dna plasmid pcdna3 flag pten  (Addgene inc)

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis (left panel) showing PTEN and PMP22 protein levels in sciatic nerve lysates of Pmp22 +/- mice at postnatal day 6 (P6), postnatal day 18 (P18) and 9 weeks of age compared to wildtype (WT) control ( n = 2 per time point and group). Fast green whole protein staining was used as loading control for the quantification (right panel). ( B ) Western Blot analysis (left panel) showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 +/- mice ( n = 3) at postnatal day 6 and an increase in S6 phosphorylation compared to wildtype (WT) control ( n = 4). Whole protein staining was used as loading control for the quantification (right panel). ( C ) Western Blot analysis (left panel) of PTEN and PMP22 protein levels in sciatic nerve lysates at P6, P18 and 9 weeks of age in Pmp22 tg rats compared to WT control. Whole protein staining served as loading control for the quantification (right panel). ( D ) Western Blot analysis (left panel) showing an increase of PTEN protein levels in sciatic nerve lysates of Pmp22 tg rats at postnatal day 6 and a decrease in S6K phosphorylation compared to wildtype (WT) control ( n = 4). Whole protein staining was used as loading control for the quantification (right panel). ( E ) Quantitative RT-PCR analysis in tibial nerves from n = 4 WT and n = 7 PMP22 +/- mice shows decreased mRNA levels of Pmp22 and Pten in Pmp22 +/- mice at P18. Rplp0 and Ppia served as housekeeping genes. ( F ) Quantitative RT-PCR analysis in tibial nerves from n = 5 WT and n = 5 PMP22 tg rats shows increased mRNA levels of Pmp22 and Pten in Pmp22 tg rats at P18. Rplp0 and Ppia served as housekeeping genes. ( G ) Immunoblot of WT P18 rat whole sciatic nerve lysate and purified myelin. PTEN and TUJ1 are enriched in the lysate while PMP22 and P0 are enriched in the myelin fraction. ( H ) Femoral nerve cross section of 9-week-old WT rats shows PTEN (green) localization to the axon (magenta, TUJ1), nuclei (blue DAPI) and bands of Cajal (indicated by arrows). Scale bar is 5 µm. ( I ) Graphical overview of Pmp22 gene-dosage dependent alterations in the PI3K/Akt/mTOR signaling pathway in animal models of CMT1A and HNPP. PMP22 overexpression leads to increased PTEN protein levels, reduced activation of the downstream PI3K/Akt/mTOR growth signaling pathway and subsequently demyelination (red). In contrast, PMP22 heterozygosity results in decreased PTEN levels, increased activation of the PI3K/Akt/mTOR signaling cascade and hypermyelination (blue). Data information: Mean numbers are displayed ±standard deviation. Statistical analysis was performed using Student’s t test, * p < 0.05,** p < 0.01, *** p < 0.001. .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Western Blot, Control, Staining, Phospho-proteomics, Quantitative RT-PCR, Purification, Over Expression, Activation Assay, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 +/- mice at 9 weeks ( n = 4, left panel) and at postnatal day 21 ( n = 4, right panel) compared to wildtype (WT) control. Fast green whole protein staining was used as loading control for the quantification. ( B ) Western Blot analysis showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 tg rats at 9 weeks ( n = 4, left panel) and at postnatal day 18 ( n = 3, right panel) compared to wildtype (WT) control. Whole protein staining was used as loading control for the quantification. ( C ) Sciatic nerve semi-thin sections of WT ( n = 3) and Pmp22 +/- mice ( n = 3) at postnatal day 6; myelin aberrations are highlighted with yellow arrowheads (left panel). Quantification shows increased percentage of axons with myelin aberrations in sciatic nerves from PMP22 +/- mice (left panel). Scale bar is 50 µm. ( D ) Quadriceps motor nerve semi-thin sections of WT ( n = 3) and Pmp22 +/- mice ( n = 3) at postnatal day 18; myelin aberrations are highlighted with yellow arrowheads (left panel). Quantification shows increased percentage of axons with myelin aberrations in sciatic nerves from PMP22 +/- mice (left panel). Scale bar is 25 µm. Data information: Means are displayed ± standard deviation. Statistical analysis was performed using Student’s t test (* p < 0.05, ** p < 0.01).

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Western Blot, Control, Staining, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Immunoprecipitation of PMP22 from rat sciatic nerve (P18). Western Blot (WB) analysis shows unspecific binding of PTEN, while PMP22 was specifically detected in PMP22 immunoprecipitation eluate (IP) and not in control eluate (ctrl IP). Input nerve homogenate (I) was diluted 50× for WB analysis. ( B ) Immunoprecipitation from HEK293T cells after transfection of PMP22-ALFA. WB analysis shows endogenous PTEN in the cell lysate (I) and in the supernatant after the binding step (unbound (Ub)), but not in the immunoprecipitation eluate. I and Ub were diluted 10× for WB analysis. ( C ) Immunoprecipitation from HEK293T cells after co-transfection of PMP22-ALFA with FLAG-PTEN or untagged PTEN. WB analysis shows specific immunoprecipitation of FLAG-PTEN but not of PMP22-ALFA or untagged PTEN. ( D ) Pull-down assay on rat sciatic nerve (P18) using purified PMP22-ALFA as prey. WB analysis shows PTEN in the nerve lysate (I) but not in the PMP22-ALFA eluate (PMP22-ALFA) or control eluate (ctrl) in both WT and CMT1A, while P0 was pulled down by PMP22-ALFA. I was diluted 3.33× for WB analysis.

