Quiz 1 answer class

Cellular Energy Metabolism in Brain

• Metabolic processes in cytosol and mitochondria

• Glycolysis and cytosolic metabolism: not oxygen-dependent; occurs under aerobic and anaerobic conditions alike

• Pyruvate formed in glycolysis enters mitochondria via the mitochondrial pyruvate carrier; fate depends on oxygen availability

Aerobic vs. Anaerobic Energy Pathways

• Aerobic condition (with oxygen)

  • Pyruvate is converted by pyruvate dehydrogenase into acetyl-CoA

  • Acetyl-CoA enters the Krebs cycle (TCA) in the mitochondrial matrix

  • TCA yields NADH and FADH2 (reduced electron carriers)

  • Electron Transport Chain (ETC) pumps protons across the mitochondrial membrane; oxidative phosphorylation produces ATP

  • Oxygen acts as the final electron acceptor, forms water with protons

  • Net ATP yield from ETC-driven metabolism: ATP_{ETC} \,\approx\;30\text{-}32\,

• Anaerobic condition (no oxygen)

  • ETC cannot accept electrons; pyruvate is reduced to lactate by lactate dehydrogenase in the cytosol

  • Regenerates NAD+ to sustain glycolysis

  • Net ATP yield from glycolysis + lactate production: 2\;ATP per glucose

  • Lactate can diffuse into blood and be transported to neurons or mitochondria and reconverted to pyruvate if oxygen becomes available

Pyruvate Transport and Mitochondrial Entry

• Pyruvate enters mitochondrial matrix via the mitochondrial pyruvate carrier

• Inside mitochondria, fate of pyruvate depends on oxygen availability and metabolic state

Fatty Acid Metabolism in Brain

• Activation in cytosol: fatty acids are activated to fatty acyl-CoA (e.g., palmitoyl-CoA)

• Carnitine shuttle required to cross mitochondrial membranes: carnitine palmitoyltransferase (CPT) system

  • Outer membrane CPT system transports the acyl group into the matrix as acyl-carnitine; CPT I/II regulate entry

• Beta-oxidation in mitochondrial matrix: cleaves two-carbon units from the fatty acyl chain

  • Each cycle yields: 1\;\text{acetyl-CoA},\;1\;NADH,\;1\;FADH_2

  • Acetyl-CoA enters the TCA cycle; NADH and FADH2 feed the ETC to generate ATP

• Note on oxygen requirement

  • Beta-oxidation occurs in mitochondria and ultimately relies on oxidative phosphorylation; oxygen is required for efficient ATP production, although beta-oxidation itself is a mitochondrial process

Cellular Compartments and Transport Carriers: Conceptual Emphasis for the Exam

• Cytosol: glycolysis; fatty acid activation to acyl-CoA; initial transport steps

• Mitochondrial matrix: pyruvate dehydrogenase, TCA cycle, beta-oxidation (in a cycle-by-cycle fashion), ETC linkage

• Membrane transporters: mitochondrial pyruvate carrier; carnitine shuttle components; GLUT transporters (see below)

• Highlighted carriers to remember: Pyruvate carrier, CPT system (carnitine shuttle), NADH/FADH2 shuttles across compartments (e.g., malate-aspartate shuttle; see below)

Glucose Transport and Brain Imaging Context

• Glucose transporters

  • GLUT3: high-capacity transporter primarily in neurons

  • GLUT1: endothelial blood-brain barrier and astrocyte/neuron interfaces

  • Transport distribution affects regional brain energetics and imaging signals

• Imaging modalities and what they reveal

  • PET with FDG (fluorodeoxyglucose): measures glucose uptake/metabolic activity

  • Blood-oxygen-level-dependent (BOLD) fMRI: relies on differences between oxygenated and deoxygenated hemoglobin; deoxyhemoglobin is paramagnetic and reduces MRI signal (T2* effects)

  • Functional hyperemia and neurovascular coupling: hemodynamic response lags neuronal activity; sensitivity considerations and delays in signal interpretation

• Practical note: use these imaging resources to infer regional energy use and neurotransmitter-related activity in the brain, not just raw ATP counts

