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Single-Cell Transcriptomic and Functional Characterization Of Departamento de Fisiología Médica y Biofísica Universidad de Sevilla Single‐cell transcriptomic and functional characterization of cortical parvalbumin interneurons in a novel conditional knock‐out mouse lacking CSPα/DNAJC5 Marina Valenzuela Villatoro Advisors: Dr. Rafael Fernández Chacón Dr. Pablo García‐Junco Clemente Doctoral Thesis Sevilla, 2019 Departamento de Fisiología Médica y Biofísica Universidad de Sevilla Avd. Sánchez Pizjuán, 4 41009, Sevilla España Dr. Rafael Fernández Chacón, Catedrático del Departamento de Fisiología Médica y Biofísica de la Universidad de Sevilla y Dr. Pablo García‐Junco Clemente, Investigador Ramón y Cajal del Departamento de Fisiología Médica y Biofísica de la Universidad de Sevilla, CERTIFICAN que Marina Valenzuela Villatoro ha realizado bajo su dirección el trabajo titulado “Single‐cell transcriptomic and functional characterization of cortical parvalbumin interneurons in a novel conditional knock‐out mouse lacking CSPα/DNAJC5” que presenta para optar al grado de Doctor por la Universidad de Sevilla. Fdo: Dr. Rafael Fernández Chacón Fdo: Dr. Pablo García‐Junco Clemente Sevilla, 2019 TABLE OF CONTENTS TABLE OF CONTENTS ...................................................................................................................................... I SUMMARY OF TABLES ................................................................................................................................... II SUMMARY OF FIGURES ................................................................................................................................ III SUMMARY OF BOXES ................................................................................................................................... IV ABBREVIATIONS ............................................................................................................................................. V ABSTRACT ...................................................................................................................................................... 1 INTRODUCTION ............................................................................................................................................. 3 1. Background: the synapse and the synaptic vesicle ................................................................................... 5 2. The synaptic vesicle cycle .......................................................................................................................... 7 3. Membrane fusion by SNARE proteins ....................................................................................................... 8 4. Cysteine string protein alpha (CSPα) ......................................................................................................... 9 4.1. Localization and molecular structure ................................................................................................. 9 4.2. The trimeric complex CSPα‐Hsc70‐SGT is essential for chaperone activity ..................................... 10 4.3. Phenotypic characterization of CSPα knock‐out (KO) mice ............................................................. 11 4.4. SNAP25 is an important substrate of CSPα ...................................................................................... 12 4.5. Role of CSPα in GABAergic synapses firing at high frequency ......................................................... 13 4.6. Importance of CSPα in human disease ............................................................................................. 14 5. The cerebral cortex .................................................................................................................................. 15 5.1. Cortical organization ........................................................................................................................ 15 5.2. Cortical glutamatergic neurons ........................................................................................................ 17 5.3. Cortical GABAergic interneurons ...................................................................................................... 17 5.3.1. Cortical interneuron classification ............................................................................................. 17 5.4. Cortical connectivity: pyramidal neurons versus GABAergic interneurons ..................................... 19 6. Parvalbumin interneurons ....................................................................................................................... 20 6.1. Morphological and functional properties of PV dendrites ............................................................... 21 6.2. Morphological and functional properties of PV axon ...................................................................... 22 6.3. Specializations of PV cells for fast synaptic signaling ....................................................................... 23 6.4. PV‐derived feedforward and feedback inhibition ............................................................................ 24 6.5. Role of PV interneurons in modulating animal behavior ................................................................. 24 6.6. Role of PV interneurons in neurological disorders ........................................................................... 25 7. Single‐cell RNA sequencing ..................................................................................................................... 25 7.1. First milestones ................................................................................................................................ 25 7.2. Cell isolation and sequencing methods ............................................................................................ 26 7.3. Up‐to‐date PV classification by scRNA‐seq ...................................................................................... 27 I 7.4. Transcriptomics in neurodegenerative diseases .............................................................................. 27 GOALS .......................................................................................................................................................... 29 MATERIALS AND METHODS......................................................................................................................... 33 1. Mice ......................................................................................................................................................... 35 1.1. Generation of conditional Dnajc5flox/flox mice ................................................................................... 35 1.2. Generation of UBC‐Cre‐ERT2:Dnajc5flox mice ................................................................................... 36 1.3. Generation of PVcre:Ai27D:Dnajc5flox mice ........................................................................................ 36 2. Fluorescent‐activated cell sorting (FACS) ................................................................................................ 37 3. Validation of conditional Dnajc5flox/flox mice ............................................................................................ 39 3.1. Genomic DNA (gDNA) validation ...................................................................................................... 40 3.1.1. gDNA extraction ........................................................................................................................ 40 3.1.2. PCR for Dnajc5 gDNA ................................................................................................................. 41 3.2. RNA validation .................................................................................................................................. 41 3.2.1. RNA extraction ........................................................................................................................... 41 3.2.2. Reverse‐transcription (RT) to cDNA .......................................................................................... 42 3.2.3. PCR for Dnajc5 cDNA ................................................................................................................. 43 3.3. Protein validation ............................................................................................................................. 43 4. Real‐time quantitative reverse transcription‐PCR (real‐time qRT‐PCR) .................................................. 44 4.1. RNA extraction .................................................................................................................................. 44 4.2. Reverse‐transcription and cDNA amplification ................................................................................ 44 4.3. Real‐time qRT‐PCR and quantification ............................................................................................. 45 5. Survival analysis ....................................................................................................................................... 45 6. Body weight curve ................................................................................................................................... 45 7. Open field ................................................................................................................................................ 46 8. Immunoblotting ......................................................................................................................................
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