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Control, Transfection, Cotransfection, Pull Down Assay, Purification

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Pmp22 +/- and wildtype (WT) control mice were injected i.p. with placebo solution or 5 mg Rapamycin per kg bodyweight two times per week from P21 until P148 to reduce mTOR activity. Grip strength analysis, electrophysiology, protein expression analysis and histology were performed at P148. ( B ) Western Blot analysis of PTEN protein ( n = 3 animals per group) and phosphorylated and total S6 ( n = 4 animals per group) (left panel) in whole sciatic nerve lysates. Quantification using whole protein staining as loading control shows increased PTEN protein levels and decreased S6 phosphorylation after Rapamycin treatment in Pmp22 +/- mice (right panel). ( C ) Sciatic nerve semi-thin sections of Pmp22 +/- placebo and Rapamycin treated mice. Tomacula are encircled in black and marked with asterisks. Yellow arrowheads point to recurrent loops. Scale bar is 20 µm. ( D ) The percentage of axons showing tomacula is decreased in whole sciatic nerves of Rapamycin treated Pmp22 +/- mice (triangle) compared to placebo controls (circles) at P148, n = 15 animals per group. ( E ) The percentage of axons showing recurrent loops is decreased in whole sciatic nerves of Rapamycin treated Pmp22 +/- mice (triangle) compared to placebo controls (circles) at P148, n = 15 animals per group. ( F ) Total axon number in sciatic nerves is unaltered between Rapamycin treated Pmp22 +/- mice (triangle) and placebo controls (circles) at P148, n = 15 animals per group. ( G ) Forelimb grip strength is decreased in Pmp22 +/- placebo mice ( n = 12, blue circles) compared to WT placebo mice ( n = 10, gray circles), whereas Rapamycin treatment improves strength in Pmp22 +/- mice ( n = 13, blue triangles) and does not affect WT mice ( n = 12, gray triangles) at P148. ( H ) Rapamycin-treated Pmp22 +/- and WT animals (triangles) gained less weight than placebo controls (circles). Groups of n = 10 WT placebo, n = 12 WT Rapamycin, n = 19 Pmp22 +/- placebo and n = 16 Pmp22 +/- Rapamycin. ( I ) Electrophysiological analysis shows reduced compound muscle action potential amplitudes (CMAP) in Pmp22 +/- placebo mice and increased CMAP after Rapamycin treatment. n = 8 WT placebo, n = 10 WT Rapamycin, n = 10 Pmp22 +/- placebo and n = 8 Pmp22 +/- Rapamycin. ( J ) Electrophysiological analysis shows reduced nerve conduction velocities (NCV) in Pmp22 +/- placebo mice and no alterations after Rapamycin treatment. n = 8 WT placebo, n = 10 WT Rapamycin, n = 10 Pmp22 +/- placebo and n = 8 Pmp22 +/- Rapamycin. Data information: Mean numbers are displayed ±standard deviation. Statistical analysis was performed using Student’s t test ( D – F ) and one-way ANOVA with Sidak’s multiple comparison test ( B , G – J ; * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Control, Injection, Activity Assay, Expressing, Western Blot, Staining, Phospho-proteomics, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) PTEN inhibitor VO-OHpic was used to disinhibit the PI3K/Akt/mTOR signaling pathway in CMT1A. ( B ) Example images of Schwann cell-dorsal root ganglia neuron co-cultures from wildtype (WT) and Pmp22 tg rats treated with DMSO as control (Ctrl) or PTEN inhibitor VO-OHpic (500 nM). Cells were stained for myelin basic protein (MBP) as a marker for myelinated segments (gray/ green) and TUJ1 for neurons (magenta) as well as DAPI for cell nuclei (blue). ( C ) Quantification of ( B ) shows a dose-dependent decrease of myelinated segments in WT co-cultures treated with DMSO and different concentrations of VO-OHpic (50 nM, 500 nM, 5 µM) and an increase of myelinated segments in Pmp22 tg co-cultures with 500 nM VO-OHpic (WT n = 5, CMT1A n = 7 animals). Groups were compared using two-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, **** p ≤ 0.0001). .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Control, Staining, Marker, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: Example images of SC-DRG co-cultures from wildtype (WT) and Pmp22 tg rats, treated with different dosages of the PTEN inhibitor VO-OHpic for 14 days. The number of myelinated segments (MBP; gray/green) decreases in WT cultures with increasing inhibitor dosage. In Pmp22 tg co-cultures an increase is observed up to 500 nM VO-OHpic but a decrease with 5 µM VO-OHpic. Scale bar is 50 µm. Images for 500 nM VO-OHpic treatment are the same as used in Fig. .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Rationale to breed Schwann cell specific heterozygous Pten knockout mice ( Pten fl/+ Dhh cre/+ ) with CMT1A mice ( Pmp22 tg ) to lower Pten expression in CMT1A mice ( Pten fl/+ Dhh cre/+ Pmp22 tg ). ( B ) In order to reduce Pten genetically in Schwann cells, heterozygous Pten floxed mice under the Dhh cre driver ( Pten fl/+ Dhh cre/+ ) were crossbred with Pmp22 tg mice. ( C ) Western Blot analysis of sciatic nerve lysate from WT, PMP22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg at postnatal day 18 against PTEN, P-S6, S6, P-S6K and S6K with whole protein staining as the loading control. ( D ) Quantification of ( C ) reveals increased PTEN protein levels in PMP22 tg sciatic nerve lysates compared to WT controls as well as decreased PTEN protein levels in Pten fl/+ Dhh cre/+ Pmp22 tg sciatic nerve lysates compared to Pmp22 tg mice (upper panel) and downregulation of P-S6 (middle panel) activation in PMP22 tg mice compared to WT mice, while P-S6K (lower panel) activation is increased in Pten fl/+ Dhh cre/+ Pmp22 tg mice as compared to PMP22 tg mice. Groups were compared using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01). ( E ) Paraffin cross sections of femoral nerves from 18 days old WT, Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice show an increased signal for Phospho-S6 in double mutants (magenta, indicated by yellow arrowheads). Myelin is visualized by P0 (green) and nuclei by DAPI (blue). Scale bar is 10 µm. ( F ) Representative example images of Schwann cell dorsal root ganglia neuron co-cultures 14 days after induction of myelination. Myelin basic protein (MBP) indicates myelinated segments (gray/green), TUJ1 neurons (magenta) and DAPI nuclei (blue). Scale bar is 50 µm. ( G ) Quantification of ( F ) shows increased numbers of myelinated segments in Pten fl/+ Dhh cre/+ Pmp22 tg compared to Pten fl/+ Dhh cre/+ co-cultures. Shown are means of 5 fields of view (500 × 500 µm) for each animal ( n = 2–3) ±standard deviation. .