Subcellular compartmentalization of CD38 in non hematopoietic cells a study to characterize its functional role in mitochondria 3

Here, for the first time, enzymatically active CD38 was shown to be present intracellularly on the outer mitochondrial membrane OMM of CD38+mitochondria isolated from COS-7 cells transie

to conclusively show the functional role of CD38 on mitochondria Here, for the first time, enzymatically active CD38 was shown to be present intracellularly on the outer mitochondrial membrane (OMM) of CD38+mitochondria isolated from COS-7 cells

transiently transfected with Mito-CD38 (CHAPTER 3) and supported by

experimental results acquired using mouse brain tissue Immunolabeling on mouse brain vibratome sections and isolated mitochondria using both TEM and SEM determined the localization of CD38 to the outer mitochondrial membrane with its C-terminal region (domain that contains the catalytic site) facing the cytosolic site This specific topology of CD38 was further verified and supported by performing

when exposed to cADPR, indicating the presence of the complex receptor machinery that is responsible for the opening of the Ca2+ channels in these stores

In mouse brain, CD38 was found in both neurons and glial cells, showing a

predominant intracellular location, and was enriched in neuronal perikarya (Ceni et

al., 2003; Jin et al., 2007) In human brain, CD38 immunoreactivity was demonstrated in the perikarya and dendrites of many neurons (Mizuguchi et al.,

1995) In rat astrocytes, ADP-ribosyl cyclase has been reported to have both

intracellular and extracellular actions (Hotta et al., 2000) Moreover, co-culture of

astrocytes with neurons resulted in significant overexpression of astrocyte CD38 both

on the plasma membrane and intracellularly, and this effect was attributed to

neuron-released glutamate action on astrocytes (Bruzzone et al., 2004)

Interestingly, Mizuguchi group’s immunohistochemical investigations using human brain tissues localized most of the brain CD38 immunoreactivity into the perikaryal and dendritic cytoplasm of neurons The granular staining profiles suggest

an association with intracellular organelles (Mizuguchi et al., 1995) In a separate

membrane as well as cell organelles such as rough ER, small vesicles, mitochondria

and nuclear envelope (Yamada et al., 1997)

Yamada group observed that in the rat cerebral cortex, CD38 immunoreactivity was demonstrated in a subset of pyramidal neurons, and was distributed preferentially in the perikarya and dendritic arbors The subcellular labeling was in the region close to the plasmalemma, including the postsynaptic densities, implying that CD38 is involved in signal transduction via the plasma membrane of certain CNS neurons In the cerebellar cortex, the immunoreactivity was recognized in a rather wide range of neuronal types such as granule, Golgi and basket cells CD38 expression in rat Purkinje cell bodies and dendrites were quite weak compared to those in the human cerebellum, probably due to species differences Except for the Purkinje cells, the subcellular localization of CD38 in cerebellar neurons was noted in the perikarya, axon terminals and dendrites, in order

of decreasing intensity Although the labeling of the plasmalemma and several organelles in these structures was similar to that in the cerebrum, the association of immunoreactivity with synaptic vesicles is rather unique to cerebellar neurons, suggesting an additional functional role of CD38 in certain types of presynaptic region

The present study also demonstrated the expression of CD38 in astrocytes The immunolabeling was more intense compared to that in the neurons, and was

Aksoy group presented the latest finding providing evidence that high cADPR levels were indicated in brain tissues as compared to other tissues such as heart, liver, spleen and kidney, conforming to the past findings (Aksoy et al., 2006) This group then proposed CD38 as the major NAD+ glycohydrolase/NADase in mouse brain The term NAD+ glycohydrolase /NADase was used for the reason that the majority of CD38 catalytic activity is the degradation of NAD+ to the final product, ADPR (Introduction 1.3) Using separate techniques they have observed that the NAD+glycohydrolase activity was present in WT brain extracts but was significantly reduced in CD38 KO mouse The finding was further supported by the observation that a majority of the NAD+ glycohydrolase activity in the WT brain extracts can be immunoprecipitated by CD38 antibody In addition, they also found that CD38 are not present solely in the plasma membrane but localized in intracellular membranes

of organelles such as mitochondria and nuclei (Aksoy et al., 2006)

