (D). Cell death was determined by trypan blue exclusion in live cells (E). F Cell cycle analysis performed by propidium iodide staining followed by FACS analyses. The representative cell cycle histograms (left panel) and quantification of the data are presented (right panel). n = 3/group. The experiments were repeated twice independently. G Oxygen consumption rate (OCR) of the parental and Dusp26-deficient SH-SY5Y cells was determined using XF96 Seahorse analyzer. The basal, ATP-linked and uncoupled respiration-linked OCR are presented. H Extracellular acidification rate (ECAR) of parental and Dusp26-deficient SH-SY5Y cells was determined using XF96 Seahorse analyzer. I The level of cellular ROS (left), mitochondrial ROS (middle), and ΔΨm (right) in parental and Dusp26-deficient SH-SY5Y cells were determined using DHE, mitosox and TMRE followed by FACS analyses. MFI (mean fluorescence intensity). J ROS in control and FCCP-treated parental and Dusp26-deficient cells was measured as in I. K, L The parental and Dusp26-deficient SH-SY5Y cells were treated with MitoQ to reduce ROS. The mitochondrial ROS and ΔΨm were measured as in I. All experiments were performed two times and in triplicates. In all relevant panels, red bars are the parental line and blue bars are Dusp26-KO clone #2 or as indicated in the panels and bars indicate mean ± SEM, p values are indicated in the panels

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Expressing, Clone Assay, Western Blot, Generated, CRISPR, Control, Cell Cycle Assay, Staining, Fluorescence

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Dusp26 loss leads to MAPK activation in different brain regions including the TH-positive DA neurons. A, B Immunoblotting analyses of isolated cortex, STR and MB to detect the indicated proteins in 4-month-old WT and dusp26−/− mice. β-actin is loading control. n = 4 mice/group. Quantification of the immunoblots is indicated (B). C, D Immunoblotting analyses of isolated cortex, STR and MB to detect the indicated proteins in 4-month-old WT and dusp26−/− mice. α-tubulin is loading control. n = 4 mice/group. Quantification of the p-ERK in cortex and STR is indicated (D). Increases in p-JNK1/2 in above brain regions were not significant. E RT-qPCR analyses to detect the expression of different Dusps in MB of WT and dusp26−/− mice. 2–3 months of age. n = 5 mice/group. F Immunofluorescent staining of SNpc region from WT and dusp26−/− mice showing coexpression of TH and p-p38 (arrows). Bars = 50 µm left, and 5 µm right panels. Quantification of TH and p-p38 -positive cells is presented in the right panel. n = 5 mice/group. G, H Cortical/STR neuronal cultures from WT and dusp26−/− mice were exposed to 6-OHDA (100 µM) for the indicated hours. Cell lysates were analyzed by immunoblotting using the indicated antibodies. β-Actin is loading control. Quantification of a representative blot is indicated in the right panel. Cells in H were treated as in G, and number of dead cells were quantified using dye exclusion. In all relevant panels, bars are mean ± SEM. p value is indicated in each panel

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Activation Assay, Western Blot, Isolation, Control, Quantitative RT-PCR, Expressing, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Generation of mice with the targeted deletion of the dusp26 gene. A Genomic locus, targeting vector and targeted locus of the dusp26 gene. p1, p2, p3 and p4 represent the location of the primers used to detect WT and mutant Dusp26 genes. Positions of the probe that was used to identify targeted ES clones are indicated. LacZ is the β-gal gene. Neo represents neomycin, and TK represents the thymidine kinase gene, which were used for positive and negative selection, respectively. Black boxes represent exons. B Southern blot and PCR analyses of tail DNA that were used to confirm the genotype of Dusp26 WT (+/+), heterozygous (+/−) and homozygous (−/−) mutant mice. kb = kilobase pairs; bp = base pairs. C IHC staining of the cortical brain region of mice showing co-expression Dusp26 and NeuN. Nuclei were stained with DAPI. Scale bar 10 µm. D WT and dusp26−/− primary neuronal cell cultures were stained for the expression of β-gal (arrow shows positive β-gal staining). E Coronal brain tissue sections of the dusp26+/β-gal mice stained to detect the β-gal expression. Dusp26 expression is apparent in the COR cortex, CPU caudate putamen, AC anterior commissure, ZI zona incerta, AMG amygdala, HT hypothalamus, SuC superior colliculus, AH anterior horn of the spinal cord, RET retina, ON optic nerve. *midbrain, Scale bar 500 µm. All experiments were performed three times or with 5 mice/group, which included both males and females

