Functions of Lysosomes

Transcription

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2 Functions of Lysosomes Intro - Degradation of cell components -Cell death -Autophagy 1st - Exocytosis / Secretory pathway - Calcium signalling 2nd O BayDH., (2009)

3 Phosphatidylcholine Intro Phosphatidylcholine (PtdCho) is the major membrane phospholipid. It has a prominent structural role, but is also a source of lipid second messengers.

4 The Kennedy pathway for PtdCho synthesis Intro ATP ADP CTP P P (CCT) CCT is the rate-limiting enzyme in PtdCho biosynthesis (CPT) CMP DAG

5 Disrupting PtdCho synthesis induces cell death Intro What type of cell death does occur? Choline deficiency Yen et al. (1999) Shin et al.,(1997) Streptococcus pneumoniae infection Zweigner et al., (2004) Drugs (CCT inhibitors) Hexadecylphosphocholine Baburina and Jackowski (1998) Van der Sanden et al., (2004) ET-18-OCH 3 Boggs et al., (1995) C 2 -Ceramide Ramos et al., (2000; 2002; 2003) CHO-MT58 cell line Cui et al., (1996)

6 The CHO MT58 cell model Intro ATP CHO MT58 cells have a thermosensitive CCT, fully active at 33 ºC ADP 33 o C CTP P P (CCT) (CPT) CMP DAG

7 The CHO MT58 cell model Intro ATP ADP 40 o C CHO MT58 cells have a thermosensitive CCT that is inactive at 40 ºC CTP P P (CCT) (CPT) CMP DAG

8 Objectives I To characterize the cell death induced by inhibition of PtdCho synthesis in CHO-MT58 cells and to compare it to the canonical apoptosis induced by Actinomycin D. To elucidate whether lysosomes and autophagy play a role in cell death caused by inhibition of PtdCho synthesis.

9 Cell death after thermal inhibition of CCT Results I

10 What is our apoptotic control? Results I ActimomycinD isa transcriptionalinhibitorthatinduces a canonical apoptotic death

11 Little nuclear condensation Results - I

12 Little DNA fragmentation Results - I G1 2n S G2/M 4n SubG1 <2n

13 The role of caspase-3 in apoptosis Intro mitochondria EXTRINSIC PATHWAY INTRINSIC PATHWAY Makand Yeh(2002)

14 Caspase-3 is not activated Results - I Caspase-3 activity Cell viability

15 The role of cathepsins in cell death Results-I Increased Cathepsin D is a marker of autophagy Jäättelä(2004)

16 Lysosomes are auto- and heterodigesters Intro Wanker E., website

17 The mechanism of (macro)autophagy Intro

18 Autophagic vacuoles appear after CCT inhibition Results - I

19 Some of them are huge Results - I M N

20 Autophagic vacuoles appear after CCT inhibition Results - I

21 CCT inhibition blocks the progress of autophagy Results - I

22 Flux of Autophagy: productive or blockade Intro Klionsky et al., (2008). Autophagy

23 CCT inhibition blocks the progress of autophagy Results - I

24 Temperature alone does not recapitulate the MT58 phenotype Results - I LC3-II / loadi ing control LC3-II western blot analysis *** CHO-MT58 CHO-K (+3MA) hours at 40 ºC

25 Our working model Discussion-I New synthesis of PtdCho is required for autophagy to progress.

26 Why do cells die? Discussion-I Autophagy is a constitutive survival mechanism

27 Does it all have any interest for clinical science? Discussion-I Autophagy defends cells against invading Group A Streptococcus (2004) Science 306, Bacterial inhibition of phosphatidylcholinesynthesis triggers apoptosis in the brain (2004) J. Exp. Med. 200, Our results show that inhibition of PtdCho synthesis may well underlie bacterial escaping from the autophagic machinery

28 Functions of Lysosomes Intro - Degradation of cell components -Cell death -Autophagy 1st - Exocytosis / Secretory pathway - Calcium signalling 2nd O BayDH., (2009)