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Knock-Out, Expressing, Western Blot, Staining, Control, Activation Assay, Comparison, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Example images of femoral nerve semi-thin sections from wildtype (WT), Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice at P18 (upper panels) and 16 weeks of age (lower panels). Yellow arrowheads indicate amyelinated axons. Scale bar = 10 µm. ( B ) Quantification of (a) displays a decreased amount of myelinated axons in Pmp22 tg whole femoral nerves and an increase in Pten fl/+ Dhh cre/+ Pmp22 tg double mutants (upper panel) at P18 while numbers of myelinated axons are similarly decreased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice at 16 weeks of age (lower panel). WT n = 3–4, Pten fl/+ Dhh cre/+ n = 3–4, Pmp22 tg n = 3–5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3–4 animals. ( C ) G -ratio plotted against axon diameter of WT (gray) and Pmp22 tg (red) mice (left panel) and Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg (purple) mice (right panel) at P18. WT n = 3, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. ( D ) Distribution of g -ratios shown in the left panel displays Pten fl/+ Dhh cre/+ Pmp22 tg femoral nerves have more axons with low g-ratios (0.4–0.5) and less axons with higher g-ratios (0.8–0.9, 0.9–1.0) compared to Pmp22 tg nerves. Mean g -ratio (right panel) showed a trend to be decreased in Pmp22 tg femoral nerves compared to WT and were significantly decreased in Pten fl/+ Dhh cre/+ Pmp22 tg femoral nerves. WT n = 3, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. Groups were compared using two-way ANOVA with Tukey’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). ( E ) G -ratio plotted against axon diameter of WT (gray) and Pmp22 tg (red) mice (left panel) and Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg (purple) mice (right panel) at 16 weeks of age. WT n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. ( F ) No alteration in the distribution of axons over the g-ratio (left panel) as well as mean g -ratio (right panel) is observed comparing femoral nerves from Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. Groups were compared using two-way ANOVA with Tukey’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). ( G ) The grip strength of hindlimbs is reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to WT controls and Pten fl/+ Dhh cre/+ mice. Behavioral analysis was done at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9–14 mice were analyzed. ( H ) Nerve conduction velocity (NCV, left panel) and compound muscle action potential amplitudes (CMAP, right panel) are similarly lower in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to WT controls. For electrophysiology measurements WT n = 10, Pten fl/+ Dhh cre/+ n = 8, Pmp22 tg n = 11 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 8 mice were analyzed. Data information: Means are displayed ±standard deviation. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) if not indicated otherwise. .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Comparison, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis shows PTEN and PMP22 protein amounts in whole sciatic nerve lysates from 16 weeks old WT, PTEN heterozygous knockout ( Pten fl/+ Dhh cre/+ ), CMT1A ( Pmp22 tg ) and double mutant ( Pten fl/+ Dhh cre/+ Pmp22 tg ) mice using whole protein staining as loading control. ( B ) G -ratio plotted against axon diameter of wildtype (WT, gray) and Pten fl/+ Dhh cre/+ (turquoise) femoral nerves at P18. Mean g -ratio is unaltered in Pten fl/+ Dhh cre/+ and Pmp22 tg mice compared to WT controls and decreased in Pten fl/+ Dhh cre/+ Pmp22 tg mice (left panel). Mean axon diameters are reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (middle panel). The number of Schwann cell nuclei per femoral nerve cross section is increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 3, Pten fl/+ Dhh cre/+ n = 4, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. ( C ) G -ratio plotted against axon diameter of WT (gray) and Pten fl/+ Dhh cre/+ (turquoise) femoral nerves at 16 weeks of age. Mean g-ratio is unaltered in Pten fl/+ Dhh cre/+ mice compared to WT controls and decreased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (left panel). Mean axon diameters are reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (middle panel). The number of Schwann cell nuclei per femoral nerve cross section is increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 4, Pten fl/+ Dhh cre/+ n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. ( D ) The number of slips on the elevated beam is similarly increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to wildtype controls at all time points. Behavioral analysis was done at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9–14 mice were analyzed. ( E ) Neither the weight of Pmp22 tg nor Pten fl/+ Dhh cre/+ Pmp22 tg mice is altered compared to wildtype controls at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9-14 mice were analyzed. ( F ) Sensory nerve action potential amplitudes (SNAP) are decreased in the tail of Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to wildtype controls. For electrophysiology measurements WT n = 10, Pten fl/+ Dhh cre/+ n = 8, Pmp22 tg n = 11 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 8 mice were analyzed. ( G ) Example images of teased fiber preparations of WT, Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg double mutants stained for MAG (green), NaV1.6 (magenta) and DAPI (blue) at P18. Internodes between two nodes (magenta arrowheads) are underlined in yellow and respective Schwann cell nuclei are marked by white stars. Mean internodal length (left panel) is significantly reduced in Pmp22 tg teased fibers compared to wildtype controls at P18, whereas Pten fl/+ Dhh cre/+ Pmp22 tg mice do not differ in internodal length compared to Pmp22 tg mice. Mean fiber diameters are not significantly altered (right panel). Analysis was performed on 100 internodes of n = 3–4 animals per group. ( H ) Example images of teased fiber preparations of WT, Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg double mutants stained for MAG (green), NaV1.6 (magenta) and DAPI (blue) at 16 weeks of age. Internodes between two nodes (magenta arrowheads) are underlined in yellow and respective Schwann cell nuclei are marked by white stars. Mean internodal length (left panel) and fiber diameter (right panel) are significantly reduced in Pmp22 tg teased fibers compared to wildtype controls at 16 weeks of age, whereas Pten fl/+ Dhh cre/+ Pmp22 tg mice do not differ in internodal length compared to Pmp22 tg mice. Analysis was performed on 100 internodes of n = 3–4 animals per group. Data information: Means are displayed ± standard deviation. Statistical analysis was done using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Western Blot, Knock-Out, Mutagenesis, Staining, Control, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Quantitative RT-PCR from P18 tibial nerves of wildtype (WT) ( n = 4), Pten fl/+ Dhh cre/+ ( n = 5), Pmp22 tg ( n = 5) and Pten fl/+ Dhh cre/+ Pmp22 tg ( n = 5) mice shows relative mRNA expression of Pten, Hmgcr, Nrg1-I, Pou3f1, Ngfr , and Sox2 . Rplp0 and Ppia were used as housekeeping genes. ( B ) Quantitative RT-PCR from P18 tibial nerves of WT ( n = 4) and Pmp22 +/- ( n = 8) mice shows relative mRNA expression of Pten, Hmgcr, Nrg1-I, Pou3f1, Ngfr , and Sox2 . Rplp0 and Ppia were used as housekeeping genes. ( C ) Semi-thin sections of P18 femoral nerves of Pmp22 +/- (left), WT (middle) and Pmp22 tg (right) mice. Pmp22 +/- nerves show myelin overgrowth, the so-called tomacula (to, yellow) and Pmp22 tg nerves are characterized by amyelinated (a, pink) and thinly myelinated (th, green) big axons as well as hypermyelinated (h, orange) small axons. Quantification shows g-ratios of Pmp22 +/- mice (blue, without tomacula) are similar to the WT (gray) distribution, while Pmp22 tg mice (red, without amyelinated) show the hypermyelination of small axons and demyelination of big axons. Data information: Means are displayed ±standard deviation. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). .

