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Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures

一种在原代海马神经元培养能辅助因子的转染和激活的光和应激门控的光遗传学工具

WB Wyatt P. Bunner
RD Rachel Dodson
RH Robert M. Hughes
ES Erzsebet M. Szatmari

发布: 2021年04月20日第11卷第8期 DOI: 10.21769/BioProtoc.3990 浏览次数: 2740

评审: Naushaba HasinAnonymous reviewer(s)

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Abstract

Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a light-activated, genetically encoded redox sensor that can be activated with precise spatial and temporal control. Here we describe a protocol for the expression and activation of the CofActor system in dissociated hippocampal neuron cultures prepared from newborn mice. Cultures were transfected with Lipofectamine on the fifth day in vitro (DIV5), then exposed to cellular stress inducing stimuli, leading to the formation of actin-cofilin rods that can be observed using live cell imaging techniques. The protocol described here allows for studies of stress-related cytoskeletal dysregulation in live neurons exposed to neurodegenerative stimuli, such as toxic Aβ42 oligomers. Moreover, expression of the sensor in neurons isolated from transgenic mouse models of AD and/or mice KO for proteins involved in AD can advance our understanding of the molecular basis of early cytoskeletal dysfunctions associated with neurodegeneration.

Keywords: Cofilin-actin rods (丝切蛋白-肌动蛋白小结) search Cytoskeletal dysregulation (细胞骨架调节异常) search Optogenetics (光遗传学) search Hippocampal neurons (海马神经元) search Cellular stress (细胞应激) search Neurodegeneration (神经退行性变) search

Background

The biochemical hallmarks of neurodegenerative disorders (neural fibrils, clumps and tangles, heightened reactive oxygen species (ROS)), have presented numerous obstacles to clinicians as potential diagnostic tools (Kanazawa, 2001; Lee et al., 2001; Polidori et al., 2007; McKhann et al., 2011; López-Otín et al., 2013; Laske et al., 2015; Sweeney et al., 2017). These markers, which include Aβ fibrils and tau tangles in Alzheimer’s Disease (AD), are only accessible via an invasive cerebrospinal fluid assay for peptide mass fingerprinting or post mortem diagnosis (Blennow and Zetterberg, 2009; Anoop et al., 2010; Johansson et al., 2011; Kitamura et al., 2017), ROS can be fleeting and thus challenging to monitor in vivo (Halliwell and Whiteman, 2004; Owusu-Ansah et al., 2008; Kalyanaraman et al., 2012; Wang et al., 2013). An alternative approach, however, is to incorporate these disease-related biochemical elements into pathology-sensitive genetically encoded switches. Such switches enable rapid characterization of critical, early stage events in neurodegeneration using cellular and animal models of disease (Ganesh et al., 2016). Researchers are currently faced with limited options for studying the formation of cofilin-actin rods (a form of cytoskeletal dysregulation that occurs in oxidatively and energetically stressed neurons during the progression of neurodegenerative disease) in living cells. Endogenous populations of cofilin and actin can be coaxed into forming rods through various chemical treatments of cells (azide, glutamate, etc.), but these methods require cell fixation and immunostaining for later characterization (Minamide et al., 2010). Furthermore, isolation and extracellular characterization of cofilin-actin rods is challenging, as they are not stable in the presence of detergents (Bamburg and Bernstein, 2016). Overexpression of cofilin, in particular cofilin-GFP, provides a more reliable means of monitoring rod formation, but is hampered by the tendency of overexpressed cofilin to spontaneously form cofilin-actin rods in the absence of cellular stress (Kim et al., 2009). Most recently, a mutant of cofilin, cofilin R21Q, was combined with red fluorescent protein (CofR21Q-mRFP), and used to monitor the induction of cofilin-actin rods in real time (Mi et al., 2013). This reagent represents an important advance in the study of rod formation and longevity. However, there are drawbacks to its implementation, including irreversibility in the presence of energetic stress and a long half-life (30-60 min) once the cell culture medium is restored to homeostasis. We recently reported the CofActor system, which capitalizes on the formation of cofilin-actin rods, by coupling cofilin and actin to the light-responsive Cry2/Cib protein pair (Salem et al., 2020). The CofActor optogenetic system enables the induction of cofilin-actin rods rapidly and reversibly in response to light and stress. We have also created gain-of-function CofActor mutants (actin S14V) that induce rods in the absence of harsh manipulation of the glycolytic pathway and oxidative environment of the cell. We have previously demonstrated that the CofActor system responds to elevated stress levels in immortalized cell lines and in cultured hippocampal neurons from mice. Rodent neuron cultures originating from different brain areas (cortex, hippocampus, striatum, dorsal root ganglia and retina) have been established and used extensively in studies of protein-protein interactions and localization (Kaech and Banker, 2006). In this protocol, we describe in detail the procedures required to transfect and manipulate an optogenetic tool we recently developed (CofActor system) in dissociated hippocampal neuron cultures, prepared from newborn mice. The procedures described here can also be used for preparation of postnatal rodent neuron cultures from other brain areas, such as the somatosensory cortex and can be applied for expression of other optogenetic tools with low toxicity for neurons. Dissociated hippocampal neuron cultures from C57BL/6 newborn mice were prepared according to a modified standard protocol (Szatmari et al., 2005; Yang et al., 2017). The number of mice from a litter that is used for culture preparation depends on the number of planned experimental conditions and replicates. Using the described method, the CofActor system and other optogenetic tools can be applied to studies of neuronal processes associated with synaptic plasticity and intracellular transport.


