Journal: Journal of Cell Science

Article Title: EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

doi: 10.1242/jcs.207423

Figure Lengend Snippet: ARF6 is regulated differently in CNS versus PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of the fluorescence signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (10 DIV) labelled with GGA3–ABD–GST to detect active ARF protein; spectrum colouring of the highlighted section indicates the highest signals. The asterisk and red colour in the CNS panel indicate neurofascin labelling of the AIS to identify the axon. Graph shows quantification of mean±s.e.m. ARF protein activation in the two axon types. n =58 (DRG) and 60 (cortical). *** P <0.001 (two-tailed t -test).

Article Snippet: DRG neuronal cultures were obtained from adult male Sprague Dawley rats.

Techniques: Comparison, Fluorescence, Activation Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Pushing myelination – developmental regulation of myosin expression drives oligodendrocyte morphological differentiation

doi: 10.1242/jcs.232264

Figure Lengend Snippet: The myosinome of developing OLs – transcriptomic and proteomic datasets reveal contrasting temporal expression patterns. (A,B) Data mining for the mRNA and protein spatiotemporal expression profile of the different members of the myosin network during OL differentiation. This information was collected from available in vitro, ex vivo and in vivo high-throughput transcriptomic and proteomic datasets or specific studies in human, mouse and rat. It reports the presence of specific myosins in myelinated fractions isolated from in vivo CNS tissue, at the mRNA and protein levels (De Monasterio-Schrader et al., 2012; Thakurela et al., 2016). Also, it describes quantitatively the temporal expression pattern for mRNA and protein during different stages of ex vivo and in vitro OL differentiation (unchanged, upregulated or downregulated) (Cahoy et al., 2008; Wang et al., 2012; Zhang et al., 2014; Marques et al., 2016; Yamazaki et al., 2017). Finally, it identifies myosin mRNAs subcellularly enriched in OPC protrusions (Azevedo et al., 2018). In light gray are highlighted seven myosins consistently identified in several studies: Myo1d, Myh9, Myh10, Myh14, Myo5a, Myo6 and Myo18a. Their transcript expression profiles during different stages of mouse OL differentiation (OPC, newly formed OL and myelinating OL) are graphically represented for one of the studies (Zhang et al., 2014; http://www.brainrnaseq.org) (B). These were reported as fragments per kilobase of transcript sequence per million mapped fragments (FPKM) and plotted according to their expression pattern during OL differentiation: mRNA expression of Myh9, Myh10 and Myo5a is markedly down-regulated (left graph), while the expression of Myo1d, Myh14, Myo6 and Myo18a transcripts is up-regulated (right graph). Data are presented as mean±s.e.m. of two replicates of pooled animals for each cell type, according to Zhang et al., 2014. (C) Western blot from OPCs (PDGF condition) and OL under differentiation (days 1, 2 and 3 after differentiation in T3-containing medium) showing the correlation between protein levels of NM2A, NM2B (downregulation) and NM2C, Myo18a (up-regulation) and MBP protein expression. Data in graphs represent the ratio of each protein relative to actin band (mean±s.e.m.; from three lysates). Myosin ratios were normalized to the baseline PDGF value. *P<0.05; ****P<0.0001 [Krustal–Wallis non-parametric test followed by Dunn's multiple comparison (PDGF vs 3dT3)].

Article Snippet: Purified OPC cultures and OPC–DRG myelinating co-cultures Primary OPCs were purified by immunopanning from mixed glial cultures of postnatal day 1 rat (Sprague Dawley) cerebral cortices as previously described ( Wang et al., 2008 , 2012 ).

Techniques: Expressing, In Vitro, Ex Vivo, In Vivo, High Throughput Screening Assay, Isolation, Sequencing, Western Blot, Comparison

Journal: Journal of Cell Science

Article Title: Pushing myelination – developmental regulation of myosin expression drives oligodendrocyte morphological differentiation

doi: 10.1242/jcs.232264

Figure Lengend Snippet: Knockdown of NM2B and NM2C in OPC–DRG co-cultures have opposite effects in OL differentiation and myelin formation. Knockdown of NM2B and NM2C in OPC co-cultured with DRG neurons. (A) Representative images of 3-week-old myelinating 3D OPC–DRG co-cultures stained for MBP, Olig2 and NF. Co-cultures were infected with a lentiviral vector expressing shRNA against the NM2B, NM2C (shNM2B, shNM2C) or non-targeting sequence (shCtrl). Images on the right are magnified views of the boxed area showing representative myelinated segments for each of the conditions. Scale bars: 20 μm. (B) Quantification of MBP-positive (MBP+) myelin segments in OPC–DRG co-cultures. Data are mean±s.e.m. values obtained from the quantification of three independent experiments (10–12 fields per culture and three or four cultures per condition/per experiment). *P=0.02 (shCTRL vs shNM2C); ****P<0.0001 (shCTRL vs shNM2B) (Krustal–Wallis non-parametric test followed by Dunn's multiple comparison test). (C) Histogram showing the distribution of MBP+ segments across all experimental conditions.

Article Snippet: Purified OPC cultures and OPC–DRG myelinating co-cultures Primary OPCs were purified by immunopanning from mixed glial cultures of postnatal day 1 rat (Sprague Dawley) cerebral cortices as previously described ( Wang et al., 2008 , 2012 ).

Techniques: Knockdown, Cell Culture, Staining, Infection, Plasmid Preparation, Expressing, shRNA, Sequencing, Comparison

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: An Apolipoprotein E-Mimetic Stimulates Axonal Regeneration and Remyelination after Peripheral Nerve Injury

doi: 10.1124/jpet.110.167882

Figure Lengend Snippet: COG112 stimulates neurite outgrowth from cultured DRG neurons. Left, the representative examples of primary DRG neurons from P2 Sprague-Dawley rat pups after treatment with either COG112 (0.1 μM) or Antp (0.1 μM), COG133 (1 μM, not shown) or NGF (2.5S, 200 ng/ml) for 5 days. On day 5, cells on coverslips were stained overnight with primary antibody TUJ1 (Abcam Inc.), followed by secondary antibody for 1 h (Alexa Fluor 488; Invitrogen). Axonal length (the longest axon of each neuron) was measured using Image-Pro Plus software, and the quantitative data were presented in adjacent graph. The columns present axonal length as means ± S.E.M. ANOVA analysis followed by post-hoc Newman Keuls test was used to compare the differences among groups. NS, not significant; *, p < 0.05; ***, p < 0.001 versus control.

Article Snippet: DRG Culture Isolated primary rat DRG from P2 Sprague-Dawley rat pups were mechanically dissociated with use of an 18-gauge needle before resuspending in culture media (DMEM/Neurobasal A, 1× B27 supplement, 0.5% FBS, penicillin/streptomycin, 10 ng/ml NGF).

Techniques: Cell Culture, Staining, Software, Control