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Crossing scheme of Schwann cell specific full Pten knockout mice ( Pten fl/fl Dhh cre/+ ) with CMT1A mice ( Pmp22 tg ) to generate a full Pten knockout in CMT1A mice ( Pten fl/fl Dhh cre/+ Pmp22 tg ). ( B ) Teased fiber preparations of 8 weeks old PTEN fl/fl Dhh cre/+ (upper panel) and Pten fl/fl Dhh cre/+ Pmp22 tg mice (lower panel) show focal myelin thickening at paranodal loops as indicated by yellow arrows. Scale bar = 20 µm. ( C ) Semi-thin cross section of femoral nerves from WT, Pten fl/fl Dhh cre/+ , Pmp22 tg and Pten fl/fl Dhh cre/+ Pmp22 tg mice at 8 weeks of age. Red arrows indicate myelin abnormalities such as outfoldings and tomacula, asterisks indicate amyelinated axons. Scale bar = 10 µm. ( D – F ) Quantification of ( C ) displays reduced axon numbers in Pten fl/fl Dhh cre/+ Pmp22 tg mice compared to wildtype controls ( D ). The percentage of amyelinated axons is increased in Pmp22 tg and further elevated in Pten fl/fl Dhh cre/+ Pmp22 tg mice ( E ). Pten depletion alone and in Pmp22 tg leads to an increase in axons with aberrant myelin profiles ( F ). WT n = 5, PTEN fl/fl Dhh cre/+ n = 4, Pmp22 tg n = 6 and Pten fl/fl Dhh cre/+ Pmp22 tg n = 3 animals were analyzed. Data information: Means are displayed ± standard deviation. Statistical analysis was done using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Knock-Out, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: Genotyping primers.

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Sequencing

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: qRT-PCR primers.

Article Snippet: Cells were transiently transfected with ALFA-tagged (Gotzke et al, ) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.

