Neurochemistry 1 2011 Tim Murphy

1. Neurochemistry 1 2011 Tim Murphy Objective: To understand the metabolic processes underlying the synthesis and metabolism of amino acid and peptide neurotransmitters. Major points to be covered:   -regulation of metabolism by enzymes -metabolic processes neurons share with other cells and organs -properties and functions of enzymes and pumps (transporters). -metabolic contingencies imposed by the existence of a blood-brain- barrier, i.e. the central role of glucose -synthesis and metabolism of amino acid transmitters and GABA. -glutamate -aspartate -glycine -neuropeptide synthesis and the pathway to regulated release

2. Neuronal metabolism. • Neurons share with other cells the need and ability to synthesize nucleic acids, proteins, carbohydrates and lipids. • Likewise they share the metabolic processes required to generate chemical energy for these processes: glycolysis, pentose-phosphate shunt, citric acid cycle, oxidative phosphorylation. • Neurons must be able to synthesize and metabolize neurotransmitters. • Neurons must also synthesize second messenger molecules needed to mediate signal transduction.

3. The brain makes use of general metabolism to find precursors and in some cases the finished products for synaptic physiology. glycine

4. Enzymes • Help processes within neurons overcome activation energy, and provide a site of regulation. • Essentially all chemical reactions in cells are mediated by enzyme, protein catalysts. •  A catalyst acts by bringing together the reactants, and thereby increasing the rate of a chemical reaction, without being permanently changed in the reaction. • Enzymes also allow the coupling of energetically unfavourable reactions with reactions that release free energy. If together the two reactions result in a negative G, the coupled reaction can occur.

5. Enzymes lower activation energy for reactions. Mol. Biol. of the Cell

6. Enzymes permit coupled reactions, for example falling rocks turn wheel to raise water for a different type of work. Mol. Biol. of the Cell

7. ATP is a useful energy currency since it can form high-energy intermediates permitting the coupling of energetically unfavorable reactions to favorable ones, shown is the amination of glutamate. Mol. Biol. of the Cell

8. General Properties of Enzymes • Enzymes are highly specific due to the specific structure of the active site • Substrate specificity • Reaction specificity • Enzymes bind substrates in specific ways that stabilize a reactive conformation, known as the TRANSITION STATE • Some enzymes require cofactors for complete activity (vitamin B6, pyridoxyl deficiency can impact GABA synthesis).

9. Velocity (V) as a function of substrate (S) plot. Saturation pseudo 1st order Km

10. Michaelis-Menton Equation, describes saturable enzyme kinetics, also applicable to binding of ligands to receptors. know this, it describes many interactions: enzymes, receptors, protein-protein. V=Vmax* [S]/([S]+Km) With a competitive inhibitor, the Km is increased but the Vmax is not effected. Km’=Km*(1+[I]/Ki), note when I= Ki the Km doubles With a noncompetitive inhibitor only the Vmax is reduced. Vmax’=Vmax*(1-[I]/([I]+Ki)), note when I= Ki the Vmax halves

11. Km and Vmax • The activity of enzymes can be discussed in terms of their Km, a measure of the affinity of the enzyme for its substrate, and the Vmax, which is the maximal velocity of the enzymatic reaction. • Km has two meanings: 1) the concentration of substrate at which 1/2 the active sites on an enzyme are filled. 2) the ratio of dissociation to association rates for enzyme substrate interactions. Km=kdissoc/kassoc. Since the association rates of many reactions at going the speed of diffusion, the strength of binding and rates of reaction are often determined by the dissociation rate. • Although these terms are associated with enzymes they are related to other saturable systems such as transporters (Kt, Vmax) and receptors (Kd, Bmax).

12. Competitive inhibitors. • Action: at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate. • Effect: Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

13. Noncompetitive inhibitors. • Action:Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate. . • Effect: Vmax is reduced; Km, as defined by [S] required for 1/2 maximal activity, is unchanged. • Knowing if something is competitive or non-competitive is important since it determines how much inhibitor you need relative to substrate (practical implication!!)

14. Substrate or ligand concentration

15. Transport can be saturable.

16. Relative scales, simple diffusion rates will be low for polar substances.

17. Channels and carriers.

19. Since many transported compounds are charged their movement is governed by electrical and chemical gradients just like small ions such as K+, Na+, Cl-, and Ca2+.