4.1.2 Brain Mitochondria and CD38

Mitochondria are unique organelles central for various cellular processes that include ATP production via oxidative phosphorylation, intracellular Ca2+homeostasis, steroid synthesis, forms of apoptotic cell death and generation of reactive oxygen species Neurons critically depend on mitochondrial functions to establish membrane excitability and to execute the complex processes of neurotransmission and plasticity (Wallace, 2005)

The cell modulates increased cytosolic calcium either by extruding it across

compared with that of the endoplasmic reticulum (Carafoli, 1987), mitochondria possess a very high capacity for calcium and thus may be of significance when a

sustained rise in intracellular calcium above 1µM occurs (Burgess et al., 1983)

So, normal Ca2+ cycling [at low resting mitochondrial Ca2+ concentration ([Ca2+]m)] occurs by the movement of Ca2+ into mitochondria via the Ca2+ uniporter and slow extrusion via the Na+/Ca2+ exchanger or by Na+-independent mechanisms involved mitochondrial H+/Ca2+ exchanger (Nicholls et al., 2000; Bernardi, 1999)

Both antiporters are of low capacity thus the transport rate can be easily surpassed by the Ca2+ uniporter, leading to net Ca2+ accumulation in mitochondria Isolated mitochondria in the presence of phosphate take up Ca2+ to a fixed capacity, in a membrane potential (∆ψm) - dependent fashion (Nicholls and Akerman, 1982; Gunter

et al., 1994; Chalmers et al., 2003)

An alternative pathway for Ca2+ is via the transient low-conductance opening

of the mitochondrial permeability transition pore (mPTP), which may release the toxic Ca2+ loads from mitochondria (Zoratti et al., 1995; Ichas et al., 1997)

Mitochondrial Ca2+ uptake was observed in intact cells at cytosolic Ca2+concentration ([Ca2+]c ) as low as 150-300nM (Pitter et al., 2002) In the event of high

local Ca2+ domains of tens of micromolar in the vicinity of voltage-operated Ca2+channels or Ca2+ release sites of the endoplasmic reticulum (Rizzuto and Pozzan, 2006), rapid uptake Ca2+ into mitochondria might be necessary (Pacher et al., 2002;

The pathways involved in the movement of calcium from the matrix space are less well characterized It is clear that efflux of accumulated calcium from respiring

mitochondria can be induced by a variety of prooxidants (Lotscher et al., 1979; Bellomo et al., 1982; Boquist, 1984; Graf et al., 1985; Frei and Richter, 1986; Richter

et al., 1987; Fagian et al., 1990) Several hypotheses have been presented to explain

the efflux of calcium from respiring mitochondria, including changes in the redox

status of the mitochondrial pyridine nucleotide pool (Lehninger et al., 1978),

oxidant-mediated formation of a nonspecific pore in the inner mitochondrial membrane

(Crompton et al., 1988), or hydrolysis of intramitochondrial oxidized pyridine

nucleotides by an NAD+ glycohydrolase /NADase (Lotscher et al., 1979; Lotscher et

al , 1980; Richter et al., 1987)

NAD+ has recently emerged as a crucial regulator of the signaling pathways implicated in multiple physiological conditions (refer to review by Chini, 2009) Important signaling roles of NAD+ such as its direct consumption for the synthesis of

ADPR/ADPR polymers (ADP-ribosylation) (Jacobson et al., 1983; Shah et al., 1996;