Glutamate–Glutamine Cycle and GABA Metabolism: Compartmentalization in Neurons and Astrocytes

• Core idea: Glutamate released from glutamatergic neurons is cleared and recycled via astrocytes; astrocytes convert glutamate to glutamine; glutamine shuttled back to neurons and converted back to glutamate

• Key enzymes and their cellular localization

  • PAG (phosphate-activated glutaminase): converts glutamine to glutamate; predominantly mitochondrial in neurons (mostly mitochondria; localized to mitochondrial membranes; largely neuronal with minor astrocytic presence)

  • GDH (glutamate dehydrogenase): mitochondria; catalyzes deamination/amination of glutamate; localized to mitochondria

  • AAT (aspartate aminotransferase): found in mitochondria and cytosol; supports transamination reactions across compartments

  • Glutamine synthetase (GS): astrocytic enzyme converting glutamate to glutamine in astrocytes

• Glutamatergic neuron side (glutamate cycle)

  • Glutamate packaged into synaptic vesicles; released into synapse

  • Taken up by neighboring astrocyte or the releasing neuron

  • Astrocyte converts glutamate → glutamine (glutamine synthetase)

  • Glutamine shuttled back to neuron; converted to glutamate via PAG (neuron-specific) to re-enter cycle

• GABAergic side (GABA cycle)

  • GABA packaged into vesicles; released into synapse

  • Reuptake into neurons or astrocytes; catabolic conversion pathways

  • GABA transaminase (GABA-T) and SSADH (succinic semialdehyde dehydrogenase) convert GABA to alpha-ketoglutarate (via transamination), feeding into the TCA cycle

  • GABA transaminase pathway can connect to alpha-ketoglutarate and glutamate pools, enabling glutamate↔GABA cycling

• Astrocyte–neuron synergy (glutamate-glutamine cycle) and slow regeneration considerations

  • Glutamate clearance is critical to prevent excitotoxicity; astrocytes regulate extracellular glutamate via EAAT transporters

  • The cycle enables neurotransmitter recycling and supports neuronal metabolic demand

Malate–Aspartate Shuttle: A Transport Mechanism for Reducing Equivalents (NADH) into the Mitochondria

• Purpose: Move cytosolic NADH equivalents into the mitochondrial matrix to feed the ETC

• Core components and flow (described conceptually in the slides)

  • Cytosolic oxaloacetate is reduced to malate by cytosolic malate dehydrogenase, consuming NADH to NAD+ (NADH + H+ → NAD+ in cytosol; malate carries reducing equivalents into mitochondria)

  • Malate enters mitochondria and is oxidized back to oxaloacetate by mitochondrial malate dehydrogenase, generating NADH inside the matrix

  • Oxaloacetate in mitochondria transaminates with glutamate to form aspartate and alpha-ketoglutarate via mitochondrial aspartate aminotransferase (AAT)

  • Aspartate is transported back to cytosol, where AAT regenerates oxaloacetate, continuing the shuttle loop

• Transporter directionality and translocation

  • The shuttle operates as an antiporter-like system: malate moves into the mitochondria while oxaloacetate/aspartate cycle moves as needed across the membrane

  • The overall effect: cytosolic NADH contributes to mitochondrial NADH supply, enabling efficient ATP production via the ETC

• Relation to neurotransmitter metabolism and energy demand

  • The shuttle supports high-energy demand during cognitive tasks by delivering reducing equivalents to mitochondria in neurons

Question Focus: Transporters, Shuttles, and the TCA Cycle Roles

• Malate–aspartate shuttle is essential for energy production when cognition demands high ATP

• Transamination and dehydrogenase reactions create a network that links amino-acid metabolism, the TCA cycle, and NADH shuttling

• Anaplerosis and metabolic flexibility (three roles of the TCA cycle):
  1) Energy production via NADH/FADH2 to ETC
  2) Generation of neurotransmitter precursors: α-ketoglutarate → glutamate (glutamatergic neurotransmission), oxaloacetate → aspartate, acetyl-CoA → acetylcholine
  3) Anaplerosis and metabolic flexibility: replenishing cycle intermediates; enabling brain to utilize diverse fuels (fatty acids, ketone bodies) via acetyl-CoA entry

Three Key Roles of the TCA Cycle in the Brain (Summarized)

• Role 1: Energy production

  • NADH and FADH2 feed the ETC to produce ATP; ATP supports Na+/K+-ATPase and vesicle recycling, signaling, etc.