Materials and Reagents

  1. Tissue culture dishes-35 mm (VWR, catalog number: 10861-656)

  2. Tissue culture dishes-60 mm (VWR, catalog number: 10861-658)

  3. #1.5 thickness round cover glass (VWR, catalog number: 72230-01)

  4. Serological Pipet, Sterile 2 ml (Fisher Scientific, catalog number: 13-675-17)

  5. Serological Pipet, Sterile 5 ml (Fisher Scientific, catalog number: 13-675-22 )

  6. Serological Pipet, Sterile 10 ml (Fisher Scientific, catalog number: 13-675-17)

  7. Serological Pipet, Sterile 25 ml (Fisher Scientific, catalog number: 13-675-30)

  8. Conical centrifuge tube, 15 ml (Fisher Scientific, catalog number: 12-565-269)

  9. Conical centrifuge tube, 50 ml (Fisher Scientific, catalog number: 12-565-270 )

  10. Sterile syringe filters with 0.45 µm pore size (VWR, catalog number: 28145-505 )

  11. Sterile bottle filters with 0.8 μm pore size (Fisher Scientific, catalog number: 09-740-1C)

  12. 10 ml sterile syringes (Fisher Scientific, catalog number: 14-817-54 )

  13. C57BL/6 newborn mice (postnatal day 0-1; P0-P1)

  14. Na2SO4 (Fisher, catalog number: S421), stored at room temperature (RT)

  15. K2SO4 (Avantor, catalog number: 3282), stored at RT

  16. MgCl2 (Fisher, catalog number: AM9530G), stored at RT

  17. HEPES (Gibco, catalog number: 15630080 ), stored at 4 °C

  18. Glucose (Gibco, catalog number: 15023-021 ), stored at RT

  19. 0.5% Phenol Red (Sigma, catalog number: P0290-100ml ), stored at RT

  20. NaOH (Fisher, catalog number: SS266-1), stored at RT

  21. Kynurenic acid (Sigma, catalog number: K3375 ), stored at RT

  22. Laminin from mouse (Corning, catalog number: 354232 ), stored at -80 °C

  23. Poly-D-Lysine (Gibco, catalog number: A3890401), stored at 4 °C

  24. Pen/Strep (Thermo Fisher, catalog number: 15140122 ), stored at -20 °C

  25. GlutaMAX (Gibco, catalog number: 35050-061 ), stored at RT

  26. Bovine Calf Serum (BCS), heat inactivated (Gibco, catalog number: 16030074 ), stored at -20 °C

  27. L-(+)-Cysteine Hydrochloride (Thermo Fisher, catalog number: C562-25), stored at RT

  28. Papain latex (Worthington Biochemical Corporation, catalog number: LS003126 ), stored at 4 °C

  29. Trypsin Inhibitor from chicken egg white (Millipore Sigma, catalog number: T2011), stored at 4 °C

  30. Basal Medium Eagle (BME; Gibco, catalog number: 21-010-046), stored at 4 °C

  31. Neurobasal A (Gibco, catalog number: 10888022), stored at 4 °C

  32. B27 plus (Gibco, catalog number: 17504044 ), stored at -20 °C

  33. 70% Ethanol (VWR, catalog number: 97064-768 )

