Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A and B ) Increased Phe concentration in the serum ( A ) and CSF ( B ) of adult Pah Enu2 mouse. ( C ) Experimental design for NMDAR-EPSC (top) and I NMDA (bottom) recordings. Stim., stimulating electrode; Rec., recording electrode. ( D ) Representative traces of NMDAR-EPSCs (top) obtained at the indicated time points ( , ), and the time course of the peak amplitudes (bottom left) of NMDAR-EPSCs measured at −40 mV in CA1 neurons. Bottom right: l -Phe had no effect on the peak amplitudes of NMDAR-EPSCs. ( E ) In the presence of NBQX and picrotoxin, I NMDA was induced by bath application of NMDA (5−10 μM). Different concentrations of l -Phe were perfused with NMDA for 5 minutes. ( F ) l -Phe exhibits concentration-dependent bidirectional effects on I NMDA . ( G ) I NMDA was induced by 3−12 μM NMDA in the presence of GluN2A or GluN2B blockers. ( H ) l -Phe-induced facilitation of I NMDA was blocked by Ro (2 μM) or ifen (6 μM) but not by PEAQX (0.5 μM). ( I and J ) A sample trace (left) and summary (right) of I NMDA measured before and during l -Phe perfusion in HEK293 cells expressing GluN2A ( I ) or GluN2B ( J ). ( K ) Representative traces (top) and the peak amplitudes (bottom left) of NMDAR-EPSCs measured in the presence of TBOA (10 μM). l -Phe induced facilitation of NMDAR-EPSCs in each condition (bottom right). ( L ) Addition of 5, 10, and 20 μM glycine attenuated l -Phe-induced I NMDA facilitation. I NMDA was induced by 5 μM NMDA. ( M ) The concentration relationship between l -Phe-induced facilitation of I NMDA and added glycine (Gly) concentration. A Student’s t test ( A , B , D , F , I , and J ) or a 1-way ANOVA with a post hoc Tukey’s test ( H and K ) was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05. Scale bars: 50 ms and 50 pA ( D and K ) Veh, vehicle.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Concentration Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A – D ) Representative Western blots and expression levels of NMDAR subunits in the total ( A and B ) and synaptosomal fractions ( C and D ) of WT and Pah Enu2 hippocampi. ( E ) Representative AMPAR- and NMDAR-EPSCs measured at −70 and +40 mV, respectively, in CA1 pyramidal neurons. ( F ) The peak amplitudes of NMDAR-EPSCs were plotted against AMPAR-EPSCs. ( G ) Normal AMPA to NMDA ratios in the CA1 pyramidal cells of Pah Enu2 mice. ( H ) Bath application of NMDA (5 μM) induced similar magnitudes of inward current in CA1 pyramidal neurons of WT and Pah Enu2 mice. I NMDA was measured in the presence of NBQX and picrotoxin. PEAQX (0.5 μM) reduced I NMDA , and subsequent AP-5 (50 μM) perfusion blocked the remnant. ( I ) Total I NMDA and PEAQX-sensitive and -insensitive components after sequential application of PEAQX and AP-5 in CA1 pyramidal cells. ( J ) Hippocampal expression levels (bottom) of α5-GABA A R were determined by Western blotting (top). ( K ) Representative traces of tonic currents measured from WT and Pah Enu2 hippocampal CA1 neurons in the presence and absence of Phe. Vehicle (Veh) or Phe (250 μM) were perfused throughout the recordings. Scale bar: 30 seconds and 100 pA. ( L ) Magnitudes of bicuculline (40 μM)-sensitive tonic current in each condition are summarized. Statistical analysis was performed using Student’s t test ( B , D , G , I , and J ) and 2-way ANOVA ( L ). ** P < 0.01 and NS, P ≥ 0.05.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A ) l -Phe reduces the magnitude of LTP in both WT and Pah Enu2 mice. Top: representative traces of fEPSP obtained at the indicated time points. Bottom left: fEPSP slopes were normalized to those obtained in the baseline and plotted against time. l -Phe was perfused from 5 minutes before to 1 minutes after TBS (arrow). Bottom right: fEPSP slopes during the last 10 minutes were normalized to baseline. ( B ) GluN2B antagonists block the effect of l -Phe on the TBS (arrow)-induced LTP. Sample traces (top), time course of fEPSP slopes (bottom left), and the magnitude of LTP (bottom right) in each condition. ( C and D ) PEAQX blocks LTP induction, and l -Phe perfusion during the peri-TBS period induces an LTD-like decrease in fEPSP slopes. ( E and F ) l -Phe and TBS had no effect on the slope of fEPSPs under GluN2A and GluN2B inhibition. ( G − K ) Representative Western blots ( G ), and the ratios of phosphorylated GluA1-S845 to total GluA1 ( H ), total GluA1 to -tubulin ( I ), phosphorylated GluA2-S880 to total GluA2 ( J ), and total GluA2 to -tubulin ( K ) in the CA1 region of acute hippocampal sections harvested 30 minutes after TBS. ( L ) WT and Pah Enu2 sections exhibit similar synaptic responses to LFS. ( M ) Normalized fEPSP slopes during the last 10 minutes in WT and Pah Enu2 sections. ( N ) Ro blocks the effect of l -Phe on LTD facilitation. Sample traces of fEPSPs (top). ( O ) Normalized fEPSP slopes during the last 10 minutes in each condition. ( A − C , E , L , and N ) Scale bars: 5 ms and 0.5 mV. Statistical analysis was performed using Student’s t test ( D , F , and M ), 1-way ( H − K , and O ) or 2-way ANOVA ( A ), or Kruskal-Wallis test ( B ) with post hoc Tukey’s test. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05. Con, control; Veh, vehicle.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Blocking Assay, Inhibition, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: GluN2B suppression restores phenylalanine-induced neuroplasticity and cognition impairments in a mouse model of phenylketonuria