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Selection, Southern Blot, Immunohistochemistry, Expressing, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Loss of Dusp26 leads to death of DA neurons in SNpc. A, B Coronal sections of MB of 2- or 16-month-old mice (2 m and 16 m) immunostained with TH-specific antibody and developed using Vectastain (brown). Arrow indicates general area of loss of TH-positive cells. Scale bar 10 µm. Quantification of TH/Nissl-positive neurons in SNpc using stereology (B). n = 5 mice per group. C Immunofluorescent staining of SNpc showing colocalization of TH and Dusp26 in both genotypes. Merged image shows colocalization of TH and Dusp26 in WT sample. DAPI is used to stain the nuclei. n = 5 mice. Scale bar 10 µm. D Immunoblotting analyses of MB tissue lysates using antibody to TH. Quantification of the data is presented in the right panels. β-actin is used as loading control. 4–5 mice/group. E Coronal sections of the dorsal STR immunostained to detect TH and developed using fast-red. Bar 10 µm. Quantification of the intensities of TH staining is presented in the right panel. n = 5 mice. F Muscle strength (measured by grasping test) of mice was analyzed at 4- and 20-months-of-age. n = 9 mice for WT and 14 mice for dusp26−/−. G Physical activity of WT and dusp26−/− mice at 20-months-of-age was analyzed in Oxymax/Clams monitored by infrared beam breaks. Total activity (X-TOTAL, walking, grooming), ambulatory activity (X-AMB, walking) and z-axis activity (Z-TOTAL, rearing) are presented. Data presented as light, dark and 24 h periods. n = 9 mice/group. In all relevant panels, bars indicate mean ± SEM, p values are indicated in the panels. Red bars represent WT and blue bars represent dusp26−/− mice

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Staining, Western Blot, Control, Activity Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Dusp26 interacts with p38, LRRK2, Miro and Kif1B. A HEK293 cells were cotransfected with vector alone (lane 1) or expression plasmids encoding Dusp26-Flag (lanes 2). Dusp26-Flag was pulled down using antibody to Flag (lanes 3, 4) followed by immunoblotting to detect Dusp26-Flag and endogenous p38 (lanes 3, 4). B p-p38 was immunoprecipitated from HEK293 or SH-SY5Y cells and endogenous Dusp26 was detected by immunoblotting. Presence of relevant proteins in the input are indicated. C Immunoblot analyses of cytoplasmic and mitochondrial fractions isolated from STR of WT and dusp26−/− mice using antibodies to the indicated proteins. β-Actin is loading control. D Immunofluorescent analyses using antibodies to Dusp26 and p-p38 in the primary MB neurons prepared from WT and dusp26−/− mice. Merge shows colocalization of the two proteins in some areas. Scale bar 5 µm. E HEK293 cells were transfected with plasmids encoding Flag-MKK6 (lane 2) and Myc-LRRK2 (ROC-COR-Kinase domain) (lanes 1, 2). Myc-LRRK2 was then pulled down using Myc antibody (lanes 3, 4) and complexes were immunoblotted to detect Flag-MKK6 and Myc-LRRK2. F Top panel: Schematic presentation of the full-length LRRK2 and location of different LRRK2 domains. Lower panel: HEK293 cells were cotransfected with plasmids encoding vector alone (lane 3) or Myc-LRRK2 deletion mutants (ROC-COR-Kinase, COR or Kinase domain) and Dusp26-Flag (lanes 1, 2, 4). Myc-LRRK2 was pulled-down followed by immunoblotting to detect Dusp26-Flag and Myc-LRRK2. Input presents the expression of LRRK2 mutants and Dusp26. G, H HEK293 cells were cotransfected with plasmids encoding full-length His-LRRK2 (WT) (lane 1), His-LRRK2 mutants G2019S, R1441G (lanes 2, 3) and Dusp26-Flag or vector alone (lane 4). His-LRRK2 was pulled down followed by immunoblotting to detect Dusp26 (lanes 1–4). Input shows the expression levels of His-LRRK2 in WT and mutants as well as Dusp26-Flag. Quantification of the data relative to WT LRRK2 is presented in H. I As in G, but following His-LRRK2 pulled-down, Dusp26 phosphatase activity was determined towards PNPP [7]. J Immunoblot analyses of whole cell lysates prepared from WT and dusp26−/− STR and MB were used to detect p-LRRK2, LRRK2 and Miro. β-Actin is loading control. Quantification of the p-LRRK2 expression to total LRRK2 is provided in the right panel. K HEK293 cells were transfected with vector alone (lane 1) or full-length V5-LRRK2 and Myc-Dusp26 (lane 2). LRRK2 was pulled-down from crude mitochondrial fraction using antibody to V5-LRRK2 followed by immunoblotting to detect Myc-Dusp26, V5-LRRK2 and endogenous Kif1B and Miro (lanes 3, 4). In all relevant panels, bars are mean ± SEM. p values are indicated in each panel. All experiments were performed at least two times and in duplicates