Techniques: Sequencing

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis (left panel) showing PTEN and PMP22 protein levels in sciatic nerve lysates of Pmp22 +/- mice at postnatal day 6 (P6), postnatal day 18 (P18) and 9 weeks of age compared to wildtype (WT) control ( n = 2 per time point and group). Fast green whole protein staining was used as loading control for the quantification (right panel). ( B ) Western Blot analysis (left panel) showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 +/- mice ( n = 3) at postnatal day 6 and an increase in S6 phosphorylation compared to wildtype (WT) control ( n = 4). Whole protein staining was used as loading control for the quantification (right panel). ( C ) Western Blot analysis (left panel) of PTEN and PMP22 protein levels in sciatic nerve lysates at P6, P18 and 9 weeks of age in Pmp22 tg rats compared to WT control. Whole protein staining served as loading control for the quantification (right panel). ( D ) Western Blot analysis (left panel) showing an increase of PTEN protein levels in sciatic nerve lysates of Pmp22 tg rats at postnatal day 6 and a decrease in S6K phosphorylation compared to wildtype (WT) control ( n = 4). Whole protein staining was used as loading control for the quantification (right panel). ( E ) Quantitative RT-PCR analysis in tibial nerves from n = 4 WT and n = 7 PMP22 +/- mice shows decreased mRNA levels of Pmp22 and Pten in Pmp22 +/- mice at P18. Rplp0 and Ppia served as housekeeping genes. ( F ) Quantitative RT-PCR analysis in tibial nerves from n = 5 WT and n = 5 PMP22 tg rats shows increased mRNA levels of Pmp22 and Pten in Pmp22 tg rats at P18. Rplp0 and Ppia served as housekeeping genes. ( G ) Immunoblot of WT P18 rat whole sciatic nerve lysate and purified myelin. PTEN and TUJ1 are enriched in the lysate while PMP22 and P0 are enriched in the myelin fraction. ( H ) Femoral nerve cross section of 9-week-old WT rats shows PTEN (green) localization to the axon (magenta, TUJ1), nuclei (blue DAPI) and bands of Cajal (indicated by arrows). Scale bar is 5 µm. ( I ) Graphical overview of Pmp22 gene-dosage dependent alterations in the PI3K/Akt/mTOR signaling pathway in animal models of CMT1A and HNPP. PMP22 overexpression leads to increased PTEN protein levels, reduced activation of the downstream PI3K/Akt/mTOR growth signaling pathway and subsequently demyelination (red). In contrast, PMP22 heterozygosity results in decreased PTEN levels, increased activation of the PI3K/Akt/mTOR signaling cascade and hypermyelination (blue). Data information: Mean numbers are displayed ±standard deviation. Statistical analysis was performed using Student’s t test, * p < 0.05,** p < 0.01, *** p < 0.001. .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Control, Staining, Phospho-proteomics, Quantitative RT-PCR, Purification, Over Expression, Activation Assay, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 +/- mice at 9 weeks ( n = 4, left panel) and at postnatal day 21 ( n = 4, right panel) compared to wildtype (WT) control. Fast green whole protein staining was used as loading control for the quantification. ( B ) Western Blot analysis showing a decrease of PTEN protein levels in sciatic nerve lysates of Pmp22 tg rats at 9 weeks ( n = 4, left panel) and at postnatal day 18 ( n = 3, right panel) compared to wildtype (WT) control. Whole protein staining was used as loading control for the quantification. ( C ) Sciatic nerve semi-thin sections of WT ( n = 3) and Pmp22 +/- mice ( n = 3) at postnatal day 6; myelin aberrations are highlighted with yellow arrowheads (left panel). Quantification shows increased percentage of axons with myelin aberrations in sciatic nerves from PMP22 +/- mice (left panel). Scale bar is 50 µm. ( D ) Quadriceps motor nerve semi-thin sections of WT ( n = 3) and Pmp22 +/- mice ( n = 3) at postnatal day 18; myelin aberrations are highlighted with yellow arrowheads (left panel). Quantification shows increased percentage of axons with myelin aberrations in sciatic nerves from PMP22 +/- mice (left panel). Scale bar is 25 µm. Data information: Means are displayed ± standard deviation. Statistical analysis was performed using Student’s t test (* p < 0.05, ** p < 0.01).

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Control, Staining, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Immunoprecipitation of PMP22 from rat sciatic nerve (P18). Western Blot (WB) analysis shows unspecific binding of PTEN, while PMP22 was specifically detected in PMP22 immunoprecipitation eluate (IP) and not in control eluate (ctrl IP). Input nerve homogenate (I) was diluted 50× for WB analysis. ( B ) Immunoprecipitation from HEK293T cells after transfection of PMP22-ALFA. WB analysis shows endogenous PTEN in the cell lysate (I) and in the supernatant after the binding step (unbound (Ub)), but not in the immunoprecipitation eluate. I and Ub were diluted 10× for WB analysis. ( C ) Immunoprecipitation from HEK293T cells after co-transfection of PMP22-ALFA with FLAG-PTEN or untagged PTEN. WB analysis shows specific immunoprecipitation of FLAG-PTEN but not of PMP22-ALFA or untagged PTEN. ( D ) Pull-down assay on rat sciatic nerve (P18) using purified PMP22-ALFA as prey. WB analysis shows PTEN in the nerve lysate (I) but not in the PMP22-ALFA eluate (PMP22-ALFA) or control eluate (ctrl) in both WT and CMT1A, while P0 was pulled down by PMP22-ALFA. I was diluted 3.33× for WB analysis.

