BCL-xL Regulates Synaptic Plasticity

8/7/2019 BCL-xL Regulates Synaptic Plasticity

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Elizabeth Jonas

Yale University School of Medicine, Section of Endocrinology, Department of Internal Medicine,

333 Cedar Street, PO Box 208020, New Haven, CT 06520-8020

Mitochondria are the predominant organelle within many presynaptic terminals. During times of high synapticactivity, they affect intracellular calcium homeostasis and provide the energy needed for synaptic vesiclerecycling and for the continued operation of membrane ion pumps. Recent discoveries have altered our ideas

about the role of mitochondria in the synapse. Mitochondrial localization, morphology, and docking at synaptic sitesmay indeed alter the kinetics of transmitter release and calcium homeostasis in the presynaptic terminal. In addition,the mitochondrial ion channel BCL-xL, known as a protector against programmed cell death, regulates mitochondrialmembrane conductance and bioenergetics in the synapse and can thereby alter synaptic transmitter release and therecycling of pools of synaptic vesicles. BCL-xL, therefore, not only affects the life and death of the cell soma, but its

actions in the synapse may underlie the regulation of basic synaptic processes that subtend learning, memory andsynaptic development.


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Volume 6, Issue 4

Introduction

Transmission of signals through the nervous system requires cell-

to-cell communication via neuronal synapses. The basic features

of chemical synaptic transmission include close apposition of two

nerve cells and release of a chemical neurotransmitter by one cell

into the synaptic cleft between two neurons (1). After release, neu-

rotransmitter influences responses of the second neuron via recep-

tors on the postsynaptic cell (2). Neurotransmitter is packaged into

small vesicles within the presynaptic terminal, and collections of

vesicles wait for a calcium signal produced by calcium entry into

the presynaptic cell during action potential firing (3). Elevation of

intracellular calcium during synaptic activity enhances the probabil-

ity of vesicle fusion. Many of the features of synaptic transmission

can be enhanced over the short and long term (4). These include

changes in presynaptic calcium levels, changes in vesicle numbersand probability of release, and alterations in postsynaptic receptor

numbers and function. Such changes lead to short- and long-term

modifications in synaptic strength and account in part for plasticity

of synaptic activity. Many of these phenomena require energy, and,

therefore, may be regulated by mitochondria as will be described in

this review. Mitochondria also buffer and re-release calcium inside

the synapse, altering the time course and amplitude of the change

in calcium concentration during vesicle fusion and recycling (5).

Unexpectedly, the BCL-2 family proteins that are known to regulate

apoptosis through their actions at mitochondrial membranes have

been newly identified as regulators of synaptic activity. Thus, the

actions of BCL-xLa BCL-2 family memberat mitochondria posi-

tion it to influence learning, memory, and alterations in behavior.

Mitochondria Regulate SynapticTransmission

Mitochondria are known to be important for synaptic transmission

and are the predominant organelle within presynaptic terminals that

release neurotransmitter at high rates (6).Mitochondria provide ener-gy in the form of ATP and buffer calcium at these active synapses.

Some synaptic mitochondria may buffer calcium even at the expense

of ATP production. Indeed, different types of neuronal synapses con-

tain different numbers of mitochondria with slightly different proper-

ties, depending on whether the main function of the mitochondriais to provide energy or buffer calcium. At some synapses, oxidative

metabolism by mitochondria is crucial to successful neurotransmis-

sion, which can be altered considerablyfor example, by the rapid

onset of synaptic fatigueif mitochondrial function is eliminated (7).

Moreover, mitochondrial bioenergetics are altered acutely in synapses

that have undergone conditioning, providing for enhanced oxidative

competence (7). Therefore, an interaction may exist between neuro-

nal plasticity and mitochondrial plasticity(8). In this review, we will

focus on the role of mitochondria in synaptic transmission and syn-

aptic plasticity and consider possible ways in which the mitochondri-

al protein BCL-xL brings about changes in mitochondrial properties

that may influence these important synaptic events.