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Isolation, Control, Transfection, Activity Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Mitochondrial impairment following loss of Dusp26. A OCR was measured in the primary MB neurons using XF96 Seahorse analyzer in the indicated genotypes. The basal, maximal and ATP-linked OCR are presented. n = 16/group and two independent experiments. B MB mitochondrial fractions were used to determine complex I activity in the indicated genotypes. n = 5 mice. C Native-PAGE followed by immunoblot analyses using a mixture of OXPHOS antibodies showing mitochondria complexes (I–V) and respiratory super-complexes (SC). D Immunoblot analyses of mitochondrial fractions isolated from STR and MB using antibodies to the indicated proteins. β-actin is loading control. n = 2 experiments in duplicates. E, F TEM analyses of SNpc region. To ensure the correct area was selected, consecutive sections were immunostained using antibody to TH (not shown). The green arrows represent healthy appearing mitochondria in both genotypes. In dusp26−/− TEM, which contained few clusters with aberrant organelles (the two panels below), there was evidence of intact or damaged mitochondria inside of autophagosomes, number of electron dense lamellar inclusion bodies and swollen vacuoles (red arrows) and number of structures similar to lysosomes (*). Mitochondrial length and aspect ratios (AR) were determined in > 200 mitochondria (F). In all relevant panels, bars are mean ± SEM. p values are indicated in each panel

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Activity Assay, Clear Native PAGE, Western Blot, Isolation, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Increased expression and release of HtrA2 and p-HtrA2 from the mitochondria upon loss of Dusp26. A IHC staining of SNpc region showing expression of pS142-HtrA2 (left panel) or HtrA2 (right panel). Quantification of the number of p-HtrA2-positve cells per 1000 cells is presented. n = 5 mice/group. Scale bars p-HtrA2, 10 µm, HtrA2, 20 µm. B IHC staining of SNpc region showing colocalization of HtrA2 with TH in dusp26−/− mice. Quantification of the data is presented. n = 5 mice/group. Scale bars 20 µm. C Immunoblotting analyses of MB cytoplasmic and mitochondrial fractions showing the increased expression of HtrA2 in the cytoplasmic fraction isolated from the mutant mice. The expression of Hsp60 and Hsc70 indicate organelle purity. Quantification of the HtrA2 in the cytoplasmic fraction relative to WT is indicated in the right panel. n = 4–5 mice/group. D Immunoblotting analyses to detect Xiap expression in the MB and cortex of WT and dusp26−/− mice. β-actin is loading control. Quantification of the data relative to WT are presented in the right panel. n = 4–5 mice/group. In all relevant panels, bars are mean ± SEM. p values are indicated in each panel

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Expressing, Immunohistochemistry, Western Blot, Isolation, Mutagenesis, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Reduced mitochondrial movement following loss of Dusp26. A Immunofluorescence analyses of cultured MB neurons using antibody to TH to show that the majority of cultured neurons are TH-positive DA neurons. Dapi is nuclear staining. Fluorescent-conjugated secondary antibody to show the absence of any non-specific staining. Bar 10 µm. Quantification of TH-positive cells per total cells are indicated below. B–D Primary MB neurons were transfected with Mito-dsRed to detect mitochondrial movement before, or after treatment of neurons with 40 µM Antimycin A for 30 min (B). The frame of each live imaging sequences is presented at the top of each kymograph that was generated from the movie. Position of the mitochondria is presented in the x axis and time is presented in the y-axis (0–180 s). Mitochondrial velocity before, and after addition of Antimycin A for 30 min are presented in C. Normalized mitochondrial run-length was quantified before any treatment (D). E, F Primary MB neurons were cotransfected with plasmids encoding Mito-dsRed and LC3-GFP. After 22 h, live cell imaging was performed. The intensity profile was generated using Image J before and 70 min after treatment of neurons with 40 µM of antimycin A (lower panel) (E). Quantification of the mitochondria number in axons showing co-localization of Mito-dsRed and LC3-GFP in WT and dusp26−/− neurons using Image J (F) [57]. For each condition, more than 120 mitochondria in 3 separate cultures were analyzed. Data were averaged over at least n = 10 axons and 5 transfection experiments and statistical analyses was performed using Student’s t test. In all relevant panels, bars are mean ± SEM. p values are indicated in each panel. ns not significant