Vu et al., 1997), which appears to be involved with responses that can lead to normal

cellular recovery, apoptosis or necrosis cADPR along with ADPR is potent Ca2+releasing agent involved in many signaling pathways leading to apoptosis or necrosis (refer to review by Chini, 2009) as well as a role as a substrate and regulator of the NAD+ dependent deacetylases sirtuins (Ziegler and Niere, 2004; Baur et al., 2006; Lagouge et al., 2006) Since NADP can be generated de novo from NAD+, conversion of NADP to NAADP by the NAD+ glycohydrolase serves as another Ca2+

fertilization to the cellular mechanism of aging; longevity and death (refer to review

by Chini, 2009)

It is not unexpected that a significant proportion of the cellular NAD+ pool is

reported to be compartmentalized within the mitochondria (Tischler et al., 1977; Lisa

et al., 2001) It appears that NAD+ can be synthesized within this organelle and

accumulated at much higher concentrations than seen in the cytosol (Lisa et al., 2001) The nuclear enzyme NMNAT-1 is the key enzyme in both de novo and

salvage pathways of NAD+ synthesis (Kolb et al., 1999), until recently two newly

reported isoforms, NMNAT-2 and NMNAT-3, which are located separately in the

Golgi complex and mitochondria, are added to the family (Raffaelli et al., 2002; Magni et al., 2004; Berger et al., 2005) This suggests independent NAD+ metabolism

in the nucleus, the Golgi complex and mitochondria (Figure 4.1)

It is well recognized that NAD+ can mediate calcium homeostasis (Figure 4.2) through pathways such as the following: a) ADP-ribosyl cyclases/ NAD+glycohydrolases produced cADPR from NAD+, which serves as a potent endogenous agonist of ryanodine receptor mediated calcium channels; b) ADPR, another Ca2+mobilizing molecule generated from NAD+ by NAD+ glycohydrolases can activate TRPM2 receptors leading to Ca2+ influx (Guse, 2005) as well as substrate used for ADP-ribosylation, a posttranslational protein modification process as briefly introduced above Several lines of evidence suggest that NAD+-dependent signaling

findings found otherwise that this molecule is in fact localized on the outer

mitochondrial membrane (Satrustegui et al., 1984; Boyers et al., 1993; Lisa et al.,

2001) It was then postulated that instead of the previous belief of mitochondrial Ca2+release upon the ADP-ribosylation of an intrinsic protein of the inner mitochondrial membrane following the pyridine nucleotide hydrolysis by a Ca2+-stimulated matrix NAD+ glycohydrolase (Boyer CS and Peterson DR, 1991; Masmoudi and Mandel, 1987), new evidence has shown that NAD+ hydrolysis is only possible after the PTP opening and Ca2+ release is the cause rather than consequence of NAD+ hydrolysis

(Lisa et al., 2001) Recent report has further identified that bovine liver

mitochondrial NAD+ glycohydrolase as the ADP-ribosyl cyclase (Ziegler et al., 1997)

Following the finding by Liang et al (1999) which showed a minimal amount

of CD38 localized in rat liver mitochondria fraction, it is of interest to investigate the presence of CD38 in other tissues that have been shown to have robust ADP-ribosyl

cyclase activities, i.e brain (Aksoy et al., 2006) To have a closer investigation into

the Ca2+ interplay by this molecule, the exact submitochondrial location of this activity has to be determined

Figure 4.1 NAD+ metabolisms in cells NAD+ metabolism occurs both intracellularly

in various subcellular organelles and extracellularly The key NAD+ synthesizing enzymes NMNAT-1, NMNAT-2, and NMNAT-3 are located at the nucleus, the Golgi complex, and mitochondria, respectively There are NAD+-consuming enzymes

in these organelles, including poly (ADP-ribose) polymerase-1 (PARP-1), PARP-2, and certain sirtuins in the nucleus, tankyrase in the Golgi complex, and NAD+glycohydrolases in mitochondria On plasma membranes, mono (ADP-ribosyl) transferases (ARTs) and ADP-ribosyl cyclases (ARCs) produce mono (ADP-ribosylation) on target proteins and generate cyclic ADP-ribose, respectively Nicotinamide phosphoribosyltransferase (Nampt) may exist extracellularly and produce its biological effects by generating nicotinamide mononucleotide from nicotinamide (Adapted from Ying, 2008)