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Control, Transfection, Cotransfection, Pull Down Assay, Purification

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Pmp22 +/- and wildtype (WT) control mice were injected i.p. with placebo solution or 5 mg Rapamycin per kg bodyweight two times per week from P21 until P148 to reduce mTOR activity. Grip strength analysis, electrophysiology, protein expression analysis and histology were performed at P148. ( B ) Western Blot analysis of PTEN protein ( n = 3 animals per group) and phosphorylated and total S6 ( n = 4 animals per group) (left panel) in whole sciatic nerve lysates. Quantification using whole protein staining as loading control shows increased PTEN protein levels and decreased S6 phosphorylation after Rapamycin treatment in Pmp22 +/- mice (right panel). ( C ) Sciatic nerve semi-thin sections of Pmp22 +/- placebo and Rapamycin treated mice. Tomacula are encircled in black and marked with asterisks. Yellow arrowheads point to recurrent loops. Scale bar is 20 µm. ( D ) The percentage of axons showing tomacula is decreased in whole sciatic nerves of Rapamycin treated Pmp22 +/- mice (triangle) compared to placebo controls (circles) at P148, n = 15 animals per group. ( E ) The percentage of axons showing recurrent loops is decreased in whole sciatic nerves of Rapamycin treated Pmp22 +/- mice (triangle) compared to placebo controls (circles) at P148, n = 15 animals per group. ( F ) Total axon number in sciatic nerves is unaltered between Rapamycin treated Pmp22 +/- mice (triangle) and placebo controls (circles) at P148, n = 15 animals per group. ( G ) Forelimb grip strength is decreased in Pmp22 +/- placebo mice ( n = 12, blue circles) compared to WT placebo mice ( n = 10, gray circles), whereas Rapamycin treatment improves strength in Pmp22 +/- mice ( n = 13, blue triangles) and does not affect WT mice ( n = 12, gray triangles) at P148. ( H ) Rapamycin-treated Pmp22 +/- and WT animals (triangles) gained less weight than placebo controls (circles). Groups of n = 10 WT placebo, n = 12 WT Rapamycin, n = 19 Pmp22 +/- placebo and n = 16 Pmp22 +/- Rapamycin. ( I ) Electrophysiological analysis shows reduced compound muscle action potential amplitudes (CMAP) in Pmp22 +/- placebo mice and increased CMAP after Rapamycin treatment. n = 8 WT placebo, n = 10 WT Rapamycin, n = 10 Pmp22 +/- placebo and n = 8 Pmp22 +/- Rapamycin. ( J ) Electrophysiological analysis shows reduced nerve conduction velocities (NCV) in Pmp22 +/- placebo mice and no alterations after Rapamycin treatment. n = 8 WT placebo, n = 10 WT Rapamycin, n = 10 Pmp22 +/- placebo and n = 8 Pmp22 +/- Rapamycin. Data information: Mean numbers are displayed ±standard deviation. Statistical analysis was performed using Student’s t test ( D – F ) and one-way ANOVA with Sidak’s multiple comparison test ( B , G – J ; * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Control, Injection, Activity Assay, Expressing, Western Blot, Staining, Phospho-proteomics, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) PTEN inhibitor VO-OHpic was used to disinhibit the PI3K/Akt/mTOR signaling pathway in CMT1A. ( B ) Example images of Schwann cell-dorsal root ganglia neuron co-cultures from wildtype (WT) and Pmp22 tg rats treated with DMSO as control (Ctrl) or PTEN inhibitor VO-OHpic (500 nM). Cells were stained for myelin basic protein (MBP) as a marker for myelinated segments (gray/ green) and TUJ1 for neurons (magenta) as well as DAPI for cell nuclei (blue). ( C ) Quantification of ( B ) shows a dose-dependent decrease of myelinated segments in WT co-cultures treated with DMSO and different concentrations of VO-OHpic (50 nM, 500 nM, 5 µM) and an increase of myelinated segments in Pmp22 tg co-cultures with 500 nM VO-OHpic (WT n = 5, CMT1A n = 7 animals). Groups were compared using two-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, **** p ≤ 0.0001). .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Control, Staining, Marker, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: Example images of SC-DRG co-cultures from wildtype (WT) and Pmp22 tg rats, treated with different dosages of the PTEN inhibitor VO-OHpic for 14 days. The number of myelinated segments (MBP; gray/green) decreases in WT cultures with increasing inhibitor dosage. In Pmp22 tg co-cultures an increase is observed up to 500 nM VO-OHpic but a decrease with 5 µM VO-OHpic. Scale bar is 50 µm. Images for 500 nM VO-OHpic treatment are the same as used in Fig. .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Rationale to breed Schwann cell specific heterozygous Pten knockout mice ( Pten fl/+ Dhh cre/+ ) with CMT1A mice ( Pmp22 tg ) to lower Pten expression in CMT1A mice ( Pten fl/+ Dhh cre/+ Pmp22 tg ). ( B ) In order to reduce Pten genetically in Schwann cells, heterozygous Pten floxed mice under the Dhh cre driver ( Pten fl/+ Dhh cre/+ ) were crossbred with Pmp22 tg mice. ( C ) Western Blot analysis of sciatic nerve lysate from WT, PMP22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg at postnatal day 18 against PTEN, P-S6, S6, P-S6K and S6K with whole protein staining as the loading control. ( D ) Quantification of ( C ) reveals increased PTEN protein levels in PMP22 tg sciatic nerve lysates compared to WT controls as well as decreased PTEN protein levels in Pten fl/+ Dhh cre/+ Pmp22 tg sciatic nerve lysates compared to Pmp22 tg mice (upper panel) and downregulation of P-S6 (middle panel) activation in PMP22 tg mice compared to WT mice, while P-S6K (lower panel) activation is increased in Pten fl/+ Dhh cre/+ Pmp22 tg mice as compared to PMP22 tg mice. Groups were compared using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01). ( E ) Paraffin cross sections of femoral nerves from 18 days old WT, Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice show an increased signal for Phospho-S6 in double mutants (magenta, indicated by yellow arrowheads). Myelin is visualized by P0 (green) and nuclei by DAPI (blue). Scale bar is 10 µm. ( F ) Representative example images of Schwann cell dorsal root ganglia neuron co-cultures 14 days after induction of myelination. Myelin basic protein (MBP) indicates myelinated segments (gray/green), TUJ1 neurons (magenta) and DAPI nuclei (blue). Scale bar is 50 µm. ( G ) Quantification of ( F ) shows increased numbers of myelinated segments in Pten fl/+ Dhh cre/+ Pmp22 tg compared to Pten fl/+ Dhh cre/+ co-cultures. Shown are means of 5 fields of view (500 × 500 µm) for each animal ( n = 2–3) ±standard deviation. .