Mitochondria Alter Calcium Homeostasis

During Synaptic Events

Synaptic transmission depends on mitochondria not only for energy

production but also for maintaining calcium homeostasis within the

presynaptic terminal (915). During synaptic events, calcium influx

through voltage-gated channels and the release of calcium from

intracellular stores produce elevations of cytosolic calcium that are

BCL-xL at the Synapse

Ca2+

Postsynapticpotentials

Stimulus

tetanus postetanicpotentiation

residualcalcium

no potentiation

Control No mitochondrialCa2+ uptake

A B

C Posttetanic potentiation

Ca2+

2. After thetetanus,calcium hasaccumulatedinside themitochondrialmatrix.

1. Repeatedactionpotentials(tetanus)

3. Upon repeated stimulation,the extra calcium provided bymitochondria is available forvesicle fusion.

Figure 1. Synaptic potentiation requires mitochondria.A. A diagram of

changes in postsynaptic potentials and presynaptic calcium levels during

and after tetanic stimulation to the presynaptic cell. B. In the absence of

mitochondria, the prolonged tail of residual calcium observed after the teta-

nus is not apparent and posttetanic potentiation is inhibited. C. Re-release of

calcium from mitochondria inside the presynaptic terminal controls short-term

synaptic plasticity.


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crucial for synaptic vesicle fusion (16). In the crayfish neuromuscular

junction, fast synaptic transmission is dependent on elevated calcium

levels inside the presynaptic terminal (10). During a high frequency

train of stimuli (tetanus), the amplitude of the response of the post-

synaptic cell to neurotransmitter release gradually increases, and even

after the tetanus has ceased, the ability of the synapse to release neu-

rotransmitter is increased for up to several minutes (Figure 1A).

At the very least, although there may be other factors, the abili-

ty of the synapse to increase the amount of neurotransmitter released

is based on: 1) the ability of the pool of releasable neurotransmitter-

containing vesicles to change size, 2) a change in the probability of

individual vesicle fusion, or 3) a change the amount of calcium avail-

able for release per vesicle (15). Different synapses may have different

degrees of potentiation or depression of release of neurotransmitter,

both during and after the tetanus, depending on their particular

attributes. It has been argued that synapses with a high probability ofinitial release will depress subsequent release, because they deplete

their vesicle pools more rapidly, whereas synapses with a low prob-

ability of release will augment release upon increased stimulation,

because these synapses contain abundant vesicles that, during base-

line stimulation are released less frequently(16).

Although the causes of a change in release probability are

complex, both the level of cytosolic calcium and the proximity of

sites of calcium influx into the cytosol to sites of vesicle fusion par-

ticipate in enhancing the probability of fusion events (17, 18). The

level of residual calcium during frequent synaptic activity can also

play a role in recovery from vesicle depletion (19, 20). In many syn-

apses, tetanic stimulation causes depression of synaptic responses,

whereas, as discussed above, in the crayfish neuromuscular junc-tion, neurotransmitter release is enhanced during the tetanus, in

part because vesicles may reaccumulate rapidly even during frequent

events. Other synapses have different responses to tetanic stimula-

tion. For example, the squid giant presynaptic terminal and the large

mammalian central nervous system auditory relay synapse (the calyx

of Held) of the medial nucleus of the trapezoid body (MNTB, Box

1) manifest synaptic depression during repetitive stimulation (19,

21). The depression at these synapses is most likely mediated by a

high probability of release of vesicles from multiple sites (i.e., active

zones) as well as by elevated calcium in the terminals. Under experi-

mental conditions in the squid presynaptic terminal, if extracellular

calcium concentration is decreased, then synaptic potentiation canbe elicited (21), as in the crayfish synapse.

