Metody badania RNA w neuronach. Magdalena Dziembowska

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1 Metody badania RNA w neuronach Magdalena Dziembowska

2 T cell fibroblast A Golgi-stained pyramidal cell in the parietal cortex of a rat. The high power images at the right show dendritic spines on apical and basilar dendritic branches. Photo by Grazyna Gorny

3 Polyribosomes localized in dendritic spines Steward and Levy w 1982

4 Synaptic spines are structural units that undergo plastic changes in response to synaptic stimulation Fig. 1: A segment of pyramidal cell dendrite from stratum radiatum (CA1) with thin, stubby, and mushroom-shaped spines. Spine synapses colored in red, stem (or shaft) synapses colored in blue. The dendrite was made transparent in the lower image to enable visualization of all synapses. Photo by Josef Spacek.

5 Local mrna translation in dendritic spines

6 A model of Arc-dependent LTP consolidation in the dentate gyrus

7 Metody wizualizacji mrna w neuronach MAP2 GFAP

8 In situ hybrydyzacja z sonda RNA wyznakowana digoksygeniną

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10 High-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mrna localization dynamics during early Drosophila embryogenesis. Lecuyer et al. 2007

11 Single molecule FISH to metoda in situ hybrydyzacji pozwalająca na obrazowanie pojedynczej cząsteczki mrna w komórce dzięki wykorzystaniu wielu fluorescencyjnie wyznakowanych sond zaprojektowanych do rozpoznawania sekwencji w obrębie tej samej cząsteczki mrna Przy użyciu tej metody można np. jednocześnie wykrywać dwa różne mrna w komórce lub mrna postałe w wyniku fuzji 2 transkryptów (translokacji genomowych) jak np. BCR-ABL

12 Obrazowanie mrna w dendrytach komórek nerwowych z wykorzystaniem smfish

13 Activity-dependent local translation of MMP-9 Dziembowska et al., J Neuroscience 2012 Dziembowska and Wlodarczyk, Int J Biochem Cell Biol, 2012

14 Medial perforant path LTP - a well established model of synaptic plasticity

15 In situ hybridization shows increse in MMP-9 expression in granular layer and molecular layer of dentate gyrus 2h after medial perforant path LTP

16 MMP-9 in situ hybridization Arc in situ hybridization

17 Sushi belt model Figure 2 from Michael Doyle and Michael A Kiebler The EMBO Journal online publication doi: /emboj European Molecular Biology Organization.

18 MS2 system to stain targeted mrna in the living cell Martha S. Rook, Mei Lu, and Kenneth S. Kosik

19 MS2 system to stain targeted mrna in the living cell GFP GFP GFP GFP GFP Dziembowska et al., J Neurosci. 2012

20 MMP-9 protein is enriched in the synaptoneurosomal fraction Hippocampus or cortex HOMOGENIZATION H F SN C FILTRATION (PVDF 100, 60, 30, 10 μm) Homogenate (H) PSD-95 GFAP c-jun Filtrate (F) CENTRIFUGATION (1000 rpm, 30 min) GAPDH MMP-9 Synaptoneurosomes (SN) Washing and centrifugation Cytosol (C) 1. Polyadenylation of MMP-9 in response to synaptic stimulation 2. Isolation of polyribosomes 3. Direct study of MMP-9 protein synthesis by Click-iT chemistry

21 Synaptoneurosomy w mikroskopie elektronowym

22 PAT assay

23 MMP-9 polyadenylation measured by PAT ssay in synaptoneurosomes after glutamate stimulation A PolyA I CPE I CPE II PolyA II PolyA III STOP MMP-9 CDS 3 UTR B CPE I R. norvegicus M. musculus 2416 ACCUUUUGUUUUUAUGGG ACCUUUUAUUUUUGUGUG 2519 CPE II R. norvegicus M. musculus 2500 CCCUUUUAUUUAUUAUGU CCCUUUUAUUUAUUAUGU 2609

24 Polysomal fractionation

25 Polyribosome association of MMP-9 mrna in synaptoneurosomes J. Miłek Dziembowska et al., J Neurosci. 2012

26 Dziembowska et al., J Neurosci Local synthesis of MMP-9 protein detected with click chemistry Synaptoneurosomes Incubation with glycine analogue (HPG) and stimulation Protein isolation (lysis in 1% SDS) And Click-iT reaction with AHA tagged with biotin Precipitation on streptavidin dynabeads Western blot with anti-mmp-9 antibody

27 Badanie oddziaływania białko-rna Fragile X syndrome is the most common form of inherited mental retardation in males and is also a significant cause of mental retardation in females. It affects about 1 in 4,000 males and 1 in 8,000 females. Nearly all cases of fragile X syndrome are caused by an alteration (mutation) in the FMR1 gene where a DNA segment, known as the CGG triplet repeat, is expanded.

28 Malformations of the dendritic spines may lead to cognitive disorders One of the characteristic features of FXS neurons is the abnormal dendritic spine morphology (Rudelli 1985). Immature, long and thin spines are also observed in FMR1 KO mice, a model of FXS. WT Fmr1 KO

29 Coimmunoprecipitation of FMRP protein with MMP-9 mrna Synaptoneurosome extract anti-fmrp antibody or normal IgG Dynabeads A Precleared extract Antibody-bound beads Immunoprecypitated complexes 1. Western blot 2. RNA isolation and RT-PCR

30 MMP-9 mrna is precipitated with anti-fmrp antibodies Janusz et al., J Neuroscience 2013

31 Polysomal fraction isolated from WT and Fmr1KO mice before and after stimulation with DHPG Janusz et al., J Neuroscience 2013

32 Local translation of MMP-9 is regulated by FMRP MMP-9 minocycline Nature Medicine Volume:17,Pages: (2011)

33 Dziembowska i wsp., Am J Med Genet A. 2013