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Immunofluorescence, Cell Culture, Staining, Transfection, Imaging, Generated, Live Cell Imaging

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Increased ROS and ROS-induced DNA damage in DA neurons in the SNpc. A Untreated MB or cortex/STR primary neuronal cultures from WT and dusp26−/− mice were stained with 5 µM DHE or Mitosox for 30 min followed by FACS analyses to determine the cytoplasmic or mitochondrial ROS levels, respectively. B IHC staining of SNpc showing 8-OHDG expression in 4-month-old WT and dusp26−/− mice. Tissue sections were stained with fast red to detect 8-OHDG-positive cells (arrows). Quantification of 8-OHDG-positive cells in SNpc is presented in the right panel. Scale bar 10 µm. n = 5 mice/group. C IHC immunostaining of the cortical region of 4-month-old WT and dusp26−/− mice showing the absence of 8-OHDG accumulation. Scale bar 10 µm. n = 5 mice/group. D Immunofluorescent staining showing coexpression of 8-OHDG and TH (arrows) in the SNpc in WT and dusp26−/− mice at 4 months of age. DAPI indicates nuclear staining. Quantification of 8-OHDG-positive cells expressing TH per 1000 cells (indicated by DAPI staining) is presented (right panel). Scale bar 10 µm, n = 5 mice/group. E RT-qPCR analyses of MB and STR from WT and dusp26−/− mice to show the expression of genes relevant to increased ROS. n = 5 mice/group. In all relevant panels, bars are mean ± SEM. p values are indicated in each panel. ns not significant

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Staining, Immunohistochemistry, Expressing, Immunostaining, Quantitative RT-PCR

Journal: Cellular and molecular life sciences : CMLS

Article Title: Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death

doi: 10.1007/s00018-022-04162-z

Figure Lengend Snippet: Dusp26 is expressed in TH-positive healthy human brain neurons in SNpc. A Immunofluorescent staining of SNpc obtained from healthy individuals using Dusp26 and TH antibodies. Merge shows colocalization of TH +/Dusp26 + neurons (arrows). Scale bar 50 µm. B Immunoblot analyses of SNpc obtained from healthy and PD patients using the indicated antibodies. β-Actin is the loading control. Quantification of the p-p38 is presented in the lower panel. C, D IHC staining of Dusp26 in SNpc obtained from healthy and PD patients. Quantification of the positive cells is presented in D. Bars are mean ± SEM. p values are indicated in each panel. Scale bar 50 µm. E Immunofluorescent staining of Dusp26 in SNpc obtained from healthy and PD patients. Positive cells are indicated by arrow. Scale bar 20 µm. Experiments were performed with four healthy and six PD patient samples. In all relevant panels, bars are mean ± SEM. p values are indicated in each panel

Article Snippet: Plasmids and transfection The plasmids encoding human Dusp26 (C-terminal Myc-Flag-tagged) was purchased from Origene (RC200202).

Techniques: Staining, Western Blot, Control, Immunohistochemistry

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Representative 30µm maximum projected confocal stack images of layers 1, 2/3, and 5 of the primary somatosensory cortex in mice sacrificed 1-, 4-, 12-, and 24-weeks following treatment with DSP4 (50mg/kg) or saline. Dopamine-β-hydroxylase (DBH)-cre x mTmG mice were used to selectively label NE axons, neuronal tissue was sliced along the sagittal plane, and the native signal was amplified through processing with antibodies raised against GFP. B , IMARIS software was used to quantify the total symbol represents the total axon surface area of a single sagittal section of an individual mouse (n=5/group) and vertical bars show the standard error. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Saline, Amplification, Software

