1. Synapse-to Nucleus Calcium Signalling

2. Why Calcium? Na + and Cl - are sea water – Excluded to maintain low osmotic pressure – [K + ] i kept high for electrical neutrality [Ca 2+ ] i maintained very low – Prevents precipitation of organic anions Mg 2+ helps solubilize organic anions

3. Calcium has been ‘selected’ by evolution as an intracellular messenger in preference to other monoatomic ions in the cell Divalency - stronger protein binding than monovalent ions. More flexible that smaller divalent Mg 2+ ions  more effective coordinate with protein-binding sites. Energetically favourable to use Ca 2+ as 2 nd messenger (large [Ca 2+ ] gradient) (10 -7 vs. 10 -3 M) – rel small amt needed to enter cell to incr signaling  relatively little energy needed to pump it back out of the cell. Higher [Ca 2+ ] would ppt with PO 4 3- ions  lethal.

4. How cells keep [Ca] i low All eukaryotic cells have PM Ca 2+ -ATPase – Excitable cells also have Na + /Ca 2+ exchanger (NCX) ER Ca 2+ -ATPase (against a high grad) Mitochondrial high capacity (low affinity) pump – When [Ca] i very high (dangerous) levels (>10 -5 M) – Inner mitochondrial membrane – Uses the electrochemical gradient generated during electron-transfer of oxidative-phosphorylation

5. Calcium Concentrations [Ca 2+ ] o / [Ca 2+ ] i >10 4 – [Ca 2+ ] o ~10 -3 M – [Ca 2+ ] ER ~10 -3 M – [Ca 2+ ] i <10 -7 M at rest

6. Ca 2+ - a versatile signal Target TissueSignaling MoleculeMajor Responses LiverVasopressinGlycogen breakdown PancreasAChAmylase secretion Smooth muscleAChContraction Mast cellsAntigenHistamine secretion Blood plateletsThrombinaggregation

7. Ca 2+ - a versatile signal Synaptic vesicle release (  s) Excitation-contraction coupling (ms) Smooth muscle relaxation (ms-sec) Excitation-transcription coupling (min-h) Gene transcription (h) Fertilization (h)

8. Ways the Cell (neuron) uses to Partition Ca 2+

9. Fig 5.3, Purves et al., 2001

10. How cells ↑ [Ca] i Voltage-gated Ca 2+ Channels – Membrane potential drives Ca 2+ down its chemical gradient – Different channels in different cells Different properties for different purposes

11. Ca 2+ shut-off pathways Voltage-gated Ca 2+ channels inactivate IP 3 rapidly dephosphorylated Ca 2+ rapidly pumped out

12. Ca 2+ as a 2 nd Messenger

13. Ca 2+ as a 2 nd Messenger (cont’d)

16. Gq signaling pathways and Calcium

17. Fertilization of an egg by a sperm triggering an increase in cytosolic Ca 2+ 3 major types of Ca 2+ channels: 1.Voltage dependent Ca 2+ channels on plasma membrane 2.IP3-gated Ca 2+ release channels on ER membrane 3.Ryanodine receptor on ER membrane

18. Calcium uptake and deprivation 1. Na/Ca exchanger on plasma membrane, 2. Ca pump on ER membrane, 3. Ca binding molecules, 4. Ca pump on Mitochondia

19. Ca 2+ as a 2 nd Messenger (cont’d)

24. Synaptotagmin and neurotransmitter release

25. Ca 2+ as a 2 nd Messenger (cont’d) Ca 2+ -Activated Signalling of Glu Receptor in the Postsynaptic Neuron

26. Synaptic Plasticity in the Nervous System Activity-dependent plasticity is mediated by electrochemical activity of the synapse. Activity-dependent plasticity is a change in neural connections and synaptic strength that are the hallmarks of learning and memory.

27. Ca 2+ in Synaptic Plasticity

28. Targeting molecules for Calcium Calcium binding protein Calmodulin

29. Ca 2+ /calmodulin dependent protein kinase (CaM-kinase) Memory function: 1. calmodulin dissociate after 10 sec of low calcium level; 2. remain active after calmodulin dissociation

30. Ca 2+ /calmodulin dependent protein kinase (CaM-kinase) Frequency decoder of Calcium oscillation High frequence, CaM-kinase does not return to basal level before the second wave of activation starts

31. Synaptic plasticity in the Nervous System Nervous system adapts to environmental changes. Such stimulation  activity-dependent plasticity or alterations in the number of synapses and/or in the strength of existing synapses.

