Journal: Glia

Article Title: Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling

doi: 10.1002/glia.23173

Figure Lengend Snippet: Upregulation of endosomal-lysosomal membranes and impaired autophagy in Vps34SCKO nerves. The levels of LC3-I and -II, p62, Lamp1, and EEA1 in sciatic nerve extracts were examined by immunoblotting at P3, P7, P21 and P56 (A–D). The abundance of each protein relative to that of the control is presented graphically. (A) At P3, the levels of the examined proteins were not significantly altered in mutant nerves. (B) By P7, there is a significant increase in LC3-I/II, p62, and Lamp1 levels in Vps34SCKO nerve extracts (EEA1: p = 0.27). (C & D) At P21 and P56, significant increases in the levels of LC3-I/II, p62, Lamp1, and EEA1 protein were observed in Vps34SCKO nerves. At each age, the average control value for a given protein was normalized to 1 (n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; mean ± SEM). For all ages, n = 3 independent extracts per genotype. Each sciatic nerve extract (the biological replicate) was prepared by homogenizing the (pooled) nerves from three mice of the same genotype at P3 or P7, and one mouse at P21 and P56.

Article Snippet: Rabbit monoclonal antibodies (mAb) for Vps34, EEA1, Rab5, ErbB3, pErbB3 (Tyr1289), ErbB2, S6, pS6 (Ser235/236), pAkt (Ser473), pAkt (Thr308), and pJNK (Thr183/Tyr185) were from Cell Signaling Technology.

Techniques: Western Blot, Control, Mutagenesis

Journal: Glia

Article Title: Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling

doi: 10.1002/glia.23173

Figure Lengend Snippet: Proposed model for the role of Vps34 in endo-lysosomal trafficking of ErbB2/3 in Schwann cells. Binding of axonal Nrg1-III to ErbB3 induces receptor heterodimerization and stimulates ErbB2 kinase activity. ErbB2 phosphorylates both itself and ErbB3, creating phosphotyrosine-based docking sites for downstream signaling proteins (lightning bolt). Activation of ErbB2/3 triggers internalization by clathrin-mediated endocytosis. Fusion of ErbB2/3 containing vesicles with early endosomes requires PI3P, which is produced by Vps34, and requires EEA1 and Rab5 (Step 1) (Simonsen et al., 1998). Internalized ErbB2/3 heterodimers likely continue to signal from endosomal compartments, and can be recycled from endosomes to the cell membrane. On endosomes, ubiquitination (Ub) of ErbB2/3 targets the heterodimer for sorting into multivesicular bodies (MVBs). In a second Vps34-dependent step, PI3P recruits Hrs, which binds concomitantly to ubiquitin on ErbB2/3. Hrs recruits the ESCRT complex, which drives the budding event that generates the receptor-containing internal vesicles of MVBs (Step 2) (Fernandez-Borja et al., 1999). Once in MVBs, ErbB2/3 signaling is terminated (Lee et al., 2017). PI3P also mediates early-to-late endosome maturation through the recruitment of Rab7 (Poteryaev et al., 2010) (Step 3). ErbB2/3 receptors are degraded when late endosomes fuse with LAMP1-positive lysosomes (Lee et al., 2017). We propose that, in Vps34−/− Schwann cells the depletion of PI3P results in a slowing or blockade of trafficking Steps 1, 2, and 3 (red dashed lines). Such trafficking defects are predicted to reduce the fusion of ErbB2/3-bearing endocytic vesicles with early endosomes (Step 1), and prevent the sorting of phosphorylated ErbB2/3 into MVBs (Step 2). This proposed abnormal trafficking of ErbB2/3 is consistent with our observations of both enhanced phosphorylation of ErbB3 on Y1289 and altered posttranslational modification of ErbB2 in Vps34SCKO peripheral nerves. Vps34−/− Schwann cells are also likely deficient in Rab7-dependent late endosome maturation (Step 3). Finally, abnormal MVBs/late endosomes may fuse inefficiently with lysosomes (Step 4), resulting in lysosome accumulation.

Article Snippet: Rabbit monoclonal antibodies (mAb) for Vps34, EEA1, Rab5, ErbB3, pErbB3 (Tyr1289), ErbB2, S6, pS6 (Ser235/236), pAkt (Ser473), pAkt (Thr308), and pJNK (Thr183/Tyr185) were from Cell Signaling Technology.

Techniques: Binding Assay, Activity Assay, Activation Assay, Produced, Membrane, Ubiquitin Proteomics, Phospho-proteomics, Modification

Journal: Glia

Article Title: Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling

doi: 10.1002/glia.23173

Figure Lengend Snippet: Vps34-deficient Schwann cells are hyperproliferative. (A) Longitudinal sections of sciatic nerves from control (upper) and Vps34SCKO (lower) mice were examined by immunofluorescence with an antibody for Ki67 (red) and DAPI (blue) staining at the indicated ages. (B) At P7, the percentage of proliferating (Ki67+) nuclei was not significantly different in control and Vps34SCKO nerves (p = 0.5372; n = 3 nerves for each genotype). (C) However, by P14, significant hyperproliferation of Schwann cells is evident in Vps34SCKO nerves (n = 3 and 4 for control and Vps34SCKO mice, respectively; n.s., not significant; ***p < 0.001; mean ± SEM). Scale bar: 100 μm. (D) Upregulation of ribosomal protein S6 in the nerves of Vps34SCKO mice. Phospho-S6 (Ser235/236) and total S6 protein levels in sciatic nerves were compared by immunoblotting. At P7, the levels of phospho-S6 and total S6 were not significantly different in the nerves of control and Vps34SCKO mice. Total S6 protein (arbitrary units); p = 0.1806; phospho-S6 protein (arbitrary units); p = 0.4390; n = 4 and 3 lysates for control and Vps34SCKO, respectively. However, by P21, both total S6 and phospho-S6 protein were significantly increased in Vps34SCKO nerves compared to controls (n = 2 and 3 lysates for control and Vps34SCKO, respectively).