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Representative 30µm maximum projected confocal stack images of layers 1, 2/3, and 5 of the primary motor cortex of mice sacrificed 1-, 4-, 12-, and 24-weeks following treatment with DSP4 (50mg/kg) or saline. Dopamine-β-hydroxylase (DBH)-cre x mTmG mice were used to selectively label NE axons, neuronal tissue was sliced along the sagittal plane, and the native signal was amplified through processing with antibodies raised against GFP. B , IMARIS software was used to quantify the total axon surface area within demonstrated in A . Each plot symbol represents the total axon surface area of a single sagittal section of an individual mouse (n=5/group) and the vertical bars show the standard error. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Saline, Amplification, Software

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Representative 108μm thick maximum projected stack images of layer 1 of the primary somatosensory cortex show the widespread loss and slow recovery of NE density over 16 weeks in a representative DSP4-treated mouse. In a representative saline-treated mouse, the overall axon density and individual axon location and morphology was quite stable. Specific labeling of NE axons was achieved using DBHcre x Ai14 transgenic mice. The dark, sinuous patches in these images are the shadows created by large surface blood vessels. B , (top) Population time-course data measuring total axon surface area are normalized to the value measured 1 day prior to treatment. Recovery of axon density following DSP4 treatment is dominated by new axon growth. There is almost no contribution from collateral sprouting originating from survived axons. Axons that initially survived DSP4-treatment continued to survive at a rate similar to those that were saline-treated. Vertical bars show the standard error. The number of mice imaged at each timepoint, shown in the lower panel, varied due to the deterioration of the imaging window’s clarity over time and downtime due to repair of the imaging rig from week to week. This change in the number of mice assessed at each timepoint, coupled with the variation in lesion magnitude across DSP4-treated animals (SE of Survived axons = 5.0% at week 2), led to some apparent instability across time in the population measures. However, examining survived axons within individual animals shows consistent stability over 16 weeks .

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Saline, Labeling, Transgenic Assay, Imaging

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Representative 3-by-3 tiled 30µm maximum projected confocal stack images of the locus coeruleus 24-weeks following treatment with DSP4 (50mg/kg) or saline. DBHcre x Ai14 mice were used to selectively label NE neurons with tdTomato. The brain was sliced along the sagittal plane, and the tdTomato signal was amplified using cross-reactive antibodies raised against DsRed. B , IMARIS software was used to produce an exhaustive count of the Ai14-positive cell bodies within the locus coeruleus of individual animals. No significant difference in the number of cells was detected between the saline (n=8) and DSP4 (n=9) groups (p = 0.289). The standard error is represented by vertical bars.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Saline, Amplification, Software

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Representative 108µm 3-D reconstructed images of the primary somatosensory cortex show virally induced GCaMP8s (green) and DBHcre x Ai14 (red) one day prior to and two weeks following lesion with the NE-specific neurotoxin DSP4. Viral injection 7 weeks prior to lesion induces patchy GCaMP expression which shows the tiled patterning characteristic of astrocytes. Note that the expression pattern and intensity of GCaMP was not significantly altered over the 15 day-long span encompassing the DSP4 challenge. B , 3µm-thick maximally projected z-stack images of the same GCaMP8s signal shown in A reveals the tendril-like processes characteristic of astrocytes. C , Representative single image of layer 2/3 of the primary somatosensory cortex 16 weeks following infection with AAV5-gfaABC1D-jGCaMP8s to selectively induce GCaMP8s in cortical astrocytes. Fixed brain tissue was sliced in the sagittal plane and processed with antibodies raised against the astrocyte marker S100β. As in A and B, the GCaMP8s signal demonstrates a patchy transfection pattern and tiling while S100β labels all astrocytes, highlighting particular cytoskeletal elements. White arrowheads indicate a capillary contacted by S100β-positive astrocyte endfeet.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Injection, Expressing, Infection, Marker, Transfection

