Subcellular compartmentalization of CD38 in non hematopoietic cells a study to characterize its functional role in mitochondria 8

Chapter 5 Future Studies And Conclusions intracellular localized CD38, i.e, mitochondrial CD38, could be involved in a central mechanism in the regulation of intracellular Ca2+ homeosta

Chapter 5 Future Studies And Conclusions

intracellular localized CD38, i.e, mitochondrial CD38, could be involved in a central

mechanism in the regulation of intracellular Ca2+ homeostasis, as oppose to another topologically paradoxical alternative involving cell surface CD38

The data presented in Chapter 3 showed that intracellular CD38 retains its enzymatic functions, and support the specific association of CD38 with mitochondria

In the study of using mouse brain tissues, data presented in Chapter 4 further supported the conjecture that the long known mitochondrial NAD+ glycohydrolase could be in fact, the CD38 identified in this study Its function in the mitochondria may therefore involve coupling intracellular NAD+ metabolism to cytosolic Ca2+signalling This may bring about deeper insight into the complex signaling possibly mediated by this molecule

Functional role of mitochondrial CD38 was first determined by study on the enzymatic activities of the molecule Both intact mitochondria extracted from Mito-CD38 transfected COS-7 cells and WT mouse brain tissues were capable of cyclisation of NGDto cGDPR, a fluorescent analog of cADPR (Chapter 3 Section 3.2.2 & Chapter 4 Section 4.2.4.1) as well as conversion of NAD+ to ADPR (Chapter

4 Section 4.2.4.2) CD38 localized on mitochondria thus possessed typical ribosyl cyclase activity as well as NAD+ glycohydrolase activity In agreement with

ADP-reports that mitochondria are associated with abundant NAD+ glycohydrolase activity,

it was noticed that total mitochondria fraction isolated from mouse brain tissues showed both enzymatic activities with a significant higher ratio of NAD+glycohydrolase activity as compared to ADP-ribosyl cyclase activity (Chapter 4 Section 4.2.4.2) In view of the well established functional role of CD38, that is the hydrolysis of NAD+ to ADPR, and thus its major enzymatic property as a classic NAD+ glycohydrolase (Berthelier et al., 1998), data reported in this study was in agreement with that reported by Aksoy et al (2006) This group further showed CD38 as the major regulator of in vivo NAD+ levels in the brain tissues and reaffirmed the presence of CD38 on mitochondria There was no observation of any cyclase activity from the isolated mitochondria fraction from CD38KO mouse brain tissues (Chapter 4 Section 4.2.4) in the present study, thus the conclusion that all cyclase activities observed were derived from CD38 found in mitochondria

The localization of the enzymatically active CD38 in the mitochondria was demonstrated in the present study to be specific to the outer mitochondrial membrane Topological studies with protease protection assay alone on intact CD38+mitochondria (Chapter 3 Section 3.2.4) and protease protection assay combined with digitonin titration assay on the Percoll purified intact mitochondria extracted from mouse brain tissues (Chapter 4 Section 4.2.3) further confirmed the localization of CD38 on the outer mitochondrial membrane Moreover, the present data suggested a specific topology for this mitochondrial CD38 with the enzyme’s carboxyl catalytic site extruding to the cytosol region (Figure 5.1) This observation was further supported by TEM and SEM results in Chapter 4 using antibodies that are highly specific to CD38 and the staining patterns were compared against CD38 KO mice samples Having confirmed the location of CD38 on mitochondria, the

Chapter 5 Future Studies And Conclusions

functional role of this molecule was further investigated by a Ca2+ release assay Indeed, the data presented demonstrated that the mitochondrial CD38 was able to initiate the cADPR-sensitive Ca2+ release mechanism in a well established ryanodine-

sensitive in vitro system (Chapter 3 Section 3.2.6) This specific location of CD38 on

mitochondria with a unique topology may provide a new perspective to the pathways that might be associated with this enzyme It is tempting to speculate that the

mitochondrial CD38 may serve as a vital molecule in regulating and mediating in vivo NAD+ level as well as important Ca2+ signaling (Figure 5.2) Nevertheless, gaps remain in the knowledge with regards to further defining the characteristic and functional roles of mitochondrial CD38 for future studies

5.2 Future studies

Lisa et al (2001) reported that a majority of NAD+ glycohydrolase activity (~90%) is associated with rat heart mitochondria which located on the outer mitochondria membrane This group further showed that sarcolemmal rupture during reperfusion injury of the heart results in exposure of the mitochondria to the millimolar [Ca2+]i of the extracellular milieu This in turn triggers the opening of the PTP with the subsequent efflux of NAD+ from the mitochondrial matrix which then becomes available to NAD+ glycohydrolase localized on outer mitochondria membrane It in turn results in the formation of Ca2+ promoters such as cADPR, NAADP and ADPR, which are known to trigger the release of Ca2+ from the intracellular Ca2+ stores It was hypothesized that a low density mitochondrial NAD+glycohydrolase which causes the mitochondrial hydrolysis of NAD+ could eventually induce an increase of intracellular [Ca2+]i, thus promoting further spreading of the permeability transition to all mitochondria in the cell in a positive feedback loop As a