• Role 2: Neurotransmitter precursors

  • α-ketoglutarate → glutamate (glutamatergic neurotransmitter)

  • Oxaloacetate → aspartate (glutamatergic and other amino-acid pathways)

  • Acetyl-CoA → acetylcholine (cholinergic pathways)

• Role 3: Anaplerosis and metabolic flexibility

  • Replenishes TCA cycle intermediates siphoned off for biosynthesis

  • Enables brain to utilize various fuels (fatty acids, ketone bodies) via acetyl-CoA and related metabolites

Transporters and Imaging: Imaging Depth and Receptor Context Notes

• Glucose transporters and distribution influence nutrient delivery to brain cells

  • Neurons: abundant GLUT3

  • Endothelial barriers and glial cells: GLUT1 and other isoforms

• Imaging modalities reflect metabolic states and receptor activity, not just single-pathway activity

  • PET or FDG: regional glucose uptake/metabolic rates

  • fMRI (BOLD): hemodynamic signals linked to oxygenation and blood flow changes; delays and sensitivity considerations matter when interpreting neural activity and metabolism

Receptor Pharmacology: Affinity, Occupancy, and Efficacy

• Key concepts

  • Affinity (KD): how tightly a ligand binds a receptor; higher affinity means lower KD

  • Definition: K_D = \frac{[L][R]}{[LR]}

  • Occupancy: fraction of receptors bound by ligand at a given concentration

  • Occupancy increases with ligand concentration; not necessarily equal to a physiological response

  • Efficacy (intrinsic activity): the ability of a ligand, once bound, to activate the receptor and produce a response (Emax)

• Relationships and distinctions

  • A ligand can have high affinity (low KD) but low efficacy (partial agonist or antagonist)

  • A ligand can have high efficacy but lower affinity (depends on overall binding kinetics and receptor state)

  • Orthosteric vs allosteric ligands

  • Orthosteric ligands bind the primary endogenous binding site and directly induce receptor activation (agonists) or block activation (antagonists)

  • Allosteric ligands bind a different site and modulate receptor function (positive/negative allosteric modulators); do not require endogenous ligand binding for activity

• Practical implications and examples

  • Partial agonist: high affinity but moderate efficacy (e.g., buprenorphine at opioid receptors) → can provide therapeutic benefit with ceiling effects

  • Inverse agonist: reduces constitutive activity of a receptor (basal activity below baseline)

  • Positive allosteric modulators (PAMs) increase potency or efficacy of orthosteric ligands; negative allosteric modulators (NAMs) reduce them

  • Receptor reserves and spare receptors: maximal response may occur with less than 100% receptor occupancy due to amplification and signaling cascades

• Related measurements and endpoints

  • EC50: concentration producing 50% of the maximal biological effect (Emax)

  • Potency vs efficacy: potency concerns dose to achieve effect; efficacy concerns maximal effect independent of dose

  • Biased signaling: ligands can preferentially activate certain pathways (e.g., G protein vs arrestin) leading to different EC50 values for distinct endpoints

Distinguishing KD, KI, and EC50; How They Relate and Differ

• KD (dissociation constant)

  • Measures binding affinity; independent of functional response

  • Low KD → high affinity

• KI (inhibition constant)

  • Used when competing ligands are involved; derived from competitive binding assays

  • Related to IC50 via the Cheng–Prusoff relation: Ki = \frac{IC{50}}{1 + \frac{[L]}{KD}} where [L] is the radioligand concentration and KD is its affinity

• EC50 (effective concentration 50%)

  • Concentration producing half-maximal effect; reflects signaling efficiency and receptor response

• Why KD and EC50 differ

  • Receptor reserves (spare receptors) and signal amplification can cause maximal effect at lower occupancy than 100%

  • Partial agonists may have high affinity but only partial efficacy; EC50 may be affected by receptor state and signaling coupling