Mitochondria participate in shaping the time course and

amplitude of neurotransmitter release from presynaptic nerve end-

ings after the invasion of the endings by action potentials. In the

example of the crayfish neuromuscular junction, eliminating the

ability of mitochondria to sequester calcium during influx through

voltage-gated calcium channels leads to a higher rise in intracellular

calcium inside the presynaptic terminal during a tetanus but also to

prevention of the normal potentiation of neurotransmitter release

after the tetanus (Figure 1B) (10). The findings demonstrate that

mitochondria are important for the persistent elevation in intracel-

lular calcium (residual calcium) normally found in the presynaptic

terminal after it has fired action potentials at a high rate. After mito-

chondria sequester calcium, they act as a source of persistent release

of calcium from the matrix into the cytosol (Figure 1C). In bullfrog

sympathetic neurons, mitochondria also slow the rise in intracellular

calcium that occurs during a depolarizing stimulus by removing cal-

cium from the cytosol, and slowing the recovery of normal calcium

levels after the stimulus (9). At these synapses, mitochondria act as a

high capacity buffer of cytosolic calcium and also re-release calcium

rapidly in response to a calcium load in the mitochondrial matrix. At

the synapse of the MNTB, however, mitochondria play a slightly dif-

ferent role; the effect of mitochondrial calcium sequestration here is

to speed the recovery from synaptic depression (20).

Mitochondrial Presence At Presynaptic Sites

Regulates Intense Synaptic Activity

We have so far suggested that mitochondria play an important role

in regulating neurotransmission in several well-studied models.

Another invertebrate model, that of the Drosophila melanogaster

neuromuscular junction, provides an ideal system for studying

mutations that affect mitochondria and neurotransmission. A

genetic screening technique for mutations that affect synaptic trans-

mission in the Drosophila visual system has led to the fascinating

Review

Box 1. Characteristics of the MNTB

The medial nucleus of the trapezoid body (MNTB)

is located in the auditory brainstem of mammals.It participates in neural pathways that compute thedirection of sounds in space by comparing the tim-ing and the intensity of signals that arrive at the twoears. To ensure the accuracy of this information,MNTB neurons, and certain other neurons in thesepathways, are capable of firing action potentialsat very high rates (600 Hz or more). Such ratesare about one order of magnitude faster than mosttypical neurons (115118). Moreover a very largepresynaptic terminal, termed the calyx of Held,envelops the soma of an MNTB neuron and pro-

vides the very strong and secure excitatory inputto these cells. These and other features ensure thatMNTB neurons fire with very high temporal preci-sion and allow them to lock their action potentialsto rapidly changing features of sound stimuli (119,120). The high energy demands of high frequencyactivity in both the presynaptic terminals and thepostsynaptic cells are associated with mitochondrialspecializations, such as the tethering of presynapticmitochondria directly to the active zones where neu-rotransmitter is released (36).


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Volume 6, Issue 4

finding that multiple genes for mitochondrial targeting are necessary

for normal synaptic transmission at the neuromuscular junction

(2224). The first mutated mitochondrial targeting protein to be

identified in this screen was Milton (22). Milton binds to kinesin

heavy chain, linking mitochondria to microtubules for transport

into synaptic endings (25). Animals lacking Milton have abnormal

on- and off-transients on electroretinograms, indicating a defect

in synaptic transmission to second-order neurons, not in photo-

transduction itself(22). Immunoblots and immunocytochemistry

performed with Milton-specific antibodies demonstrated that Milton

localizes to axonal endings and synaptic sites and is co-localized

with mitochondria and with kinesin heavy chain. The mutant pho-

toreceptors contain abundant somatic mitochondria but completely

lack synaptic mitochondria. In other ways synaptic morphology is

fairly normal. For example, neurotransmitter-containing vesicles are

targeted normally to synapses, as evidenced by the presence of syn-aptic vesicles at active zones, but the density of synaptic vesicles is

slightly reduced, suggesting that the lack of mitochondrial targeting

influences the establishment or maintenance of vesicle pools in the

presynaptic terminal.