doi: 10.1172/JCI184299

Figure Lengend Snippet: ( A and B ) Phe concentrations in the serum ( A ) and CSF ( B ) were measured 30 minutes after vehicle (Veh) or l -Phe (1 mg/g, i.p.) administration. ( C ) Western blot analyses for the protein levels of p-eEF2 and eEF2 in Veh- and l -Phe–treated mice. Whole brains (WB) and hippocampi (HP) were collected 30 minutes after Veh or l -Phe (1 mg/g, i.p.) administration. Bottom: Quantification of the p-eEF2 to eEF2 ratio. ( D ) Enhanced eEF2 phosphorylation (top) and increased p-eEF2 to eEF2 ratio (bottom) in the Pah Enu2 hippocampus. ( E ) Experimental design for fiber photometry recording in the mPFC of Camk2a-Cre mice. Bottom: immunohistochemical staining of an mPFC section showing the GCaMP6s-expressing cells (green) and canula placement. DAPI (blue) was used to identify the brain structures. Calibration, 200 μm. ( F ) Neuronal activity of CaMKII-expressing cells in the mPFC was decreased by l -Phe administration. The bottom panel shows the ΔF/F signals obtained during the indicated periods (1, 2, and 3) on an expanded time scale. ( G ) Quantification of the frequency of Ca 2+ transients obtained from 5 mice. ( H ) Ro blocks l -Phe–induced eEF2 phosphorylation in the hippocampus. Ro (3 mg/kg, i.p.) was administered 30 minutes before l -Phe or vehicle injection. ( I ) l -Phe and Ro were sequentially administered to Camk2a-Cre mice during the recording. ( J ) Summary of the frequency of F transients during the baseline and perfusion of l -Phe and Ro. Statistical analysis was performed using the Mann-Whitney U test ( A ), Student’s t test ( B − D and H ), or 1-way ANOVA with post hoc Tukey’s test ( G and J ). * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, P ≥ 0.05.

Article Snippet: Pah Enu2 mice (Jackson Laboratory; catalog 002232) were provided by Sung-Chul Jung (Ewha Womans University) and backcrossed with C57BL/6N mice for at least 5 generations before use.

Techniques: Western Blot, Phospho-proteomics, Immunohistochemical staining, Staining, Expressing, Activity Assay, Injection, MANN-WHITNEY

Journal: Experimental Biology and Medicine

Article Title: Gender difference in pre-clinical liver-directed gene therapy with lentiviral vectors

doi: 10.3389/ebm.2025.10422

Figure Lengend Snippet: Differential transduction efficiencies in male vs. female Pah enu2 mice dosed IV with either OXB-401 or OXB-Null. Median VCN is shown for each group at day 85 post dosing. No significant differences were observed between the groups (p > 0.05).

