genetically encoded fluorescent sensors for studying brain ... · •mruby-iatpsnfr1.0 •designed...
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• Outline• Background (what is energy metabolism and why is it important?).
• Imaging tools for measuring metabolites/mitochondrial function.
• My project: Ca2+ signaling to the mitochondria regulates neuronal energy homeostasis and shapes neuronal excitability.
Genetically encoded fluorescent sensors for studying brain energy metabolism
Imaging key components of energy metabolism• Energy metabolism-examining the pathways that consume substrates to generate ATP.
• Neurons in the brain- oxidative phosphorylation:
• Glucose & derivatives + O2 ATP + CO2 + H2O + heat
Brain energy metabolism
• The brain is energy demanding • e.g. brain only comprises 2% of body weight but contributes 20% of whole
body O2 consumption in the resting conscious state (Belanger et al 2011).
• Brain energy consumption is largely driven by information processesing:• ‘Signalling’ and ‘Housekeeping’
Brain energy metabolism• Main Question: What are the cellular/tissue level systems that maintain neuronal ATP production
despite dramatic variation in energy demand?
• Neuronal ATP production• Oxidative phosphorylation:
• Glucose & derivatives + O2 ATP + CO2 + H2O + heat
• Areas of study:• Neurovascular coupling
• Neuroenergetic coupling
Magistretti & Allaman (2015).
Brain energy metabolism
• Why should we care?
• Energy metabolism is critical for maintaining brain function.• Normal brain function is critically dependent on a stable/adaptable energy supply.
• Disruption of metabolism is implicated in acute/chronic brain disorders.
• Stroke, Parkinson's, Alzheimer’s, etc.
Imaging Glucose
• Obligatory energy substrate for the adult brain
• Glucose uptake has been quantified indirectly using 2-DG uptake• 2DG: glucose analog that is transported but not metabolized.• fluorescent 2-DG or radioactive tracer 2-DG (PET imaging).
• Limitations: • does not discriminate between distinct cell types • indirect measure of endogenous glucose utilization.
• Study of glucose uptake/metabolism would benefit from development of a fluorescent sensor of endogenous glucose.
Imaging Glucose• New – IGlucoSnFR
• Developed by Janelia Research Campus, Jacob Keller et al (2019).
• Genetically encoded, cpGFP-based, intensiometric fluorescence increase upon binding of glucose (strongest interaction), D-galactose, or 2-DG.
• Variants with different affinities and targeting (intracellular or extracellular)
• In vivo imaging of extracellular or intracellular glucose.
• Not yet available on Addgene, direct query to Janelia likely needed.
BioRxiv (2019)
• Lactate• can be produced/released by astrocytes upon activation and can be utilized by neurons as fuel
(astrocyte-neuron lactate shuttle).• evidence for a direct role (energy independent) in regulating neuronal excitability and plasticity.
• Controversies that require clarification: • When and where is lactate produced and released in the brain?• What is its functional role?
• Field would benefit from tools to visualize lactate with high temporal and spatial resolution.
Imaging key components of energy metabolism: Lactate/Pyruvate
Astrocyte-neuron lactate shuttle hypothesis (ANLS)
• Pyronic (San Martin et al 2014).• FRET pyruvate sensor based on the transcriptional regulator PdhR.
• Laconic (San Martin et al 2013).• Based on a bacterial transcription regulator (LldR) consisting of two modules: L-lactate binding and DNA binding
domain.
• Reduced FRET between TFP & YFP with increasing lactate.
• Designed for intracellular, but not extracellular sensing (?).
Imaging key components of energy metabolism: Lactate/Pyruvate
San Martin et al (2013)
Machler et al 2016 (Cell Metabolism) Machler et al 2016 (Cell Metabolism)
ATP sensors
ATP-cellular energy currency and extracellular signal molecule.
• Genetically encoded fluorescent sensors• Perceval – cpYFP coupled to bacterial GlnK1, excitation shift with changes in ATP/ADP ratio
Berg et al (2009)• Perceval HR – modified to have ideal sensitivity to physiological ATP/ADP ratios Tantama et al
(2013).• ATEAM- FRET (CFP/YFP coupled to bacterial F0F1 ATP synthase subunit) ATP sensor-increased
FRET with ATP. Imameura et al (2009)
• Good tools, but each have limitations: • i.e. respond to ATP/ADP ratio, not ATP. • Lack single wavelength fluorescence imaging.• ATP sensitivity is poor.• Not yet designed for extracellular ATP sensing (?).
ATP sensors• iATPSnFR1.0 & 1.1 (improved sensitivity)
• Lobas et al (Nature Communications, 2019)
• cpGFP coupled to microbial FoF1-ATP synthase subunit
• Intensiometric- fluorescence increase with ATP.
• Micromolar affinity, large dF/F.
• Insensitive to ADP, AMP or adenosine at concentrations equivalent to ATP.
• Designed for intra- or extracellular ATP measurements.
ATP sensors• iATPSnFR1.0 & 1.1 (improved sensitivity)
• cpGFP coupled to microbial FoF1-ATP synthase subunit
• intensiometric
• Micromolar affinity, large dF/F.
• Insensitive to ADP, AMP or adenosine at concentrations equivalent to ATP.
Lobas et al (2019)
acute brain slice imaging of extracellular ATP (sensor expressed in astrocytes)
ATP sensors• mRuby-iATPSnFR1.0
• Designed to provide a ratiometric measure of ATP.
• Similar properties to iATPSnFR1.0
• Example: measures intracellular ATP changes in neurons vs astrocytes during oxygen-glucose deprivation in acute brain slices.
Lobas et al (2019)
acute brain slice imaging of intracellular ATP
Imaging mitochondrial structure/function
• ATP production in neurons is critically dependent on mitochondrial OxPhos. • Other interesting functions:
• ROS, apoptosis, Ca2+ signalling.
• Research questions:• How is mitochondrial function regulated?
• How does mitochondrial function influence and contribute to brain function in health and disease?
• Questions can be addressed by examining mitochondrial properties with imaging.
Imaging mitochondrial structure/function
Sun et al 2013 Cell Reports, mitoDsRed cultured hippocampal neuron axons.
.
• Mitochondrial properties amenable to imaging:• Mitochondrial dynamics- changes in motility/form. • Functional modulation by Ca2+.
• Requires targeting fluorescent proteins to the mitochondrial matrix • mitochondrial targeting sequence (MTS) of electron transport chain subunit (CytC
oxidase).• Organelle tracking
• GFP, RFP, etc.
Chen and Sheng et al 2013 JCB cultured cultured hippocampal neuron axons.
Imaging mitochondrial structure/function
.
• Measuring mitochondrial fusion/fission dynamics• Mito dynamics implicated in maintaining mitochondrial quality control
• Mitochondrial photoactivatable GFP (mitoPAGFP)• Photoactivable GFP enables detection and quantification of organelle fusion in living cells.
Archer et al 2013 NEJM, mitochondrial fusion dynamics in vitro.
Imaging mitochondrial structure/function
.
• Mitochondrial properties amenable to imaging:• The role of Ca2+ in tuning energy metabolism- mitochondrial Ca2+ imaging
The functional relevance of mitochondrial Ca2+ dynamics in the mammalian brain is unclear.
Hypothesis: Mitochondrial Ca2+ dynamics regulate neuronal Ca2+ signalling and contribute to long-lasting changes in energy metabolism required for brain function.