  • Biased agonism can produce different EC50s for different downstream endpoints even with the same receptor

• Practical figure of merit: often KD and EC50 differ; KI helps interpret competitive binding data when direct labeling of the ligand of interest is not available

Germaine Concepts: Radioligand Binding Assays and Inverse Questions

• Experimental approaches to study receptor affinity

  • With radiolabeled ligand available:

  • Perform radioligand binding assay with varying concentrations of ligand

  • Plot bound vs free ligand to determine KD (binding affinity) and Bmax

  • Without labeled ligand for the ligand of interest:

  • Use a labeled ligand with known KD for competition assays

  • Co-incubate with the ligand of interest and a fixed amount of radiolabeled ligand; measure displacement to determine IC50 and then KI via the Cheng–Prusoff equation

• Interpreting receptor specificity with KD vs KI values

  • If two receptors show KD or KI values within a tenfold range, specificity is considered limited

  • A hundredfold difference is often used as a practical threshold for useful specificity; this threshold is a rule of thumb rather than an absolute rule

  • Consider tissue context, receptor reserves, and signaling cascades when interpreting specificity

• Conceptual illustrations and data interpretation tips

  • Visualize KD/EC50 curves as hyperbolas; the plateau indicates saturation or maximal binding/response

  • When KD = EC50, the occupancy-response relationship is near one-to-one in a simplified model; real systems can deviate due to receptor reserves and signaling amplification

G Protein–Coupled Receptors (GPCRs) and Biased Signaling: Mechanistic Nuances

• GPCR basics (brief recap)

  • GPCRs respond to diverse ligands and activate heterotrimeric G proteins (Gs, Gi/o, Gq/11, etc.)

  • Downstream effects include cAMP production, PLC signaling, calcium flux, ERK activation, and more

• Biased signaling and endogenous bias

  • Ligands can favor certain signaling pathways (e.g., G protein vs arrestin pathways)

  • Bias depends on receptor conformation and ligand-induced changes; downstream EC50s may vary by endpoint

• Orthosteric vs allosteric modulation in GPCRs (revisit concepts with context)

  • Orthosteric endogenous ligands bind the primary site; allosteric modulators bind alternative sites to fine-tune signaling

  • Allosteric modulators can be positive or negative; they can avoid receptor desensitization while maintaining signaling patterns

• Practical examples and notes from discussions

  • 5-HT1A somatodendritic autoreceptors regulate serotonergic firing; their desensitization is linked to mood disorders and pharmacotherapy

  • The serotonin receptor system is complex; presynaptic autoreceptors modulate neurotransmitter release and postsynaptic signaling

  • The D2 dopamine system and autoreceptors illustrate nuanced pharmacology where antagonist effects can produce paradoxical increases in release under certain conditions

• Grasping receptor pharmacology in context

  • Important to distinguish where receptors are located (somatodendritic vs terminal) and how autoreceptors influence release dynamics

  • In practice, receptor pharmacology in clinical settings involves balancing affinity, occupancy, efficacy, and bias to achieve therapeutic goals with minimal side effects

GRAB Sensors: Real-Time Neurotransmitter Detection Tools

• GRAB sensors: GPCR–based fluorescent reporters

  • Engineered GPCRs fused to circularly permuted GFP respond to endogenous neurotransmitters

  • Provide real-time optical readouts of neurotransmitter release with high temporal resolution (milliseconds to seconds)

• Key advantages over older methods

  • Higher temporal resolution than microdialysis (minutes) and better molecular specificity than general electrochemistry approaches

  • Can be cell-type and region-specific, enabling precise mapping of neurotransmitter dynamics

• Evaluation criteria for good neurotransmitter detectors (from class discussion)

  • Selectivity: must respond predominantly to the targeted neurotransmitter

  • Sensitivity: capable of detecting physiological release levels

  • Spatial resolution: precise localization to cell types/regions

  • Temporal resolution: fast enough to track rapid signaling events

  • Noninvasiveness: minimal perturbation to normal physiology

• Contextual cautions and scientific discussion

  • Potential concerns about perturbation: some GPCR-based sensors might affect native signaling if overexpressed or mislocalized