Figure 4.2 Pathways by which NAD+ and NADP can affect calcium homeostasis ADP-ribosyl cyclases (ARCs), poly(ADPribose) polymerases (PARPs)/poly(ADP- ribose) glycohydrolase (PARG), and sirtuins use NAD+ as a substrate to generate

several Ca2+-mobilizing second messengers, including cADPR, ADP-ribose, and acetyl-ADP-ribose (O-acetyl-ADPR), which can activate TRPM2 receptors and ryanodine receptors (RyR) NAD+-dependent mono (ADP-ribosyl) transferases (ARTs) can also affect Ca2+ homeostasis by producing mono-ADP-ribosylation of P2X7 receptors (ADPR- P2X7R) NADH could modulate Ca2+ homeostasis by affecting IP3-gated Ca2+ channels, mitochondrial permeability transition (mPTP) and RyR NAADP generated from NADP can also mobilize intracellular NAADP-dependent Ca2+stores NADPH may affect calcium homeostasis by its major effects

O-on antioxidatiO-on and ROS generatiO-on, which can affect Ca2+pumps and Ca2+channels

(Adapted from Ying, 2008) The encircled area is the major focus in this study

4.2 Results

4.2.1 Mitochondria Isolation from mouse tissues

A well-characterized Percoll density gradient method for purifying mitochondria from mouse brain tissues with negligible contamination from other cellular organelles in particular microsomal membranes was adopted The Percoll density gradient method has been shown to provide the optimum recovery of active

and pure brain mitochondria (Nishadi et al., 2001) The isolation of mitochondria

based on this method has been shown to result in an enriched mitochondrial preparation with negligible microsomal, nucleus, endoplasmic reticulum and plasma membrane contamination as well as microsomal-mitochondrial association

Mitochondria were isolated from mouse brain tissues using the combination of gravity and gradient centrifugation After gradient centrifugation using 30% Percoll solution, two distinct bands with white upper layer and yellowish brown lower layer could be observed in the continuous gradient body, with diffuse floating material between the two bands for brain tissues (Figure 4.3) There were 4 faintly distinct bands observed for liver tissues instead (data not shown) All these fractions were collected and investigated using SDS PAGE, Western blot and electron microscopy

Figure.4.3 Image of a Ti70.1 centrifuge tube after Percoll gradient centrifugation

during the mitochondrial isolation procedure on mouse brain tissues Two bands were found, with some diffuse materials in the intermediate area The upper band contained membranous structure and lysosomes, whereas the lower band was proved later to be highly pure intact mitochondria

4.2.2 Determination of the purity of Percoll purified brain mitochondria

At the end of the washing step, the resulting mitochondrial fraction obtained for mouse brain tissues was brown in color, instead of dark brown for liver mitochondrial pellet (data not shown) The fact that there was no contamination of the mitochondrial fraction by elements of the plasma membrane and microsomes was

Membranous structure

Pure mitochondrial extract

microsomal fractions (Figure 4.4), it can be seen that there was prominent presence of the marker enzymes in all fractions except Percoll purified mitochondria thus further attesting to the purity of the Percoll purified mitochondrial fraction This is expected

as crude mitochondria are normally contaminated with microsomal fraction (Boyers

et al., 1993)

Using an antibody against mitochondrial heat shock protein 70 (mtHSP70), a mitochondrial resident protein, it was observed that the Percoll purified mitochondrial fraction as well as crude mitochondrial fraction have been enriched for mitochondrial components Antibodies against the dominant endoplasmic reticulum and nucleus markers consisting of calreticulin and nucleoporin p62 were then used to assess the purity of the Percoll purified mitochondrial fraction with respect to microsomal and nucleus contamination Again, the presence of the two above-mentioned proteins in the Percoll purified mitochondrial fraction could not be detected in comparison with the homogenates, microsomal and crude mitochondrial fractions