29 is a universal messenger Intro - II

30 Basic mechanisms of regulation Intro - II NCX PMCA Receptor channel Na + ATP ADP PLC ARC ARC? [ ] i ~ nM IP 3 cadpr NAADP IP 3 R RyR NAADPR ATP ATP H + ADP V-ATPasa ADP SERCA Endoplasmic Reticulum Lysosome HCX

31 What is NAADP? Intro-II NAADP is a new potent second messenger derived from NADP. NAADP receptor is localised in lysosomes. The NAADP receptor is not known yet. Recently, up to four putative receptors have been proposed: TRP-ML1; TPC1; TPC2 and RyR. NAADP-R is inactive at high agonist concentrations, generating a bell-shaped concentration-response curve. NAADP signalling is thought to be a trigger signal that is subsequently amplified by CICR and influx.

32 Some physiological functions of NAADP Intro - II Insulin secretion Egg fertilization Neuron differentiation Neurite outgrowth Has NAADP a role in astrocytes?

33 Astrocytes Intro-II Traditional functions of Astrocytes: Structural and Metabolic support of neurons Neurotransmitter reuptake Regulation of ion concentration in the extracellular space New evidences: Astrocytes contain many neurotransmitter receptors Astrocytesrespond to agonist by increasing concentrations, which can expand in neighbouring astroyctesto produce waves Astrocyteexcitability is based on changes in concentrations New functions (tripartite synapse): Release of neurotransmitters (gliotransmission) Modulation of synaptic transmission

34 Objectives II To determine whether NAADP mobilises from lysosome-related vesicles in cortical astrocytes. To find out whether NAADP and lysosome-related vesicles mediate neurotransmitter-induced responses in cortical astrocytes. To elucidate the participation of NAADP and lysosome-related vesicles in mechanically induced waves between cortical astrocytes.

35 Measurementof concentration Methods II dyes: Fura-2-AM Fluo-4-AM imagingsystem

36 Astrocyte lysosomes contain Lysotracker Red staining GPN 50 µm 120s 600s Results - II

37 Astrocyte lysosomes express NAADP-R Results - II 100 µm Ned µm LysoTracker red overlay bright field

38 Making NAADP permeable Intro - II AM group NAADP Charge: -5 NAADP-AM (permeable) Parkesh et al., 2008

39 NAADP releases from astrocyte lysosomes Results - II Maximum peak height NAADP-AM 500nM control 50 µm GPN 0,5 RU 5 min

40 Neurotransmitters induce rise in Astrocytes Intro -II Can neurotransmitters release from lysosomes through activation of NAADP receptors?

41 ATP-induced responses are mediated by NAADP and lysosomes Results - II 100 µm Ned-19 NAADP 2 µm Bafilomycin A1 100 µm ATP NAADPR ATP 50 µm GPN H + Lysosome ADP V-ATPasa HCX

42 100 µm Ned-19 Endothelin-1-induced responses are mediated by NAADP and lysosomes NAADP 2 µm Bafilomycin A1 10 nmet-1 Results - II NAADPR ATP 50 µm GPN H + Lysosome ADP V-ATPasa HCX

43 Acetylcholine-induced responses are mediated by NAADP and lysosomes NAADP 100 µm Acetylcholine Results - II 100 µm Ned-19 NAADPR 2 µm Bafilomycin A1 ATP 50 µm GPN H + Lysosome ADP V-ATPasa HCX

44 Bradykinin-induced responses ARE NOT mediated by NAADP and lysosomes NAADP Results - II 100 µm Ned-19 NAADPR 2 µm Bafilomycin A1 ATP 50 µm GPN H + Lysosome ADP V-ATPasa HCX

45 Some Neurotransmitters use NAADP signalling to induce responses in astrocytes Discussion - II External Stimuli ATP ET-1 Ach Internal Signal NAADP Lysosome NAADPR