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knock-Out, Expressing, Western Blot, Staining, Control, Activation Assay, Comparison, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Example images of femoral nerve semi-thin sections from wildtype (WT), Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice at P18 (upper panels) and 16 weeks of age (lower panels). Yellow arrowheads indicate amyelinated axons. Scale bar = 10 µm. ( B ) Quantification of (a) displays a decreased amount of myelinated axons in Pmp22 tg whole femoral nerves and an increase in Pten fl/+ Dhh cre/+ Pmp22 tg double mutants (upper panel) at P18 while numbers of myelinated axons are similarly decreased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice at 16 weeks of age (lower panel). WT n = 3–4, Pten fl/+ Dhh cre/+ n = 3–4, Pmp22 tg n = 3–5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3–4 animals. ( C ) G -ratio plotted against axon diameter of WT (gray) and Pmp22 tg (red) mice (left panel) and Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg (purple) mice (right panel) at P18. WT n = 3, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. ( D ) Distribution of g -ratios shown in the left panel displays Pten fl/+ Dhh cre/+ Pmp22 tg femoral nerves have more axons with low g-ratios (0.4–0.5) and less axons with higher g-ratios (0.8–0.9, 0.9–1.0) compared to Pmp22 tg nerves. Mean g -ratio (right panel) showed a trend to be decreased in Pmp22 tg femoral nerves compared to WT and were significantly decreased in Pten fl/+ Dhh cre/+ Pmp22 tg femoral nerves. WT n = 3, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. Groups were compared using two-way ANOVA with Tukey’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). ( E ) G -ratio plotted against axon diameter of WT (gray) and Pmp22 tg (red) mice (left panel) and Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg (purple) mice (right panel) at 16 weeks of age. WT n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. ( F ) No alteration in the distribution of axons over the g-ratio (left panel) as well as mean g -ratio (right panel) is observed comparing femoral nerves from Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. Groups were compared using two-way ANOVA with Tukey’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). ( G ) The grip strength of hindlimbs is reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to WT controls and Pten fl/+ Dhh cre/+ mice. Behavioral analysis was done at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9–14 mice were analyzed. ( H ) Nerve conduction velocity (NCV, left panel) and compound muscle action potential amplitudes (CMAP, right panel) are similarly lower in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to WT controls. For electrophysiology measurements WT n = 10, Pten fl/+ Dhh cre/+ n = 8, Pmp22 tg n = 11 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 8 mice were analyzed. Data information: Means are displayed ±standard deviation. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) if not indicated otherwise. .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Comparison, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Western Blot analysis shows PTEN and PMP22 protein amounts in whole sciatic nerve lysates from 16 weeks old WT, PTEN heterozygous knockout ( Pten fl/+ Dhh cre/+ ), CMT1A ( Pmp22 tg ) and double mutant ( Pten fl/+ Dhh cre/+ Pmp22 tg ) mice using whole protein staining as loading control. ( B ) G -ratio plotted against axon diameter of wildtype (WT, gray) and Pten fl/+ Dhh cre/+ (turquoise) femoral nerves at P18. Mean g -ratio is unaltered in Pten fl/+ Dhh cre/+ and Pmp22 tg mice compared to WT controls and decreased in Pten fl/+ Dhh cre/+ Pmp22 tg mice (left panel). Mean axon diameters are reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (middle panel). The number of Schwann cell nuclei per femoral nerve cross section is increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 3, Pten fl/+ Dhh cre/+ n = 4, Pmp22 tg n = 5 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 3 animals. ( C ) G -ratio plotted against axon diameter of WT (gray) and Pten fl/+ Dhh cre/+ (turquoise) femoral nerves at 16 weeks of age. Mean g-ratio is unaltered in Pten fl/+ Dhh cre/+ mice compared to WT controls and decreased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (left panel). Mean axon diameters are reduced in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice (middle panel). The number of Schwann cell nuclei per femoral nerve cross section is increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice. WT n = 4, Pten fl/+ Dhh cre/+ n = 4, Pmp22 tg n = 3 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 4 animals. ( D ) The number of slips on the elevated beam is similarly increased in Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to wildtype controls at all time points. Behavioral analysis was done at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9–14 mice were analyzed. ( E ) Neither the weight of Pmp22 tg nor Pten fl/+ Dhh cre/+ Pmp22 tg mice is altered compared to wildtype controls at 12, 16 and 24 weeks of age. WT n = 10–14, Pten fl/+ Dhh cre/+ n = 6–14, Pmp22 tg n = 9–19 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 9-14 mice were analyzed. ( F ) Sensory nerve action potential amplitudes (SNAP) are decreased in the tail of Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg mice compared to wildtype controls. For electrophysiology measurements WT n = 10, Pten fl/+ Dhh cre/+ n = 8, Pmp22 tg n = 11 and Pten fl/+ Dhh cre/+ Pmp22 tg n = 8 mice were analyzed. ( G ) Example images of teased fiber preparations of WT, Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg double mutants stained for MAG (green), NaV1.6 (magenta) and DAPI (blue) at P18. Internodes between two nodes (magenta arrowheads) are underlined in yellow and respective Schwann cell nuclei are marked by white stars. Mean internodal length (left panel) is significantly reduced in Pmp22 tg teased fibers compared to wildtype controls at P18, whereas Pten fl/+ Dhh cre/+ Pmp22 tg mice do not differ in internodal length compared to Pmp22 tg mice. Mean fiber diameters are not significantly altered (right panel). Analysis was performed on 100 internodes of n = 3–4 animals per group. ( H ) Example images of teased fiber preparations of WT, Pten fl/+ Dhh cre/+ , Pmp22 tg and Pten fl/+ Dhh cre/+ Pmp22 tg double mutants stained for MAG (green), NaV1.6 (magenta) and DAPI (blue) at 16 weeks of age. Internodes between two nodes (magenta arrowheads) are underlined in yellow and respective Schwann cell nuclei are marked by white stars. Mean internodal length (left panel) and fiber diameter (right panel) are significantly reduced in Pmp22 tg teased fibers compared to wildtype controls at 16 weeks of age, whereas Pten fl/+ Dhh cre/+ Pmp22 tg mice do not differ in internodal length compared to Pmp22 tg mice. Analysis was performed on 100 internodes of n = 3–4 animals per group. Data information: Means are displayed ± standard deviation. Statistical analysis was done using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Knock-Out, Mutagenesis, Staining, Control, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Quantitative RT-PCR from P18 tibial nerves of wildtype (WT) ( n = 4), Pten fl/+ Dhh cre/+ ( n = 5), Pmp22 tg ( n = 5) and Pten fl/+ Dhh cre/+ Pmp22 tg ( n = 5) mice shows relative mRNA expression of Pten, Hmgcr, Nrg1-I, Pou3f1, Ngfr , and Sox2 . Rplp0 and Ppia were used as housekeeping genes. ( B ) Quantitative RT-PCR from P18 tibial nerves of WT ( n = 4) and Pmp22 +/- ( n = 8) mice shows relative mRNA expression of Pten, Hmgcr, Nrg1-I, Pou3f1, Ngfr , and Sox2 . Rplp0 and Ppia were used as housekeeping genes. ( C ) Semi-thin sections of P18 femoral nerves of Pmp22 +/- (left), WT (middle) and Pmp22 tg (right) mice. Pmp22 +/- nerves show myelin overgrowth, the so-called tomacula (to, yellow) and Pmp22 tg nerves are characterized by amyelinated (a, pink) and thinly myelinated (th, green) big axons as well as hypermyelinated (h, orange) small axons. Quantification shows g-ratios of Pmp22 +/- mice (blue, without tomacula) are similar to the WT (gray) distribution, while Pmp22 tg mice (red, without amyelinated) show the hypermyelination of small axons and demyelination of big axons. Data information: Means are displayed ±standard deviation. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). .