Two recent studies have shed further light on the role of mito-

chondria in vesicle pool dynamics. A genetic screening of Drosophila

yielded two other mutants for synaptic transmission, one of which

is a mutation in the GTPase dMiro, a protein that participates in the

anterograde transport of mitochondria to presynaptic terminals (24,

25). As observed with the Milton mutation, the dMiro-mutated flies

lack mitochondria in the presynaptic terminals of neuromuscular

junctions. The flies exhibit defects in locomotion and die prema-

turely. It is fascinating to note that in these dMiro mutants the mito-chondria line up in regular rows in the soma and cannot be escorted

out to the neuritic processes. The result is a defect in synaptic bou-

ton shape and size and an absence of the normal microtubule loops

that form in mature synapses. During high frequency activity at these

terminals, there is a slight increase in levels of intracellular calcium

compared to controls, and a more rapid fatigue of neurotransmitter

release. Calcium is rapidly cleared, however, from the terminals after

stimulation has ceased, and this clearance is no different from that

of controls. Another striking finding in these synapses is the desyn-

chronization of neurotransmitter release, such that activity causes a

barrage of miniature excitatory postsynaptic currents (EPSCs) after

the stimuli have ceased. These minis are unlikely to be related tocalcium homeostasis, which appears to be normal after the stimuli,

but may be related to inadequate or delayed functioning of vesicle

mobilization inside the presynaptic terminal.

Mitochondrial ATP Production Regulates Normal

Functioning Of Synaptic Vesicle Pools

Many studies in synaptic physiology have contributed to the idea

that distinct pools of vesicles have different probabilities of release,

thoroughly reviewed in Rizzoli and Betz (26). Several different

nomenclatures have been employed to describe the pools, but one

will be used here (26). The readily releasable pool is defined as the

vesicles that are immediately available for release, or docked at the

active zone. In hippocampal synapses, for example, there appear to

be approximately 510 vesicles that are docked at each active zone,

but a single brief stimulus (such as an action potential) may release

only one vesicle. The recycling pool is defined as the pool of vesicles

that continue to release and reaccumulate during moderate or

physiological stimulation. This pool contains 520% of all vesicles,

but these estimates vary in different synapses. The reserve pool is

defined as those vesicles that only release upon extremely frequent

stimulation. The reserve pool of vesicles makes up about 8090% of

the vesicles in most terminals.

Experiments on the temperature-sensitive Drosophila shi-

bire mutant (27) demonstrated that the reserve pool of vesicles is

normally mobilized only after the recycling pool is depleted. This

mutant exhibits defective endocytosis at high temperatures, leadingto an inability of vesicles to re-accumulate after exocytosis. In con-

ditions of mild or moderate stimulation, which would not usually

mobilize the reserve pool in controls, the reserve pool is neverthe-

less mobilized at the high temperatures in the mutant, suggesting

that the reserve pool must be used under circumstances where the

recycling pool has been depleted. The recycling pool therefore may

contain vesicles that are privileged for release, either by their interac-

tion with specific cytoskeletal elements, or their location, or both

(28). Surprisingly, however, as observed in synapses where vesicles

were fluorescently labeled and then photoconverted for electron

microscopy, the recycling pool is not located adjacent to the active

zone. Rather, the vesicles of the recycling pool are distributed widely

throughout the vesicle cluster(28).ATP is required for a myriad of cellular processes, and certain

steps in synaptic vesicle mobilization, release, and recycling, could

be compromised by the lack of locally and rapidly generated ATP.

Specific enzyme-dependent steps in synaptic transmission include

refilling single vesicles with neurotransmitter(29),membrane fis-

sion during endocytosis (30), and coated pit formation (31, 32).

Using whole-terminal capacitance measurements of goldfish retinal

bipolar neurons, Heidelberger showed that ATP was required for fast

compensatory membrane retrieval after exocytosis because dialysis

of a non-hydrolyzable form of ATP into the terminal completely and

rapidly inhibited endocytosis (30).

Recent evidence suggests that ATP is required for normalfunctioning of vesicle pools (23). Studies of another mutation in

Drosophila that prevents normal synaptic transmission suggest that

ATP is needed for mobilizing the reserve pool. In this set of experi-

mental findings, homozygous mutations in the eye for a gene that

encodes the dynamin GTPase family mitochondrial fission protein

DRP1 (dynamin-related protein 1) caused abnormal synaptic trans-

mission, as evidenced by abnormal electroretinograms. In the mutat-

ed flies, mitochondrial movement into presynaptic sites at the photo-

receptor synapses was absent, but presynaptic morphology appeared

normal in other ways. Photoreceptor somata contained numerous

mitochondria that were functional. In the neuromuscular junction,



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however, mitochondria were conspicuously absent, and resting cal-

cium levels were twice as high as those observed in controls.