result, generalized mitochondrial dysfunction and irreversible contracture and sarcolemmal rupture would follow In combination with the present results, having concluded that mitochondrial CD38 is an active enzyme which is capable of catalyzing both ADP-ribosyl cyclase and NAD+ glycohydrolase activities, it is therefore interesting to determine whether the same process could apply to mitochondrial CD38 observed in brain tissues (Figures 5.1 and 5.2) A simulated

post-ischemic/hypoxia model of brain in vitro system can be established as future

study to characterize the functional role (s) of brain mitochondrial CD38

A study of CD38KO mice (Jin et al., 2007) was conducted and showed that

transmembrane CD38 has an essential role in regulating the secretion of oxytocin (OT) via cADPR signaling pathways However, it was observed that cADPR was only effective at stimulating OT release in CD38 KO neuron terminals when the tissue was permeabilised by digitonin as well as the availability of extracellular NAD+ The group also observed that there was no increase of intracellular cADPR when the intact cells were incubated with NAD+ Since there were no indication of the presence of cADPR transporters on the respective OT and hypothalamic neurons, therefore in order for CD38 to be involved in the OT secretion pathway, cADPR must

be present in the intracellular milieu It was proposed that CD38 could act as the transporter for transporting the cADPR to the intracellular region (Chapter 3 Introduction); the present data could also serve as an alternate model whereby

intracellular CD38 i.e, mitochondrial CD38 would fill in the missing link that bridges

CD38 and the intracellular cADPR-mediated calcium signalling in responsive cells Future study can be carried out to investigate this

Generally most studies to date had focused on the ectocellular CD38 mechanism in cellular physiology This study is the first to describe an expression of

Chapter 5 Future Studies And Conclusions

functional CD38 in a fully glycosylated form observed in mitochondria (Chapter 3), which is further supported by results obtained from mouse brain mitochondria (Chapter 4) It is not unreasonable to postulate that the locale of CD38 may be a key factor in determining the specific function(s) it will perform in a particular site It would then be interesting to investigate the mechanism of the ubiquitous expression

of CD38 in different cellular compartments i.e, mitochondria, nucleus, ER Two

possible areas could be ventured in order to explain this ubiquity First, the molecule may undergo post-translational modification and thus the isoforms are routed to different locales in the cell This is not without precedence It was reported that multiple IP3 receptor isoforms have been shown to be present both on plasma

membrane and internal membranes (Quinton and Dean, 1996; Yule et al., 1997)

Second, significant data reported beginning in the 1990s indicate that lipid movement between intracellular organelles can occur through contacts and close physical

association of membranes (Discussion of Chapter 3; Vance et al., 1991; Camici and

Corazzi, 1997)

Recent studies reporting the processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum (ER) lumen prior to mitochondrial

importation (Mavinakere et al., 2006), as well as observing mitochondrial and

secretory human cytomegalovirus UL37 proteins traffic into

mitochondrion-associated membranes (MAM) of human cells (Bozidis et al., 2008), further support

the above statements Because of the well documented role of MAM trafficking

membrane-bound molecules from ER to mitochondria (Vance, 1991; Stone et al., 2000; Ardail et al., 2003; Bionda et al., 2004) as well as taking in the consideration of

as a type II membrane glycoprotein, CD38 may subject to the ER-Golgi route; it is then very tempting to speculate that CD38 would be shuttling between organelles via

the membrane contact point, for example, the mitochondrion-associated membranes,

a subdomain of the ER acting as membrane bridges and thus provides direct physical contact to mitochondria It would be interesting to investigate the mechanism of the shuttling of molecule between different cellular compartment as well as its specific role involved in the particular locale

It is interesting to note that liver mitochondrial CD38 has demonstrated a role

in NAADP sysnthesis, as observed by Liang et al (1999) It was also reported that

the presence of specific NAADP binding sites in the brain on both neuronal and

non-neuronal cells (Bezin et al., 2006) In view of the results observed in current studies,

it would be interesting to investigate the synthesis of NAADP by brain mitochondrial CD38 as well as its Ca2+ mobilizing property, i.e, whether it can act in a similar manner as cADPR It was reported by Cancela et al (1999) that pancreatic acinar

cells are more sensitive to NAADP than either cADPR and IP3 with regard to induced Ca2+-signaling It remains to be seen if NAADP can have a role in brain mitochondrial Ca2+ signaling

agonist-The next important question to be addressed regarding CD38-mediated mitochondria Ca2+ signaling is the mechanism involved in regulating the enzymatic activities of mitochondrial CD38 A novel intracellular soluble ADP-ribosyl cyclase

can be regulated by tyrosine-phosphorylation, as reported by Guse et al., (1999) It

would therefore be tempting to investigate if similar mechanism applies to the mitochondria CD38 as well, given that it has been reported that rat CD38 contains a tyrosine residue in the cytoplasmic tail, which is conserved in mouse CD38 but not human CD38 (Shubinsky and Schlesinger, 1997)