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: A , Schematic of the functional imaging configuration. The primary somatosensory cortex of DBHcre x Ai14 mice was infected with AAV5-gfaABC1D-jGCaMP8s (designed to drive GCaMP8s expression in astrocytes) 5-7 weeks prior to recording. The awake mouse was placed on a treadmill (not depicted) and head fixed. Compressed air (30PSI) was delivered through a tube to the back of the neck to elicit a startle-response and, following a short delay, NE release. B , Representative single trial showing a Ca 2+ transient measured in a group of adjacent astrocytes evoked by a 1s long air-puff (grey bars) to the back of the mouse’s neck. Treatment with the selective α1-adrenoreceptor antagonist prazosin (7.5mg/kg) 15 minutes prior to the trial abolishes the startle-evoked astrocytic Ca 2+ transient, indicating that it is mediated by NE release. C , Representative individual (non-averaged) false-color thermal-coded false color images of GCaMP8s fluorescence before and after treatment with 7.5mg/kg Prazosin. D-F , Select response characteristics before and after treatment with the NE-specific neurotoxin DSP4 (50mg/kg) or saline. During each assessment week, animals underwent 4 individual trials with a 5-minute inter-trial interval. Individual trials were excluded from analysis using a baseline stability criterion (see methods). Each plot point represents an individual mouse and vertical bars show the standard error. D , The proportion of trials each assessment week that elicited a startle-evoked Ca 2+ response in neocortical astrocytes. E , The peak amplitude of all GCaMP8s response traces each week. F , The latency from stimulus onset to 25% of the maximum amplitude of all GCaMP8s response traces each week. * = P < 0.05; *** = P < 0.001.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: Functional Assay, Imaging, Infection, Expressing, Fluorescence, Saline

Journal: bioRxiv

Article Title: Functional Regrowth of Norepinephrine Axons in the Adult Mouse Brain Following Injury

doi: 10.1101/2024.08.19.608684

Figure Lengend Snippet: Long-term in vivo 2-photon imaging shows the regrowth of NE axons innervating the adult mouse somatosensory cortex following DSP4 treatment. Individual mouse (shaded) and population mean +/- standard error (solid) measurements of the axon surface area are shown. The population data are the same as illustrated in and are reproduced here to allow for comparison with the individual mouse measurements. Weeks during which data was able to be collected are indicated by shaded closed circles. Collection was sometimes limited by repair of the imaging rig and lines representing individual mice sometimes terminate due to deterioration of the optical quality of the imaging window. ** = P < 0.01; *** = P < 0.001.

Article Snippet: 4-6 weeks following viral injection and cranial window installation surgery, mice received a single 50mg/kg intraperitoneal dose of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, Tocris Bioscience #2958, PubChem ID: 38533) reconstituted in sterile water or an injection of volume-matched saline as a control.

Techniques: In Vivo, Imaging, Comparison

Journal: Journal of Neural Transmission

Article Title: The connections of Locus Coeruleus with hypothalamus: potential involvement in Alzheimer’s disease

doi: 10.1007/s00702-021-02338-8

Figure Lengend Snippet: The Locus Coeruleus. The figure in panel a shows two pictures at different magnifications ( a and b ) of a 10 µm-thick paraffin-embedded coronal section cut at the level of the pons from the brain of an adult C57 Black male mouse (Charles River). The section is collected at approximately at − 5.3 mm from the Bregma, according to the stereotactic mouse brain atlas by Paxinos and Franklin . The section has been immune-stained with a primary antibody (#T1299 Sigma, U.S.A.) against the enzyme tyrosine hydroxylase (TH). Neurons immune-positive for the enzyme TH (brown color in the figure, due to DAB staining of biotin-coupled anti-mouse antibodies followed by exposure to Horseradish peroxidase streptavidin; Vector Laboratories), are neurons belonging to the nucleus Locus Coeruleus (LC); the section is counter-stained with Nissl Staining (Cresyl violet). The LC nucleus is placed right below the floor of the fourth ventricle of the pons (abbreviated as “f.v.” in the pictures) (scale bar: 200 µm). The graph in panel b shows the effects of the experimental lesion of LC-hypothalamic projections by the neurotoxin N -(2-chloroethyl)- N -ethyl-2-bromobenzylamin (DSP-4). The systemic administration of DSP-4 selectively lesions NA terminals originating from the LC in rodents. The figure shows the effect of the administration of DSP-4 50 mg/kg i.p. in adult Sprague Dawley Rats (DSP-4 N = 5; controls N = 5) on NA levels in homogenates collected from the hypothalamus (see legend to Table for details on methodology). The NA levels (ng/mg protein) of the group “DSP-4” are expressed as % of “controls”. * p < 0.01 vs controls

Article Snippet: The figure shows the effect of the administration of DSP-4 50 mg/kg i.p. in adult Sprague Dawley Rats (DSP-4 N = 5; controls N = 5) on NA levels in homogenates collected from the hypothalamus (see legend to Table for details on methodology).

Techniques: Staining, Plasmid Preparation