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: ( A ) Crossing scheme of Schwann cell specific full Pten knockout mice ( Pten fl/fl Dhh cre/+ ) with CMT1A mice ( Pmp22 tg ) to generate a full Pten knockout in CMT1A mice ( Pten fl/fl Dhh cre/+ Pmp22 tg ). ( B ) Teased fiber preparations of 8 weeks old PTEN fl/fl Dhh cre/+ (upper panel) and Pten fl/fl Dhh cre/+ Pmp22 tg mice (lower panel) show focal myelin thickening at paranodal loops as indicated by yellow arrows. Scale bar = 20 µm. ( C ) Semi-thin cross section of femoral nerves from WT, Pten fl/fl Dhh cre/+ , Pmp22 tg and Pten fl/fl Dhh cre/+ Pmp22 tg mice at 8 weeks of age. Red arrows indicate myelin abnormalities such as outfoldings and tomacula, asterisks indicate amyelinated axons. Scale bar = 10 µm. ( D – F ) Quantification of ( C ) displays reduced axon numbers in Pten fl/fl Dhh cre/+ Pmp22 tg mice compared to wildtype controls ( D ). The percentage of amyelinated axons is increased in Pmp22 tg and further elevated in Pten fl/fl Dhh cre/+ Pmp22 tg mice ( E ). Pten depletion alone and in Pmp22 tg leads to an increase in axons with aberrant myelin profiles ( F ). WT n = 5, PTEN fl/fl Dhh cre/+ n = 4, Pmp22 tg n = 6 and Pten fl/fl Dhh cre/+ Pmp22 tg n = 3 animals were analyzed. Data information: Means are displayed ± standard deviation. Statistical analysis was done using one-way ANOVA with Sidak’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knock-Out, Standard Deviation, Comparison

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: Genotyping primers.

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Sequencing

Journal: EMBO Molecular Medicine

Article Title: Targeting PI3K/Akt/mTOR signaling in rodent models of PMP22 gene-dosage diseases

doi: 10.1038/s44321-023-00019-5

Figure Lengend Snippet: qRT-PCR primers.

Article Snippet: For interaction analysis of heterologously expressed proteins, plasmids encoding for PTEN with an N-terminal FLAG-tag (Vectorbuilder, #VB220531-1217bxp), untagged PTEN (#VB220603-1029rjt) or PMP22 with a C-terminal ALFA-tag (#VB210322-1182nwx) were transfected into HE293T cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Sequencing