46 ATP-induced responses are also mediated by IP 3 signalling and extracellular EGTA: extracellular chelator 100 µm ATP Results - II 20 µm CPA 100 µm 2-APB IP 3 R 100 µm 8-Br-cADPR IP 3 cadpr RyR 25 µm Dantrolene ATP ADP SERCA Endoplasmic Reticulum

47 20 µm CPA ATP-induced responses are slightly influenced by cadpr signalling and Ryanodine receptors 100 µm 2-APB 100 µm 8-Br-cADPR IP 3 R IP 3 cadpr 25 µm Dantrolene RyR 100 µm ATP Results - II ATP SERCA ADP Endoplasmic Reticulum

48 Not all neurotransmitters signal through NAADP Discussion - II External Stimuli BK ATP ET-1 Ach Internal Signal IP 3 / cadpr NAADP Endoplasmic Reticulum IP 3 R RyR Lysosome NAADPR

49 Mechanically-induced waves Results - III Analysed Parameters: magnitude of response of each cell number of responding cells velocity of propagation (Δ time from estimulated cell)

50 ATP release from astrocytes mediates the propagation of waves Intro- III Bennett et al., (2003)

51 Purinergicreceptor activation is needed for wave propagation Results - III (Non selective P2) P2X antagonist P2Y 1 antagonist

52 No need for ET-1, Ach or BK receptor activation in wave propagation Results - III

53 NAADP signalling and lysosomal participate in wave propagation 100 µm Ned-19 NAADP Lysosome 2 µm Bafilomycin A1 Results - III NAADPR ATP H + HCX ADP V-ATPasa 50 µm GPN

54 NAADP signalling and lysosomal participate in wave propagation 1 st wave 2 nd wave Results - III 100 µm Ned µm GPN 2 µm Bafilomycin A1

55 Extracellular is needed for wave propagation Results - III

56 IP 3 signalling mediates wave propagation Results - III 20 µm CPA 100 µm 2-APB IP 3 R 100 µm 8-Br-cADPR IP 3 cadpr RyR 25 µm Dantrolene ATP SERCA ADP Endoplasmic Reticulum

57 Ryr/cADPR signalling does not seem to take part in wave propagation 100 µm 8-Br-cADPR Results - III 20 µm CPA 100 µm 2-APB IP 3 R IP 3 cadpr 25 µm Dantrolene RyR ATP SERCA ADP Endoplasmic Reticulum

58 NAADP signalling participates in wave propagation through purinergic receptors Discussion - III

59 Does it all have any interest for clinical science? Discussion - III Astrocytic activation spectrum: a biological model NAADP modulates astrocyte excitability NAADP and lysosomal may be effective pharmacological targets

60 Conclusions - I 1. CHO MT58 cells at 40 o C undergo a rapid inhibition of PtdCho synthesis, followed byadecreaseinthemassofptdchoandalossofviability. 2. Cell death caused by inhibition of PtdCho synthesis takes place with very little chromatin condensation and DNA degradation, together with no activation of caspase-3. Therefore, cells die by a non apoptotic mechanism. 3. Inhibition of PtdCho synthesis involves overexpression of cathepsin D and the appearance of big autophagic vesicles with high levels of LC3-II. High LC3-II over time shows that inhibition of PtdCho synthesis blocks the progress of autophagy.

61 Conclusions- II 4. Astrocyte lysosomes contain and express NAADP receptors that can be activated by a cell permeable analogue of NAADP, NAADP-AM. 5. TheneurotransmittersATP,Endothelin-1andAcetylcholineinduce responses thatinpartareduetonaadp-mediated releasefromlysosomes. 6. Thepropagationof wavesinratcorticalastrocytesrequirestheactivationof purinergic receptors, but it does not depend on endothelin, cholinergic or bradykinin receptors. 7. NAADP-mediated release from lysosomes has a key role in the astrocytic excitability by modulating the velocity of propagation of waves, as well as the strength of response.