Synaptic transmission at the neuromuscular junction failed dur-

ing intense stimulation, and this effect was temperature-dependent,

suggesting that transmission is normally mediated by a metabolic

change within the synapses. In addition, the effect on failure of syn-

aptic transmission during intense stimulation was partially rescued

by perfusion of ATP into the synapse. Verstrecken et al. reasoned

that during intense stimulation, mobilization of vesicles from the

reserve pool might require local ATP synthesis. Relying on a previ-

ous finding that the recycling pool of vesicles refills constantly dur-

ing stimulation, but that the reserve pool fills only after stimulation

has ceased (27), Verstrecken and colleagues were able to use the

styryl dye FM1-43 to differentiate between effects of the mutation

on the two different pools (Figure 2A). FM1-43 is taken up into

synaptic vesicles during vesicle recycling, where it fluorescently

labels collections of vesicles. By stimulating the nerve in a way that

produced exocytosis of vesicles from, and endocytosis of vesicles to,

the recycling pool alone, the authors demonstrated that there were

no differences in the properties of the recycling vesicle pool between

the mutants and the controls (Figure 2A, B).

Although recycling pool endocytosisexocytosis kinetics

appeared to be normal, the endocytosisexocytosis kinetics of the

reserve pool were not. The authors determined that the difference

in the mutants was in the ability of the reserve pool to take up dye

(Figure 2B). By adding the dye to the bath after strong depletion ofall pools, and letting the cells re-accumulate their vesicle pools in

the presence of dye, they discovered that the size of the filled pool

in controls was much larger than that of the mutants, and, when

they unloaded only the recycling pool of vesicles with a brief stimu-

lus, dye remained in the controls, but not in the mutant synapses,

suggesting that the mutant synapses contained a poorly functioning

reserve pool. The mutants could be rescued by overexpression of

the normal DRP1 protein, or by perfusion of ATP into the synapse.

Furthermore, the authors found that control reserve pools could be

functionally altered by treatment of synapses with inhibitors of mito-

chondrial function.

Additional experiments enabled the authors to conclude that

the defect in the drp1 mutants was in mobilization of vesicles fromthe reserve pool, not in the size of the reserve pool. They deter-

mined that an ATP-sensitive site was an intracellular motor that

moved vesicles from pool to pool in an energy-dependent manner.

The ATP sensitive motor turned out to be the myosin light chain

because: 1) inhibitors of the mysosin light chain kinase caused the

same defect in reserve pool cycling in controls as that seen in the

mutants, and 2) in the presence of the myosin light chain kinase

inhibitor, the reserve pool defect could no longer be rescued by per-

fusion of ATP into the synapse.

It is clear that mitochondria need to be targeted to the syn-

apse for synaptic transmission to function normally during intense

stimulation. Many questions remain, however. For example, what isthe mechanism of mitochondrial targeting to the synapse? When a

new synaptic connection is made, what is the role of mitochondria?

Does mitochondrial fission help target mitochondria to new synap-

tic sites? What is the signal that a mitochondrion is needed? How

does the release of ATP from mitochondria increase at the time it is

needed during intense stimulation? What are other ATP-dependent

steps in vesicle pool management?

Review

PostPre

Synaptic depletion After synaptic depletion,only the recycling and readilyreleasable pools label with dye

PrePost

PostPre

Synaptic depletion After synaptic depletionall three pools label with dye

PrePost

PostPre

Recycling Pool

Reserve Pool

Readily releasable pool

Calcium channel

During moderate stimulation,recycling and readily releasablepools lable with dye

PrePost

A Wild-type synapse

B Drp1 mutant synapse

Figure 2. Drosophila drp1 mutation prevents mobilization of neu-

rotransmitter-containing vesicles from the reserve pool.A. Labeling of

distinct pools of synaptic vesicles with FM 1-43 in the wild-type synapse is

achieved with different stimulation paradigms. B. In the mutant synapse, lack

of mobilization of reserve pool vesicles prevents dye uptake into the reserve

pool during stimulation as compared to control in A. See text for details.