20. Uniports-facilitative or uncoupled transport •  Molecules or ions move down their concentration gradient via a specific carrier. •  In contrast to a channel which will allow movement of thousands of ions per millisecond and whose specificity is primarily mediated by pore size, a facilitative carrier requires binding of a specific substrate which induces conformational changes in the carrier through which the substrate is moved, and then released, restoring the carrier to its original conformation.

21. Carrier-Mediated Transport, Uniporters. •  Carrier types at the blood brain barrier: hexose, monocarboxylic acid, large neutral amino acid, basic amino acid, acidic amino acid, choline, purine, and nucleoside carriers. •  These substances serve as building blocks for all brain macromolecules and neurochemicals.

23. Symports and antiports • Couple movement of one molecule with that of one or more other substrates. Energy is derived from concentration gradients no ATP needed (directly) although indirectly to establish gradient. •  The high-affinity pumps for amino acids, and neurotransmitters are principally Na+-symporters, i.e. the movement of Na+ down its electrochemical gradient provides the free-energy required to move another substrate (neurotransmitter) up its concentration gradient • Na+/Ca++ antiporters, and Na+/H+ antiporters move these ions out of cells as Na+ enters.

24. Na+, Ca2+ exchange protons Glutamate

25. The Na+ gradient can be used to pump glucose uphill.

27. Primary active transport • Systems utilize the free-energy obtained by ATP hydrolysis to move ions against concentration gradients (uphill), i.e. Na+-, K+-ATPase or the Ca2+ ATPase. • Estimated to require up to half the brain ATP, while other biochemical processes including protein, lipid and neurotransmitter synthesis together use perhaps 10%. • Other primary pumps, such as Ca2+-ATPases and proton pumps probably account for the rest. The brain uses 20% of total body oxygen consumption, thus 10% of total is used primarily to maintain neuronal ionic gradients via this pump.

28. Na+, K+ ATPase

29. Na+, K+ ATPase • Energy is directed into the pumping process by the 3Na+-dependent phosphorylation, followed by the 2K+-dependent dephosphorylation. Phosphorylation induces a conformational change that moves 3Na+ to the outside of the cell. • Pump stoichiometry is 3/2 making it electrogenic.

30. Fundamental Neurosci. 2002 Zigmond et al.

31. Role of the pump in resting membrane potential. • If pump is blocked with ouabain (blocks binding of K+) an immediate small depolarization occurs (only a few mV), however membrane will remain relatively constant as it is largely determined by K+ permeability, however the membrane is also slightly permeable to Na+ and over time the membrane potential will depolarize if Na+ diffuses in unchecked by the pump.

32. Glucose • Is the major fuel of the brain because it is the only fuel which enters in sufficient amounts to support the energy requirements. • Glucose gains access to brain and into cells by specific carriers - blood levels much higher than brain levels, thus glucose moves down its concentration gradient via facilitative transport. • Glucose utilization of tied to neuronal activity and increased blood flow, basis of PET functional imaging with 2-deoxyglucose. • Isolated neurons can use other fuels such as pyruvate and lactate, but they normally are not BBB permeable.

33. Blood (~6 mM glucose). CSF (~4 mM glucose). 4X Glut-1 expressed on the ab-lumenal side Farrell and Pardridge 1991 Fundamental Neurosci. 2002 Zigmond et al.

34. Glucose transport • The Km of the BBB glucose transporter is about 7 mM, which is about the level of plasma glucose, thus brain glucose varies directly with changes in blood levels. The blood brain barrier transporter is Glut-1. • Neurons possess a carrier of higher affinity, Glut3 Km = 200 M, allowing them to extract glucose from the extracellular space. Within neurons, glucose is immediately phosphorylated to a charged, impermeant metabolite, glucose-6-phosphate, thus the intracellular glucose concentration is effectively zero. Why is it advantageous to reduce the apparent free concentration of glucose.

35. Used in PET scanning. Fundamental Neurosci. 2002 Zigmond et al.

36. Glycolysis and TCA cycle • Within the cell, glucose enters the glycolysis pathway in the cytoplasm, and via pyruvate and acetyl-CoA, in the mitochondrial tri-carboxylic acid cycle (TCA) or Krebs cycle. In these systems, reducing equivalents are generated and via oxidative phosphorylation they generate ATP, the chemical fuel for the brain. •  Glycolysis and the TCA cycle are also the source of non-essential amino acid precursors used to synthesize the neurotransmitters glutamate, aspartate, GABA, and glycine.

38. Blood brain barrier. • What is the blood brain barrier (BBB)? • The existence of a blood-brain-barrier prevents molecules in the circulation from freely entering the brain. • Prevents constant fluctuations in circulating metabolites, ions, and hormones from directly influencing neuronal activity. • Diffusion allows passage of gases, i.e. (O2 and CO2) and lipid soluble compounds, i.e. psychoactive drugs.

39. Endothelium The blood brain barrier largely occurs at capillaries through astrocyte endfeet and endothelium tight junctions. • Transport across it is selective. Carrier types at the blood brain barrier: hexose,monocarboxylic acid, large neutral amino acid, basic amino acid, acidic amino acid, choline, purine, and nucleoside carriers. Drewes LR. Adv Exp Med Biol. 1999;474:111-22. • .

40. Iadecola and Nedergaard 2007 Nat. Neurosci.

41. Perivascular glia contain high levels of the antioxidant tripeptide glutathione Sun et al. 2006.

42. Fig. 1. Characteristics of the endothelium. In the muscle capillary (upper) there are pores or slits between the endothelial cells allowing bulk flow of water and smaller solutes between the blood and the extracellular space in the tissue. In contrast, the brain endothelial cells (lower) are connected by tight junctions. No pores or slits are present preventing bulk flow. Water therefore has to cross the blood–brain barrier by the mechanism of diffusion. Paulson, European Neuropsychopharmacology 12, 2002, Pg. 495

43. Brain activity and blood supply are tightly linked. • It has been known for over 100 years increased neuronal activity is associated with increases in blood flow. Roy CS, Sherrington CS (January 1890). "On the Regulation of the Blood-supply of the Brain". J. of Physiol. 11 (1-2): 85–158.17. • Changes in blood flow or oxygenation are used a surrogate measure of neuronal activity.

44. Glial and neuronal control of brain blood flow Glial and neuronal control of brain blood flow David Attwell1, Alastair M. Buchan2, Serge Charpak3, Martin Lauritzen4, Brian A. MacVicar5 & Eric A. Newman6 Nature 2010 468:231

45. Imaging brain metabolism. • 2-deoxygluocose method radioactive detection or positron emission tomography (PET) scanning, need isotopes poor time resolution (Sokoloff 1977 J. of Neurochem.). • Functional magnetic resonance imaging (fMRI), second level time resolution, signals related to changes in oxy/deoxyhemoglobin potentially complicated (Ogawa et al. 1990 PNAS). • Intrinsic signal imaging more direct spectroscopy of brain signals related to changes in oxy/deoxyhemoglobin, can be performed with a video camera (Grinvald et al. 1986 Nature).

46. Synapses are on average 13 mm from capillaries. RBC supply rates are normally ~100 cells/sec. Acute reduction in supply rate by >90% leads to damage within 10 min, which can reverse if reperfusion occurs early. 10 mm Zhang et al. 2005

47. Irreversible ischemia; red vessels, green dendrites (Murphy lab). Control 10 min 30 min clot 1 hr 2 hr 3 hr Scale bar=10 um region1 ctr at 49_54 10 mm

48. Intrinsic optical signals, light scattering provides a reflection of neuronal activity. 2) General blood volume. Reflected light Stim 1 sec • Local deoxyhemo- • globin signal. 635 nm light 1) Reduced reflection, increased absorbance with elevated deoxyhemoglobin in active areas. 2) General increase in blood volume and oxyhemoglobin in surrounding areas leads to large late positive global signal.

49. Sources of intrinsic optical signals. From Grinvald and Bonhoeffer OPTICAL IMAGING OF ELECTRICAL ACTIVITY BASED ON INTRINSIC SIGNALS AND ON VOLTAGE SENSITIVE DYES THE METHODOLOGY 2001

50. Change in light scattering in response to forelimb stimulation.