32. The 3 Phases of Synaptic Plasticity 1.Early (sec-min) after electrical activity: changes in neural connections via modifications (phosphorylation) of existing proteins (ion channels) or delivery of proteins to postsynaptic membrane. 2.Intermediate (min-hr): synthesis of new proteins by existing levels of genes. 3.Late (days - longer ): changes in gene expression: txn and tln => long-lasting changes. All of these phases triggered by Ca 2+ influx.

33. Hippocampus – site of much plasticity and LTP studies. Patients with hipp lesions  anterograde and retrograde amnesia. LTP – induced into postsynaptic neuron by high-freq. train of electrical impulses into presynaptic afferents. - model for learning and memory. - activity-dependent incr in synaptic efficacy that can last days-weeks in vivo.

34. LTP in the Hippocampus. A model for plasticity - learning and memory. Is an activity-dependent increase in synaptic efficiency that can last for days – weeks. Induced in the postsynaptic neuron by repeated high- frequency stimulation of presynaptic afferents. Characterized by an early, protein synthesis independent phase and late phases, which can be blocked by protein synthesis inhibitors. During the longest phase, there is a critical period of transcription after the LTP-inducing stimuli has been applied. Induction of LTP is critically dependent on an elevation of postsynaptic Ca 2+.

35. IEGs – genes whose txn can be triggered without de novo protein synthesis (e.g., txn factors)  2ary wave of txn for other proteins required for LTP.

36. LTP in the Hippocampus (cont’d) LTP-inducing stimuli Ca 2+ IEGs zif268 c-fos c-jun NMDA receptors Secondary wave of txn, leading to the struct/func changes required for maintenance of LTP e.g.,tissue plasminogen activator; activity-regulated cytoskeletal-assoc protein

37. Synaptic plasticity in the Nervous System – Control of Gene Expression Pre-initiation complex. Histone acetylase activity. RNA pol. Transcription factors. Promoter, enhancers, silencers. REST/NRSF binding  NRSE. Signal-inducible transcription factors.

38. Control of Gene Expression Control of gene expression can occur at any stage in the process. By far, the most common point of regulation is at transcription initiation (RNA Pol II). Transcription factors

39. Transcription Factors

40. Synaptic plasticity in the Nervous System – Ca 2+ - Responsive DNA Regulatory Elements and their Txn Factors Cyclic-AMP response element (CRE). - Incr of synaptic activity  synaptic NMDA receptor- dependent transient Ca 2+ currents and long-lasting LTP in hipp (CA1 region) activate (phosphorylate) CREB txn factor  CaMKII and MAPK (ERK) signalling pathways Serum response element (SRE). - Induce expression of c-fos promotor  activation of L- type Ca 2+ channels. Nuclear Factor of Activated T cells (NFAT) response element. - NFAT activity regulated by Ca 2+ -activated calcineurin. - Calcineurin dephos cyto NFAT  transport into nucleus. - W/o Ca 2+ -activated calcineurin activity, NFAT becomes rephos by GSK and re-exported to cytoplasm.

41. Pre-initiation complex Core Promotor Element RNA Pol II Basal txn Recall: Activating txn factors bind here, upstream, enhance the rate of PIC formation by contacting and recruiting the basal txn factors via adaptors or co-activators Txn factors can also acetylate histones, disrupting/modifying chromatin structure Stimulus A wide variety of intracellular signaling pathways can influence the rate if txn initiation by many txn factors Phosphorylation Reactions There are several well- characterized DNA elements that act as binding sites for txn factors that are regulated by Ca-activated signaling pathways

42. Ca-Responsive DNA Regulatory Elements and their Transcription Factors cAMP-response element (CRE) – bound by CRE binding protein (CREB). - Ca activation of CREB is mediated by CaM KII and Ras-ERK1/2 signaling pathways.

43. Ca-Responsive DNA Regulatory Elements and their Transcription Factors Serum Response Element (SRE) – binding site for serum-response factor (SRF) SRE SRF Ternary complex factor (TCF) 5’ SAP-1 Elk-1 SAP-2 Ca signaling pathways – dependent synaptic activation Rsk 2 ERK 1/2 Ras TCF recognizes and binds SRE only with SRF bound

44. Ca 2+ as a 2 nd Messenger (cont’d) Ca-Responsive DNA Regulatory Elements and their Transcription Factors Nuclear Factor of Activated T cells (NFAT) Response Element Calcineurin Ca 2+ NFAT-P Extracellular Intracellular Cytoplasmic Nuclear NFAT P GSK-3β ATP Ca 2+ Calcineurin (decr activity) NFAT-P

45. Ca 2+ as a 2 nd Messenger (cont’d)

46. Terminology: CRE(cyclic AMP response element); CREB: CRE binding protein; CBP: CREB binding protein

47. Physiological Importance of CREB LTM Information storage (Aplysia). Confirmed by anti-sense oligonucleotides  blocked LTM, but not STM formation. Drug addiction. Circadian rhythmicity. Neuronal survival mediated by neurotrophins (BDNF). Changes in synaptic strength and efficacy. Besides BDNF, CREB-dependent pro-survival genes include nNOS, bcl-2, mcl-1 and VIP.