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Axonal Targeting ofMitochondria and Their Docking

The axonal transport of mitochondria may be important for target-

ing of mitochondria to sites of presynaptic activity. Mitochondria

appear to move along the axon via cytoskeletal motors and can

move in both directions along the axon, as well as remain stationary

for prolonged periods of time when they are presumably docked at

a site where they are needed (33). Mitochondria in cortical neurons

in culture respond to application of the neurotransmitter glutamate

by ceasing all movement and changing morphology, suggesting that

neuronal activity and elevation of cytosolic calcium concentrations

may play a role in mitochondrial docking, as well as in cessation

of movement during excitotoxicity(34). Docking can also occur in

response to changes that produce axonal growth or in response to

intracellular signaling pathways stimulated by the binding of growthfactors extracellularly(35). The anterograde movement of mito-

chondria employs microtubules and kinesin motors, and it appears

that different organelles may utilize different adapter proteins to

link them to microtubules (35). As stated above, Milton and dMiro

are proteins that bind mitochondria to what is likely to be a large

microtubule-based complex of proteinsalso including the protein

syntabulininvolved in movement (25). After traveling along the

microtubule, mitochondria arrive at the synapse, where they trans-

fer from microtubules to an actin-based complex that docks the

mitochondrion. This complex most likely includes other membrane

anchoring proteins as well as actin, but most of those proteins have

not yet been identified (33). As seen in electron micrographs, the

brainstem auditory synapse of the MNTB (the calyx of Held), whichis specialized to release neurotransmitter at extremely high frequency

and fidelity, contains a mitochondrial adherens complex. The

complex is a collection of filaments that tethers mitochondria very

closely to the synapse in a regulated fashion, orienting the matrix

cristae perpendicular to the active zone (36). It is likely that the

organization of mitochondria within this specialized synapse enables

the mitochondria to carry out precisely timed ATP release and cal-

cium buffering. In hippocampal neurons, which have considerably

different synaptic organization than that observed in the calyx of

Held, it appears that mitochondria are mostly untethered and that

they sometimes move and sometimes remain stationary. When hip-

pocampal neurons are stimulated by local application of growth fac-tors to points on the axon, mitochondria move preferentially to the

stimulated site, presumably mimicking the in vivo situation where

mitochondria might be targeted rapidly during growth or plasticity

(37).At synaptic sites, mitochondria bind actin, under the control of

phosphatidylinositol-3 kinase (PI3K) (35, 38).

Function of DRP1 inSynaptic Targeting and Localization

Fusion and fission of mitochondria are dynamic processes that occur

within many cell types (39). Whether mitochondria exist as an inter-

connected network or as individual, discrete organelles most likely

depends on the requirements of the individual cell type. The equilib-

rium between fusion of individual mitochondria and fission of mito-

chondria into two or several individual mitochondria is a complex

and highly regulated process involving the replication and segregation

of mitochondrial DNA (40). The proteins that control mitochondrial

fission in mammals include Drp1 (4143) and Fis1 (44). Proteins that

control fusion include OPA1 (for Optic Atrophy Type 1, a dynamin-

related GTPase) (39), and Mitofusin 1 and 2 (45, 46). During apop-

tosis, mitochondria fragment under the control of the mitochondrial

fission proteins (47, 48), and this fragmentation and some of the

features of cell death can be prevented (48, 49) by overexpression of

Drp1K38A, a dominant negative mutant of Drp1 (41).