  34. ActinS14V.CIB.GFP plasmid construct (Addgene, Plasmid #133620), stored at 4 °C

  35. Cry2PHR-mCh-Cof plasmid construct (Addgene, Plasmid #133619), stored at 4 °C

  36. Lipofectamine 2000 (Thermo Fisher, catalog number: 12566014 ), stored at 4 °C

  37. Sodium Azide (Fisher Scientific, catalog number: 26628-22-8), stored at RT

  38. 2-Deoxy-D-glucose (DDG) (Fisher Scientific, catalog number: 154-17-6) stored at RT

  39. Opti-MEM (Thermo Fisher, catalog number: 31985062 ), stored at 4 °C

  40. Tissue Culture water (TC water; autoclaved Milli-Q water), stored at RT

  41. Ice (for keeping working solutions at 4 °C)


Stock Solutions (see Recipes)

  1. Stock Dissociation medium, stored at 4 °C

  2. 10× Ky.Mg stock, stored at -20 °C

  3. Stock coating solutions (PDL stock, stored at -20 °C and laminin stock stored at -80 °C)

  4. 1½ solution, stored at -20 °C

  5. Cysteine.HCl stock solution, stored at -20 °C

  6. 2.5 M Glucose solution, stored at 4 °C


Working Solutions (see Recipes)

  1. Working dissociation medium (DM/Ky.Mg) – prepare 100 ml/culture

  2. Growth medium with serum (sGM) – for washing (prepare 50 ml/culture) and plating (needs 2 ml/35 mm culture dish)

  3. Serum-free Neurobasal A-B27 growth medium (GM) – needs 2 ml/35 mm culture dish

  4. Optimem/Glucose – needs 50 ml/culture

  5. Enzyme (Papain) Solution – needs 10 ml/culture

  6. Trypsin Inhibitor solution – needs 10 ml/culture

  7. Transfection medium (TM) – needs 200 µl/35 mm culture dish

  8. Working PDL solution

  9. Laminin-PDL coating solution

Equipment

  1. Large metal scissors (Fine Science Tools, catalog number: 14001-18 )

  2. Small (Bonn) scissors (Fine Science Tools, catalog number: 14184-09)

  3. Vannas Spring Scissors (Fine Science Tools, catalog number: 15000-00 )

  4. Forceps (Roboz, catalog number: RS-5050 )

  5. Curved Forceps (Fine Scientific, catalog number: 91197-00 )

  6. Spatula (Fine Science Tools, catalog number: 10090-13 )

  7. P20 PIPETMAN (Fisher Scientific, catalog number: F123600G)

  8. P200 PIPETMAN (Fisher Scientific, catalog number: F123601G)

  9. P1000 PIPETMAN (Fisher Scientific, catalog number: F123602G)

  10. Drummond Pipet-Aid (Fisher Scientific, catalog number: 13-681-08)

  11. 4 °C refrigerator

  12. Laxco Zoom Stereo Zoom Microscope, 0.67 × 4.5 × zoom (Fisher Scientific, catalog number: Z220PLS200L)

  13. Laxco iLED Series LED Light Source (Fisher Scientific, catalog number: AMPSILED22)

  14. Water bath, 37 °C (VWR, catalog number: WB02)

  15. Metallic beads for water bath (VWR, catalog number: 10158-552)

  16. Biological safety cabinet (Fisher Scientific, catalog number: 13-261-334)

  17. Water jacketed CO2 tissue culture incubator set to 5% CO2 and 37 °C (Fisher Scientific, catalog number: 11-676-600)

  18. Tissue culture microscope, such as EVOS Floid Cell imaging station (Fisher Scientific, catalog number: 4471136)

  19. Swing bucket centrifuge and rotor (Fisher Scientific, catalog number: 75-410-885) 

  20. pH meter (Fisher Scientific, catalog number: 13-636-AE153)

  21. Keyence (BZ-X800) digital microscope for live cell imaging

  22. Theraband (red, Superior Medical Equipment, catalog number: H20130)

  23. Sterilization pouches (Santa Cruz Biotechnology, catalog number: SC-363641)

Software

  1. Fiji (Schindelin et al., 2012) equipped with the Bio-Formats package (Linkert et al., 2010)

  2. GraphPad Prism (Version 8.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Bunner, W. P., Dodson, R., Hughes, R. M. and Szatmari, E. M. (2021). Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures . Bio-protocol 11(8): e3990. DOI: 10.21769/BioProtoc.3990.
  2. Salem, F. B., Bunner, W. P., Prabhu, V. V., Kuyateh, A. B., O'Bryant, C. T., Murashov, A. K., Szatmari, E. M. and Hughes, R. M. (2020). CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells. J Biol Chem 295(32): 11231-11245.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 基础技术

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