48. Mechanism of CREB Activation CREB Activation Requires a Crucial Phosphorylation Event CREB binds CRE. Ser 133. Depol  incr [Ca 2+ ] cyto  P-ser133 on CREB. A133S  abolished CREB-mediated gene expression of many IEGs.  CREB is a Ca 2+ -sensitive txn factor.

49. Mechanism of CREB Activation CREB Activation Requires a Crucial Phosphorylation Event Ca 2+ -dependent signaling molecules capable of phoshorylating CREB on ser133: CaM kinases and their role in Ca 2+ -activated, CRE-dependent gene expression: CaMKII, CaMKIV, and CaMKI. -Play roles in secretion, gene expression, LTP, cell cycle regulation, tln control. - Activate c-fos expression: - experiments with KN-62  decr L-type Ca 2+ channel-activated c-fos expression. - experiments with calmodulin antagonist, calmidazolium. - CaMKIV – the prime member for CREB-mediate gene expression by nuclear Ca 2+ signals. - experiments with anti-sense oligonucleotide disruption of CaMKIV expression  abolished Ca 2+ -acticated CREB phosphorylation in hipp neurons. - critical for LT plasticity. - Knock-out mice for CaMKIV  cognition/memory deficits related to noxious shock stimulus and related to spatial learning (hippocampus). - both inhibition of either CREB or CaMKIV function  blocked cerebellar LTD (late phase).

50. MAPK Cascade

51. Parallel Activation of CaMK and MAPK pathways by Synaptic Activity CREB – end-point of several signaling pathways

52. The Role of CREB Binding Protein in CREB- Mediated Transcription Phosphorylated CREB activates transcription by recruiting its coactivator, CREB binding protein, CBP CREB has an inducible domain, the kinase- inducible domain (KID). CBP and p300 (closely related protein) function as coactivators for many signal-dependent txn factors (e.g., c-Jun, INF-α, STAT2, ELK-1, p53, and many nuclear hormone receptors).

53. Ca-Responsive DNA Regulatory Elements and their Transcription Factors cAMP Response Element (CRE) CRE RNA Pol II (CBP has intrinsic histone acetyl transferase (HAT) activity) 5’ CBP TFIIB TATA BP Ca signaling pathways – dependent synaptic activation Rsk 2 ERK 1/2 Ras HAT p/CAF HAT SRC1

54. Cytoplasmic Ca 2+ Ras ERK 1/2 Rsk 2 Nuclear Ca 2+ CBP CREB CRE Ser133 P CaM Kinase IV P – Ser301 Slow activation, long-lasting Fast activation, Short -lasting Physiological Importance of CREB: A Model for Nuclear Ca 2+ -Regulated Txn: Regulation of CBP

55. Decoding the Ca 2+ Signal The neuron regulates Ca 2+ signals on many levels: 1.Amplitude. 2.Temporal properties (oscillatory frequency). 3.Spatial properties. 4.Site of entry. The cellular requirements for Ca 2+ will determine the extent of these 4 properties: Subcellular localization of Ca 2+ and its many effectors involves compartmentalization (i.e., nuclear vs. cytosolic proteins, e.g., CaMKII is nuclear).

56. In contrast, ERK pathway, located just adjacent to the PM inside the cell and tethered to PM- bound nt receptors, requires only a tiny amt of Ca 2+ to get things started. But what about cytosolic proteins that are not tethered to a membrane? - Calcineurin: this membranous Ca 2+ not enough; requires global incr in [Ca 2+ ] cyto to trigger NFAT nuclear translocation.

57. NEXT SLIDE: Successive recruitment of signalling molecules by 3 distinct spatially distinct Ca 2+ pools may underline differential gene expression by synaptic activity.

58. Weak Stronger Strongest MAP Kinase TCF/SRE CREB (weak) MAP Kinase TCF, CREB (weak) CalcineurinNFAT MAP Kinase TCF, CREB (weak) CalcineurinNFAT CaM Kinase IVCREB (strong) c-Jun

59. Ways the Cell (neuron) uses to Partition Ca 2+ reveals different buffering capacities or Ca 2+ clearance mechanisms in different areas of the cell