Article Snippet: Rabbit monoclonal antibodies (mAb) for Vps34, EEA1, Rab5, ErbB3, pErbB3 (Tyr1289), ErbB2, S6, pS6 (Ser235/236), pAkt (Ser473), pAkt (Thr308), and pJNK (Thr183/Tyr185) were from Cell Signaling Technology.

Techniques: Control, Immunofluorescence, Staining, Western Blot

Journal: Journal of Neuroscience

Article Title: Behavioral, Neurophysiological, and Synaptic Impairment in a Transgenic Neuregulin1 (NRG1-IV) Murine Schizophrenia Model

doi: 10.1523/jneurosci.4632-15.2016

Figure Lengend Snippet: Figure7. PreclinicalrelevanceofPIK3CDsignalingandpharmacologicalinhibitioninNRG1-IV/NSEtTAmice.A,ErbB4,p110,andrelatedNRG1signalingproteinsareincreasedinthemPFCofNRG1-IV/NSE tTAmice(*p 0.05;#p 0.01,unpairedStudent’sttest;n6WTandn5–6NRG1-IV).B,Westernblots.C,ReversalofPPIdeficitsinNRG1-IV/NSEtTAmicewiththePIK3CDinhibitor,IC87114(n13 WTveh,n11WTIC,n11NRG1-IVveh,andn11NRG1-IVIC).Therewasasignificantinteractionbetweengenotypeanddrugtreatment:(p0.002)andamaineffectofgenotype(p0.027,GLM MANOVA).IC87114treatmentrescuedNRG1-IV-dependentPPIdeficits,significantlyimprovingPPIindrug-treatedNRG1-IV/NSE-tTAmicecomparedwithvehicle-treatedtransgenicmice(db74-db90;**p 0.005;byposthocLSD).NosignificanteffectofdrugonPPIwasobservedinWTmice.D,IntraperitonealadministrationofIC87114(0.1mg/kg)30minbeforethetestphaseofthetemporalorderobjectrecognition task(n13WTveh,n10WTIC,n6NRG1-IVveh,andn9NRG1-IVIC).Therewasasignificantinteractionbetweengenotypeanddrugtreatment(p0.02).IC87114treatmentrescuedtemporalorder discriminationdeficitsfoundinNRG1-IV/NSE-tTAmice(*p 0.05).NoeffectofdrugwasevidentinWTmice(p0.5).Again,asignificanteffectofgenotypewasconfirmedonrecencydiscriminationmemory invehicle-treatedmice(*p 0.05).E–H,NosignificanteffectofIC87114treatmentinNRG1-IV/NSE-tTAmicewasobservedintestsofsociabilityorsocialnovelty.Foreachmeasure,asignificantmaineffectof chamberwasconfirmedbyGLMRM-ANOVA:E,F(1,54)55.861,p 0.001;F,F(1,54)111.992,p 0.001;G,F(1,54)20.179,p 0.001;H,F(1,54)79.834,p 0.001.Nomaineffectoftreatmentor treatmentgenotypeinteractionswasobserved(WTVeh,n16;WTICn14;NRG1-IVVeh,n14;NRG1-IVIC,n14).PosthocLSDcomparisonsrevealedthatvariancesweresignificantlydifferent betweenchambers(novelmousevsnovelobject;E,F)and(novelmouse1vsnovelmouse2;G,H).**p 0.01;***p 0.001.DataareshownasmeansSEM.

Article Snippet: The following primary antibodies were diluted in 5% milk in Tris-buffered saline 1% Tween 20: anti-AKT-1 (1:500; Millipore; #07-416), anti-pAKTser473 (1:50; Millipore; #05-669-K), anti-pAKTthr308 (1:1000; Cell Signaling Technology; #2965), antimTOR (1:1000; Cell Signaling Technology; #2972), anti-pmTOR (1: 1000; Cell Signaling Technology; #2971), anti-S6 (1:1000; Cell Signaling Technology; #2317), anti-pS6 (1:2000; Cell Signaling Technology; #4858), anti-p110 (A-8 1:500; Santa Cruz Biotechnology, #sc-55589), anti-NMDAR1 (H-300 1:200; Santa Cruz Biotechnology; #sc-9058), anti-PSD95 (1:1000; Cell Signaling Technology; #2507), anti-ErbB4 (C-18 1:500; Santa Cruz Biotechnology; #sc-283), anti-pErbB4 (Tyr1056 1:500; Santa Cruz Biotechnology; #sc-33040), anti-pErbB3 (tyr1289 1:1000; Cell Signaling Technology; #4791), and anti- -actin-peroxidase (1:25000, Sigma-Aldrich #A3854).

Techniques: