Lecture 2 Engram - Plasticity and Molecular Manipulation Tools PDF

29-9-2021 What we will cover Neuroscience of the engram: Tools for memory manipulation • Molecular basis of plasticity and learning – aplysia • Genomic tools to manipulate plasticity – RNA and DNA based • Optogenetic tools to manipulate neural activity Peter De Weerd Vincent van de Ven Jan Zimm...

29-9-2021 What we will cover Neuroscience of the engram: Tools for memory manipulation • Molecular basis of plasticity and learning – aplysia • Genomic tools to manipulate plasticity – RNA and DNA based • Optogenetic tools to manipulate neural activity Peter De Weerd Vincent van de Ven Jan Zimmermann – Many applications – Our focus: application in memory 1 Molecular basis of plasticity and learning 2 Neurophysiological basis of plasticity and learning • Non-associative forms of learning Lessons from Aplysia – Habituation: S – R (repeated S  less R) – Sensitization: S – R (intense S  enhanced R) • Associative forms of learning – Classical conditioning: pairing CS – US – Operant conditioning: pairing R – outcome 3 Habituation Before 4 Habituation After Gill withdrawal = freezing in the rat 5 6 1 29-9-2021 Habituation Sensitization Habituated Sensitized 7 8 Sensitization Sensitization Early-phase: functional effects First messenger Second messenger Presynaptic Pre to Post Signaling Postsynaptic 9 10 What is a 1st & 2nd messenger? Neuro transmitter (first messenger) In our example Neuro transmitter (first messenger) e.g. 5-HT activates intra-cellular processes activates e.g. 5-HT receptor e.g. Adenyl cyclase intra-cellular e.g. cAMP 11 e.g. processes PKA 12 2 29-9-2021 In another example Some definitions A receptor is a membrane-bound molecule Neuro transmitter from facilitating interneuron (first messenger) the activated effector stimulates synthesis of a diffusing signal called a 2nd messenger; a molecule that serves signal amplification (c) Photochemical amplification of stimulus G-proteins are transducers activates effector is another membrane-bound molecule that in turn also can be activated the molecule at the beginning of this intra-cellular process is also called Intracellular process secondary effector Note: ‘messengers’ are smaller molecules; ‘effectors’ are larger proteins/enzymes 13 Sensitization 14 Different pools of vesicles Early-phase: functional effects are calciumn dependent First messenger Receptor Transducer First effector Second messenger Second effector Presynaptic Postsynaptic Pre to Post Signaling 15 Role of SNARE proteins in priming for release and actual release of vesicles Calcium-dependent detachment from the reserve pool Depolarization of the presynaptic membrane induces a calcium ion influx into the axonal nerve terminal of neurons, and increases the intracellular concentration of calcium ions. Synapsin I was shown to be phosphorylated by this calcium influx.[12] The calcium ion, Ca2+, binds to calmodulin to form a calcium/calmodulin complex which then activates the calcium/calmodulin-dependent protein kinase, in turn triggering phosphorylation.[10] Calcium/calmodulindependent phosphorylation of synapsin I causes dissociation of synapsin I from the vesicular membrane. This allows the vesicle to detach from the reserve pool and become available for release. In the nerve terminal ending, there are two pools of synaptic vesicles, the reserve pool and the ready-release pool. The reserve pool refers to the synaptic vesicles that are not ready to release neurotransmitters and the ready-release pool refers to the vesicles which are primed to release their neurotransmitters across the presynaptic cytoplasmic membrane and into the synaptic cleft. 16 17 18 From wikipedia 3 29-9-2021 Protein kinases (A and C) interact with the SNARE proteins to regulate priming and release of vesicles SNARE proteins (an acronym derived from "SNAP (Soluble NSF Attachment Protein) REceptor") are a large protein superfamily. SNAREs are small, abundant, tail-anchored proteins which are often post-translationally inserted into or attached onto membranes NSF or N-ethylmaleimide sensitive fusion proteins, is an enzyme which in humans is encoded by the NSF gene; found in the cytoplasm of eukaryotic cells. It is a central component of the cellular machinery in the transfer of membrane vesicles from one membrane compartment to another. During this process, SNARE proteins on two joining membranes (usually a vesicle and a target membrane such as the plasma membrane) form a tight complex. 19 From wikipedia For example, phosphorylation of SNAP-25 by PKC decreases its interaction with syntaxin (Shimazaki et al. 1996), which might enhance Ca2+-dependent exocytosis by accelerating dissociation of the SNARE complex. Further, PKC-dependent phosphorylation of n-Sec1 (also called Munc-18) inhibits its interaction with syntaxin, which might enhance exocytosis by increasing the amount of syntaxin available to form SNARE complexes (Fujita et al. 1996). Synaptotagmin I, a Ca2+-binding protein thought to serve as a Ca2+ receptor for transmitter release, is a substrate for PKC both in vitro and in vivo (S. Hilfiker, V. A. Pieribone, C. Nordstedt, P. Greengard & A. J. Czernik, unpublished observations). PKC might enhance exocytosis by modulating the Ca2+ sensitivity of synaptotagmin, by enhancing its binding to either Ca2+ or to the SNARE complex. 20 Filfiker & Augustine, 1999 The dogma of molecular biology Sensitization Late-phase: Gene expression replication (DNA -> DNA) transcription (DNA -> RNA) A Polymerase Rna translation (RNA -> Protein) osome ooooooo Protein 21 22 DNA (deoxyribonucleic acid) Guanine Cytosine Thymine Adenine GC; TA Guanine Cytosine Uracil Adenine GC; UA 23 24 4 29-9-2021 Transciption: RNA polymerase in action Regulation of transcription DNA Guanine Cytosine Thymine Adenine RNA Guanine Cytosine Uracil Adenine 25 26 From transcription to translation 27 28 What do proteins do? Many different functions in different cells of the body In neurons, one function is that they form signaling cascades which allow neurons to adapt to the input they are getting https://www.google.nl/?gfe_rd=cr&ei=tBkGVcunPMKCUJvfgZAC&gws_rd=ssl#q=fr om%20rna%20to%20protein%20synthesis 29 How does it work? - There are genes that are expressed in reaction to neuronal activity! - Called IEGs - There are genes that can increase synaptic connectivity - They are LGs - Proteins expressed by IEGs control the LGs that can express other proteins that can modify connectivity among neurons 30 5 29-9-2021 The dogma of molecular biology Sensitization An application of the dogma of molecular biology Stimulation IEG expression DNA Transcription Transcription RNA, mRNA RNA, mRNA Translation Translation LG expression Transcription RNA, mRNA Translation Protein Protein Protein Late-phase: Gene expression ‘transcription factor’ 31 32 Sensitization Early-phase: functional effects Classical Conditioning Late-phase: Gene expression Tail Unpaired to US Paired to US 33 34 Classical Conditioning Classical Conditioning US = Shock [US = Shock] CS+ = Touch Selective association of CS+ with UStim and UResp 35 36 6 29-9-2021 Classical Conditioning Long-term potentiation 37 Revealing LTP at work in (amnesia of) spatial memory • LTP is a laboratory paradigm – Hypothesis: LTP is mechanism for (episodic) memory in the brain 38 Tools to manipulate contribution of single genes to plasticity Rats, mice and songbirds • LTP maintenance depends on proteine kinase activity (PKMz) • Blocking PKMz in hippocampal cells recruited in spatial memory should lead to amnesia 39 40 Blocking at the RNA level: Differential display and antisense Experimental Sample mRNA Experimental cDNA Amplification by PCR PCR : Polymerase Chain Reaction Control Sample mRNA Reverse Transcription 30 - 40 cycles of 3 steps : Control cDNA ^Tnrinrni®^nTi^^ I I 1 minut 94 °C PCR Amplification 5' Gel Electrophoresis y # 3'iWiMy S| 2 : annealing 45 seconds 54 °C 5'nmnw^3' Differentially Expressed Genes Sequencing nWOIWOTH^^ ^llllhl ^JlUjy PCR Cloning Confirmation Antisense Steps Generation 41 42 7 29-9-2021 Amplification by cloning (E.coli bacteria) Bacterial DNA Plasmids syrinx 43 Blocking (modulating) at the genomic level: Molecular tools for spatially and temporally selective manipulation of gene expression An Example of Differential Display in Song Birds C C 0 0 112 44 2 Sequencing Anti-sense Generation 45 46 Courtesy R. Pinaud Viruses Origins: regressive, escape, and co-evolution hypothesis History: It started with Pasteur/Chamberland 1884 (rabies) and Ivanovski in1892 Virus replication independent of genome Genomic Diversity: Property Nucleic acid Parameters • DNA • RNA • Both DNA and RNA (at different stages in the life cycle) Tobacco virus: coiled RNA Shape Strandedness • Linear • Circular • Segmented • Single-stranded • Double-stranded • Double-stranded with regions of singlestrandedness Diversity in shape: helix, spherical, cylindrical, complex 47 Lytic cycle 48 8 29-9-2021 Genome-dependent virus replication A specific type of virus: Bacteriophage P Insertion into genomic DNA Mitosis X Meiosis X Meiosis 49 50 From Bacteriophage P to viral vector Bacteriophage P (retrovirus) contains -LoxP codes (Lox code from bacteriophage P, 34bp) -Cre (Gene for Cyclization Recombinase) -Code for self replication -Retrovirus: requires targeted cells to be dividing (as opposed to a lentivirus) Produce a viral vector with these elements BUT without code for self-replication 2D How to do this? -Get the LoxP (flanking a gene of interest) and Cre codes -Put them in a plasmid of bacteria -One pool for Cre -Another pool for LoxP -Digest the bacteria and engineer viral vectors -Containing Cre -Containing LoxP -Inject vectros in embryonic cells -Mouse strain expressing Cre 51 -Mouse strain expressing LoxP Original gene function is disrupted, a reporter gene Is transcribed Instead. Original gene function is untouched. 52 LoxP / Cre to interfere with LTP and CA1 place fields Specificity of Cre expression Spatially specific knockout 53 54 9 29-9-2021 Spatially and temporally specific gene block to interfere with LTP and CA1 place fields Related technologies in some of the papers we will read Tetra cycline transactivator CREB-Cre This produces an ineffective version of Ca2+ Calmodulin protein kinase (phosphorilation made impossible) 55 56 Optogenetic tools to manipulate function of selected neuronal classes Myriad applications Channelrhodopsins Conducts cations and depolarizes neurons on illumination Halorhodopsins Conducts chloride ions into the cytoplasm OptoXRs Chimeras that activate biological function upon illumination dictated by the intracellular loops used in the hybrid 57 Optogenetics 58 Selective re-activation of memories • The different types of opsins possess different functions – Open natrium kalium channels  depolarization of the cell – Open chloride chanels  hyperpolarization of the cell • Next it is important to introduce the opsin gene into the target organism – This is done by genetic engineering – A promotor is coupled with the opsin gene (for specific cell targeting) and the modified gene is inserted into an engineered virus – The virus then infects the brain cells of the target organisms and alters the genetic code of the cells – Many different viruses and ways to introduce opsin genes into the organism exist, however the virus vector is the most commonly used way 59 60 Xu et al., Nature, 2012 10 29-9-2021 Optogenetics Selective re-activation of memories Iigurv I Itaxlc experimental protocol* and u-lnthvklH'IUiiK»i DG crib by ChR2 -EYFP. a. The c-fm-lTA mouac wax Injected with AAV,-TRE-ChR2EYFP and Implanted with an optical fibre targeting the DG. h, When off Dox. training induces the expression of tTA. which binds to IRE and drive* the expression of ChR2-EYFP, labelling a subpopulation of activated cells (yellow) In the DG. c, Basic experimental scheme Mice were habituated in context A with light stimulation while on Dox tor 5 days, then taken off Dox lor 2 days and fear -conditioned (IC)In context B. Mice weir put back on Dox and tested for 5 days in context A with light stimulation, d. Representative image showing the expression of ChR2-EYFP in a mouse that was taken off Dox lor 2 days and underwent PC training, e g. An image of each rectangular area In d is magnified. showing the DG (•), CAI (I) and CAJ (g). The green signal from ChR2-EYFP In the DG spreads throughout entire granule cells, including dendrites (e). whereas the green signal confined to the nuclei in CA I and CA J is due to a 2 h halt life EGFP (ihEGFP) expresssm from the c h» shEGFP construct of the transgenic mouse(f.g). Blue is nuclear marker 4’,6 dumidlno 2-phenylindolc (DA PI). Panel d is al x in magnification and panels eg arc at X 50 magnification. Opsin engineering and genomic expansion; generalizable opsin targeting strategies; brain-disease and neuroscience applications; stem cells, heart cells, muscle cells and human neurons studied 75 “I 70 - <n 65 CL - 60 - 45 40 35 - 55 s 50 - 5 Rberoptic interface (May 2007) and single-component control of freely moving mammals (October 2007) described - 30 - o 25 - 5 £> Bacteriorhodopsin described as a single-component light-activated regulator of transmembrane ion flow 20 15 - Halorhodopsin described Microbial opsins fir to neurons (August Channelrhodopsin described E 10 3 Z 5- 0 - oooooooooooooooooooiOOg^HlsiOlgggiis 61 62 Xu et al., Nature, 2012 Deisseroth (2011) What we covered Thanks for your attention ! • Molecular basis of plasticity and learning – aplysia • Genomic tools to manipulate plasticity – RNA and DNA based • Optogenetic tools to manipulate neural activity – Myriad applications – Our focus: application in memory 63 64 11