Moreover, it is interesting to note that recently published studies regarding mitochondrial nitric oxide synthase (mtNOS) was observed in various tissues

Chapter 5 Future Studies And Conclusions

including rat and mouse brain, as discussed in the excellent review by Navarro and Boveris (2008) Indeed, Nitric oxide has been shown to play a role in intracellular

Ca2+ mobilization in sea urchin eggs via the cADPR-ribose signaling pathway

(Willmot et al., 1996) Nitric oxide activates a downstream signaling pathway in

as well as regulate the ADP-ribosylation of various proteins (Zoche and Koch, 1995)

and the ADP-ribosyl cyclase activity of CD38 via S-nitrosylation (White et al., 2002)

mtNOS, which was reported to be localized at inner mitochondria membrane facing the intermembrane space, may be in close proximity to the outer mitochondrial membrane located CD38 This close apposition between the two molecules may have specific role against each other The roles of nitric oxide in the enzyme regulation such as the autoribosylation of CD38 or regulation of the ADP-ribosylation of other proteins via ADP-ribosyl transferase activity of CD38 are therefore interesting areas

to be determined and explored

5.3 Concluding remarks

Mitochondrial CD38 was first reported by Liang et al (1999) using rat liver

tissues, though the reports of mitochondrial NAD+ glycohydrolase in rat brain and liver tissues has long been established in 1980s (Discussion of Chapter 4) The enzyme was identified by its subcellular localization in mitochondria and immunoreaction towards anti-CD38 antibodies that recognize the cell surface CD38, nucleus CD38 as well as microsomal CD38 on both rat and human brain samples

(Mizuguchi et al., 1995; Yamada et al., 1997) More reports surfaced on CD38 localized in mitochondria mouse brain tissues (Aksoy et al., 2006) as well as different tissues such as pancreatic acinar cells (Sternfeld et al., 2003) It is hard to attribute

CD38 in mitochondria to contamination from other cellular compartments because it would seem unreasonable that very different tissues/ tissues from different species would show a similar level of “contamination” of the mitochondrial fraction with the membrane associated CD38 Hence the observations made in the current studies begin to shed new light on the role and involvement of CD38 in the complexities of mitochondrial signalling

In conclusion, the current study proposes that signaling through mitochondrial CD38 might represent a novel paradigm in cellular signaling processes, and is unique

in the sense that in addition to extracellular signaling, it is also involved in intracellular signaling This is particularly interesting in which mitochondria are known as central to intracellular Ca2+ homeostasis, steroid synthesis, generation of free radical species, and apoptotic cell death As a consequence, mitochondrial dysfunction has devastating effects on the integrity of cells and may thus be critically involved in aging, metabolic and degenerative diseases, as well as cancer in higher organisms and humans (Wallace, 2005) Indeed the results of the current study give

us a new platform with which to re-visit and re-evaluate the current dogma on the limits of this unusual molecule While CD38 has long been regarded to be primarily involved in surface membrane signaling events, the revelation of its presence on mitochondria and the promise of the various roles it may play in mitochondrial processes suggest to us that there is still much to learn from this fascinating molecule

Chapter 5 Future Studies And Conclusions

Figure 5.1 Schematic representations of the proposed model of structure and characteristic of CD38 located on mitochondria Mitochondrial CD38 localized on the outer mitochondrial membrane with a specific topology of its bulky carboxyl catalytic domain extruding into the cytosol In response to Ca2+, atractyloside, adenine nucleotide depletion, chemotherapeutics and pro-oxidant agents, the mitochondrial membrane permeabilization can result from the opening of PTP as a large unspecific channel and leads to the release of proapoptotic factors into the cytosol Following the release of intramitochondrial NAD+ to the cytosol, the immediate NAD+ source becomes substrate to CD38 located on the outer mitochondrial membrane Ca2+mobilizing agents, cADPR and ADPR are generated which are in turn responsible for the downstream signaling (Modified from Ayub and Hellett, 2004)

PTP

NAD + cADPR

ADPR

CD38

Permeability transition pore

IMM OMM

Ryanodine Receptor (RYR) CD38

TRPM2 Permeability transition pore

NAD +

ADPR

Chapter 5 Future Studies And Conclusions

Figure 5.2 Proposed mechanisms for possible intracellular signaling mediated by mitochondrial CD38 In response to rise in [Ca2+]i that promotes the opening of PTP causing the release of intramitochondrial NAD+ to the cytosol, this immediate NAD+source becomes substrate to CD38 located on the outer mitochondrial membrane

Ca2+ mobilizing agents, cADPR and ADPR are generated which are in turn responsible for the downstream signaling as shown In close proximity, RYR located

on endoplasmic reticulum is sensitive to activation under the action of cADPR Mitochondrial RYR was reported recently and the mechanism interplay with mitochondrial CD38 waits to be explored further Close apposition of mitochondria and plasma membrane may give rise to an immediate source of ADPR which bind on the TRPM2 cation channel located on the plasma membrane thus results in influx of

Ca2+ and Na+ from extracellular region (Ayub and Hellett, 2004)

CHAPTER 6

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