  • Debates about whether introduced receptors could alter endogenous GPCR signaling networks

  • Ongoing work to minimize interference and confirm physiological relevance across models

Five-HT1A Autoreceptors and Serotonergic Signaling (Autoreceptor Focus)

• Somatodendritic autoreceptors (5-HT1A): located on neuron cell bodies and dendrites; regulate firing rate and serotonergic tone

• Functional implications in mood disorders

  • Altered autoreceptor function linked to reduced serotonergic neurotransmission in certain conditions

  • Targeting autoreceptors with specific ligands can modulate neuronal firing and downstream signaling

• Context of broader receptor pharmacology discussion

  • The serotonergic system exemplifies autoreceptor-mediated feedback control and the complexity of pharmacological interventions

In-Depth Exam Q&A Topics Highlight (From Transcript Discussion)

• How receptor affinity, occupancy, and efficacy relate and differ

  • Affinity (KD): binding tendency; occupancy is the fraction of receptors bound; efficacy is the receptor’s ability to generate a functional response

  • Can a drug have high affinity but low efficacy? Yes; partial agonists demonstrate this phenomenon (high affinity, moderate intrinsic activity)

• KD vs KI vs EC50 conceptual differences

  • KD: binding affinity; KI: inhibition constant in competitive binding contexts; EC50: concentration for half-maximal effect

  • The Cheng–Prusoff relationship connects IC50 to KI in competitive binding assays

  • Receptor reserves, signal amplification, partial agonism, and biased signaling can separate KD from EC50

• Practical experimental design for affinity measurement (radiolabeled ligand available vs not)

  • If radiolabeled drug is available: perform radioligand binding assay to obtain KD and Bmax

  • If not available: use a labeled ligand with known KD for competition; determine IC50 and compute KI using the appropriate equation

• Specificity thresholds in drug design

  • Tenfold KD/KI difference suggests limited specificity; a 100-fold difference is a common practical target for specificity

  • Real-world considerations include tissue distribution, spare receptors, and off-target effects in different organs

Quick Reference Equations (LaTeX)

• General binding affinity: K_D = \frac{[L][R]}{[LR]}

• Intrinsic efficacy and EC50 concept: EC50 is the ligand concentration that yields 50% of the maximal effect (Emax)

• Ki calculation in competitive binding: Ki = \frac{IC{50}}{1 + \frac{[L]}{K_D}}

• ATP yield notation for oxidative phosphorylation: ATP_{ETC} \approx 30\text{-}32

• Glucose oxidation balance (simplified): glycolysis yields 2 ATP per glucose in anaerobic pathways; ETC yields ~30–32 ATP per glucose under aerobic respiration

• Malate–aspartate shuttle flow (conceptual): cytosolic NADH transfer to mitochondria via malate ↔ oxaloacetate ↔ aspartate translocation cycle; NADH generated in mitochondria enhances ATP production via ETC

Summary: Big Picture Takeaways

• Brain metabolism relies on tight coordination between glycolysis, mitochondrial oxidation, and neurotransmitter synthesis; oxygen availability governs whether glycolysis proceeds to lactate or pyruvate enters the TCA cycle

• The brain employs specialized shuttles (malate–aspartate) and transaminases (AAT, GDH, PAG) to shuttle carbon and reducing equivalents between compartments and to support neurotransmitter cycling (glutamate–glutamine and GABA cycles)

• The TCA cycle serves multi-faceted roles beyond energy production, including neurotransmitter precursor generation and metabolic flexibility to utilize diverse fuel sources

• Glucose transporters and neuroimaging techniques provide functional windows into brain metabolism and its regulation under different states of activity and disease

• Receptor pharmacology concepts (KD, KI, EC50, occupancy, efficacy) underpin drug development and interpretation of pharmacodynamic data; orthosteric vs allosteric ligands and biased signaling add layers of nuance

• GRAB sensors represent a modern toolset for real-time neurotransmitter monitoring, offering advantages in speed, specificity, and spatial resolution, while inviting careful consideration of potential perturbations to native signaling

Title

Comprehensive Neuro Metabolism and Receptor Pharmacology Notes