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Synapse-to Nucleus Calcium Signalling

Synapse-to Nucleus Calcium Signalling. 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.

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Synapse-to Nucleus Calcium Signalling

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  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 • [Ca2+]i maintained very low • Prevents precipitation of organic anions • Mg2+ 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 Mg2+ ions  more effective coordinate with protein-binding sites. • Energetically favourable to use Ca2+ as 2nd messenger (large [Ca2+] gradient) (10-7vs. 10-3M) – rel small amt needed to enter cell to incr signaling  relatively little energy needed to pump it back out of the cell. • Higher [Ca2+] would ppt with PO43- ions  lethal.

  4. How cells keep [Ca]i low • All eukaryotic cells have PM Ca2+-ATPase • Excitable cells also have Na+/Ca2+ exchanger (NCX) • ER Ca2+-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 • [Ca2+]o / [Ca2+]i >104 • [Ca2+]o ~10-3 M • [Ca2+]ER ~10-3 M • [Ca2+]i <10-7 M at rest

  6. Ca2+ - a versatile signal

  7. Ca2+ - a versatile signal • Synaptic vesicle release (ms) • 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 Ca2+

  9. Fig 5.3, Purves et al., 2001

  10. How cells ↑ [Ca]i • Voltage-gated Ca2+ Channels • Membrane potential drives Ca2+ down its chemical gradient • Different channels in different cells • Different properties for different purposes

  11. Ca2+ shut-off pathways • Voltage-gated Ca2+ channels inactivate • IP3 rapidly dephosphorylated • Ca2+ rapidly pumped out

  12. Ca2+ as a 2nd Messenger

  13. Ca2+ as a 2nd Messenger (cont’d)

  14. Ca2+ as a 2nd Messenger (cont’d)

  15. Ca2+ as a 2nd Messenger (cont’d)

  16. Gq signaling pathways and Calcium

  17. Fertilization of an egg by a sperm triggering an increase in cytosolicCa2+ • 3 major types ofCa2+channels: • Voltage dependent Ca2+channels on plasma membrane • IP3-gated Ca2+release channels on ER membrane • Ryanodine receptor on ER membrane

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

  19. Ca2+ as a 2nd Messenger (cont’d)

  20. Ca2+ as a 2nd Messenger (cont’d)

  21. Ca2+ as a 2nd Messenger (cont’d)

  22. Ca2+ as a 2nd Messenger (cont’d)

  23. Ca2+ as a 2nd Messenger (cont’d)

  24. Synaptotagmin and neurotransmitter release

  25. Ca2+ as a 2nd Messenger (cont’d)Ca2+-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. Ca2+ in Synaptic Plasticity

  28. Targeting molecules for Calcium Calcium binding protein Calmodulin

  29. Ca2+/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. Ca2+/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 • 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. • Intermediate (min-hr): synthesis of new proteins by existing levels of genes. • Late (days - longer ): changes in gene expression: txn and tln=> long-lasting changes. All of these phases triggered by Ca2+ 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 Ca2+.

  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) e.g.,tissueplasminogen activator; activity-regulated cytoskeletal-assoc protein Ca2+ LTP-inducing stimuli IEGs zif268 c-fos c-jun NMDA receptors Secondary wave of txn, leading to the struct/func changes required for maintenance of LTP

  37. Synaptic plasticity in the Nervous System – Control of Gene Expression • Pre-initiation complex. • Histoneacetylase 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 – Ca2+-Responsive DNA Regulatory Elements and their Txn Factors • Cyclic-AMP response element (CRE). - Incr of synaptic activity  synaptic NMDA receptor-dependent transient Ca2+ 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-fospromotor activation of L-type Ca2+ channels. • Nuclear Factor of Activated T cells (NFAT) response element. - NFAT activity regulated by Ca2+-activated calcineurin. - Calcineurindephoscyto NFAT  transport into nucleus. - W/o Ca2+-activated calcineurinactivity, NFAT becomes rephos by GSK and re-exported to cytoplasm.

  41. 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 acetylatehistones, disrupting/modifying chromatin structure Pre-initiation complex RNA Pol II Basal txn Core Promotor Element A wide variety of intracellular signaling pathways can influence the rate if txn initiation by many txn factors There are several well- characterized DNA elements that act as binding sites for txn factors that are regulated by Ca-activated signaling pathways Phosphorylation Reactions Stimulus

  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) Ternary complex factor (TCF) TCF recognizes and binds SRE only with SRF bound Elk-1 SAP-1 SAP-2 SRF 5’ SRE Rsk 2 ERK 1/2 Ras Ca signaling pathways – dependent synaptic activation

  44. Ca2+ as a 2nd Messenger (cont’d)Ca-Responsive DNA Regulatory Elements and their Transcription Factors • Nuclear Factor of Activated T cells (NFAT) Response Element Extracellular Intracellular Calcineurin NFAT-P Ca2+ NFAT P Cytoplasmic Nuclear NFAT NFAT-P Ca2+ Calcineurin (decr activity) GSK-3β ATP

  45. Ca2+ as a 2nd 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 [Ca2+]cyto P-ser133 on CREB. • A133S  abolished CREB-mediated gene expression of many IEGs. •  CREB is a Ca2+-sensitive txn factor.

  49. Mechanism of CREB Activation CREB Activation Requires a Crucial Phosphorylation Event Ca2+-dependent signaling molecules capable of phoshorylating CREB on ser133: CaMkinases and their role in Ca2+-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 Ca2+ channel-activated c-fos expression. - experiments with calmodulin antagonist, calmidazolium. - CaMKIV – the prime member for CREB-mediate gene expression by nuclear Ca2+ signals. - experiments with anti-sense oligonucleotide disruption of CaMKIV expression  abolished Ca2+-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

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