Article Snippet: For the second study, BTBR- Pah enu2 /J mice [ ], also referred to as Pah enu2 ) were obtained from The Jackson Laboratory and dosed IV with TSSM buffer (200 μL), OXB-Null (4 × 10 10 TU/kg) or OXB-401 (4 × 10 10 TU/kg) and observed for the duration of the study (85 days).

Techniques: Transduction

Journal: Communications Biology

Article Title: Hyperphenylalaninemia and serotonin deficiency in Dnajc12 -deficient mice

doi: 10.1038/s42003-024-07360-6

Figure Lengend Snippet: a Representative western blot for PAH in the liver of DNAJC12 KO and wild-type (WT) mice. b Quantification of PAH expression in the liver of DNAJC12 KO ( n = 7) mice in comparison to WT ( n = 7). GAPDH was used as loading control. GAPDH was used as loading control. Phe concentrations in the liver ( c ) and serum ( d ) of DNAJC12 KO and PAH enu2 (PAH KO) mice relative to wild-type (WT) mice ( n = 4). e 5-HTP in vitro generation assay in the liver of DNAJC12 KO ( n = 5), WT ( n = 5) and TPH1/TPH2-double KO ( n = 2) mice. *, p < 0.05, ** p < 0.01, *** *p < 0.0001. b : Student’s t -test; c–e: One-way ANOVA wi t h Tukey’s multiple comparisons. Data are presented as mean ± SEM.

Article Snippet: PAH enu2 mice (PAH-KO) were obtained from Jackson Laboratories, USA (#002232).

Techniques: Western Blot, Expressing, Comparison, Control, In Vitro

Journal: JCI Insight

Article Title: SLC6A19 inhibition facilitates urinary neutral amino acid excretion and lowers plasma phenylalanine

doi: 10.1172/jci.insight.182876

Figure Lengend Snippet: ( A ) JN-170 pharmacokinetics in male Pah enu2 mice after a single oral dose ( n = 6). ( B ) Changes in 12 hour urine amino acid excretion in male Pah enu2 mice treated with JN-170 ( n = 4–6 per dose) or vehicle (veh, n = 24) or in mice with heterozygous or homozygous loss of Slc6a19 (data adapted from ). Data are displayed as log FC. ( C ) 12-hour urine Phe excretion in mice treated with vehicle ( n = 24) or JN-170 ( n = 6); 1-way ANOVA with Tukey’s post hoc analysis. ( D ) Plasma Phe in male Pah enu2 mice treated with vehicle ( n = 24) or 50, 100, 200, or 250 mg/kg JN-170 ( n = 6); 2-way ANOVA with Dunnett’s post hoc analysis to compare vehicle versus JN-170 groups (statistical analysis shown for comparison of vehicle to 250 mg/kg JN-170). ( E – G ) Plasma Phe in male Pah enu2 mice fed defined amino acid diets to modulate basal plasma Phe levels. Mice were fed 0.75% Phe ( E , n = 17–22), 0.45% Phe ( F , n = 4–9) or 0.225% Phe ( G , n = 11–17) for 2 weeks prior to administration of a single 200 mg/kg dose of JN-170 or vehicle; 2-way ANOVA with Šidák’s post hoc analysis.** P < 0.01, *** P < 0.001, **** P < 0.0001; All data represent mean ± SD.

Article Snippet: Male homozygous BTBR-Pah/J ( Pah enu2 ) aged 10–13 weeks were purchased from Jackson Laboratory (no. 002232).

Techniques: Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes

doi: 10.1016/j.omtm.2023.05.004

Figure Lengend Snippet: Comparison of in vivo bioactivity of pOXB dual and triple transfection vectors BTBR Pah enu2 mice were systemically injected with (A) a low dose of 1 x 10 12 VG/kg or (B) a high dose of 1 x 10 14 VG/kg of vector produced by pOXB dual- or triple-plasmid transfection. A vehicle-only injection group was included as a negative control. Mice were monitored over 6 weeks including weekly serum Phe analysis. At endpoint, liver samples were collected to analyze (C) VGs and (D) mRNA expression. Statistical significance was determined using a Student's t test; ns, not significant.

Article Snippet: A colony of BTBR Pah enu2 was maintained in the animal facility at Homology Medicines under standard laboratory conditions, and animals were supplied with food and water ad libitum .

Techniques: Comparison, In Vivo, Transfection, Injection, Plasmid Preparation, Produced, Negative Control, Expressing