Detection of CD38 from each fraction was carried out in the process along with different markers for different cellular compartments mentioned above The presence of a single 42-45 kDa protein band in each fraction was detected with the anti-CD38 antibody, a commercial product from Santa Cruz, sc-7049 (M19) A further test to investigate the specificity of the anti-CD38 antibody was therefore essential In a separate experiment, anti-CD38 antibody was incubated with 10X in

band reappeared when the same immunoblot was relabeled with sc-7049 without peptide blocking (data not shown) This further confirmed the specificity of antibody and the band detected at 42-45kDa on this organelle is indeed CD38 and not a false positive result Previously CD38 in an 85kDa dimeric form was detected under non-reducing condition in partially purified nuclear fraction prepared from rat cerebellum (Khoo and Chang, 2002) It still remains to be seen if a dimeric form of CD38 could

be found in mitochondrial fraction

Overall, this experiment showed Percoll purified mitochondrial fraction presented a distinct but rather low density of CD38 as compared to homogenates, microsomal and crude mitochondrial fractions This result is expected considering that cellular localization of CD38 is found predominantly in the plasma membrane

(Jackson et al., 1990; Gelman et al., 1993; Hara-Yokoyama et al., 1996; Deaglio et

al., 1996; Konopleva et al., 1998; Franco et al., 1998)

Figure 4.4 Detection of marker enzymes with Western blotting Fractions prepared

from homogenates, microsome, crude mitochondrial and Percoll purified mitochondrial fractions as described in Materials & Methods were probed with (A) anti-CD38 antibody (B) anti-Na+/K+ ATPase α form (C) anti-Na+/K+ ATPase β form

Figure 4.5 Detection of CD38 from purified mitochondrial fraction prepared from

mouse brain tissues Immunoblots comprised of the purified mitochondrial fractions

at 100µg (lanes 3 and 3a) and 50µg (lanes 4 and 4a) were probed with the anti-CD38 antibody (sc-7049) and compared with microsome fractions (lanes 1 and 1a) as well

as partial purified CD38 fraction (lanes 2 and 2a) A single distinct ~45kDa protein (arrow) could be detected from all fractions as shown in immunoblot (B) The same band disappeared when the immunoblot is probed with sc7049 incubated with 10 X excess CD38 blocking peptide as described in Materials & Methods High background in immunoblot (A) is a result of lengthening the exposure time for the immunoblot with x-ray film as compare to immunoblot (B) to confirm the missing protein band Approximately 50µg loaded for microsomes and 10µg for partial purified CD38 fraction

4.2.3 Determination of the submitochondrial localization of CD38

The morphological studies using EM (electron microscopy) suggests that CD38 was successfully localized on the outer mitochondria membrane (Figures 4.11-4.22) Biochemical studies were carried out in parallel to further support this view

To determine the localization of the CD38 protein on mitochondria, the Percoll purified brain mitochondrial fractions were analyzed by treatments with increasing concentrations of digitonin along with protease digestion The outer mitochondrial membrane is known to have higher sensitivity to saponin and digitonin

as compared to the inner mitochondrial membrane (Saks et al., 1993) At low

concentrations, digitonin selectively solubilises cholesterol, thereby disrupting the cholesterol-rich outer mitochondrial membrane, whereas at higher concentrations it

acts as nonspecific detergent (Boyer et al., 1993) In other words, the outer

mitochondrial membrane solubilises at a lower concentration of digitonin as compared to the mitochondrial inner membrane, which makes it possible to selectively release intermembrane space proteins or accessible to protease digestion

(Greenswalt et al., 1974) Figure 4.6 shows the selective and progressive removal of

the outer mitochondrial membrane by increasing concentration of digitonin Upon

increasing the digitonin (dig)/protein (Prot) ratio from 0 to 0.1 the mitochondrial

outer membrane became permeable to the inter membranous space, without significant removal of the mitochondrial outer membrane, as shown by the almost