In neurons, a putative function of mitochondrial fission is pre-

sumed to create more mitochondria during growth, and particularly

to target mitochondria to nascent synapses during development ortimes of synaptic plasticity. In a study of the role of mitochondrial

targeting and fission in the postsynaptic compartment of hippocam-

pal neurons in culture, Li et al. (50) determined that 89% of the

total cellular mitochondria were found within or close to dendritic

protrusions (the site of contact with the presynaptic cell), and that

the time of greatest co-localization of mitochondria with dendritic

spines was during active phases of synaptic development. At these

developmental stages, in resting cells, approximately 10% of den-

dritic spines contained mitochondria. After repetitive depolarization

of the neurons, however, mitochondria changed shape from elon-

gated structures to aggregated clusters and 21% redistributed rapidly

to dendritic spines (at three hours after stimulation), suggesting that

acute alterations in mitochondrial morphology could play a role insynaptic plasticity. When a stimulating electrode was placed on the

cell, mitochondria were found to be more likely to change shape the

closer they were to the site of stimulation, and the morphological

changes of the mitochondria were prevented by inhibition of NMDA

receptors, suggesting that the changes in mitochondrial shape and

location were correlated with synaptic excitation. The changes in

mitochondrial morphology could also be brought on by overex-

pression of Drp1 and inhibited by overexpression of Drp1K38A.

Accordingly, the number of synapses was increased in neurons

overexpressing Drp 1. In contrast, the number of synapses was

decreased in controls overexpressing the dominant negative mutant

of Drp1K38A, indicating that Drp1 was both required and limitingfor the development and plasticity of spines and synapses. Li and

colleagues also studied the effect of activity on mitochondrial fission

and fusion by time-lapse microscopy. They found that a decrease in

neuronal activity in neurons treated with tetrodotoxin (TTX) (which

prevents action potential firing) increased the rate of fusion over fis-

sion, whereas increased activity in the setting of neuronal depolariza-

tion caused an increase in fission over fusion, presumably to make

new mitochondria that would be available for new or increasingly

active synaptic sites.



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Mitochondrial Ion Channels

Specific targeting of mitochondria is thus required for normal

synaptic transmission at high frequencies. The regulated targeting

of mitochondria to sites of high energy demand suggests that the

mechanisms of ATP production and release by mitochondria could

very well be regulated during frequent synaptic events. Mitochondria

are suggested to release ATP via the voltage-dependent ion channel

(VDAC), the most ubiquitous protein in mitochondrial outer mem-

branes. VDAC is the major pathway for the release of metabolites

across the mitochondrial outer membrane, and its regulation is

important for normal cell function as well as for cell death (51, 52).

It is predicted that during synaptic events (such as synaptic plastic-

ity), regulation of the opening of VDAC in the outer mitochondrial

membrane could occur. Another prediction is that there is likely to

be a second messenger that signals the opening of VDAC duringsynaptic events.

The first evidence that mitochondrial ion channel activity could

be regulated during synaptic events came from studies of mito-

chondrial membrane conductance during synaptic transmission in

an intact presynaptic terminal, that of the squid stellate ganglion.

Through the use of a double-barreled patch pipette (53), recordings

were made both at rest and during and after intense synaptic stimu-

lation (11).

In control recordings within the resting squid presynaptic termi-

nal, the most frequent mitochondrial ion channel activity was small,

with a conductance of less than 50 pS, but other conductances were

occasionally seen. In contrast, during frequent electrical stimulation

of the squid presynaptic nerve, there occurred a marked increasein activity and conductance of mitochondrial membrane patches

within the presynaptic terminal (11). With a delay of less than one

second after the onset of nerve stimulation, mitochondrial membrane

conductance increased by as much as sixty-fold, a change that per-

sisted for approximately a minute after the stimulus. The delay and

persistence of the mitochondrial membrane activity after stimulation

implied that the mitochondrial outer membrane channel activity was

not simultaneous with the opening of plasma membrane channels

and suggested that the increase depended on an intracellular second

messenger. Such a messenger could be calcium, which remains ele-

vated in the squid terminal for approximately one minute after stimu-

lation, just as in the crayfish neuromuscular junction and superiorcervical ganglion (9, 10, 21). In keeping with these reports, in a calci-

um-deficient bathing medium, there was no change in mitochondrial

conductance in response to stimulation of the presynaptic terminal,

demonstrating that the evoked mitochondrial membrane chan-

nel activity was dependent on calcium influx (11). Mitochondrial

membrane channel activity was also found to be dependent on an

intact mitochondrial membrane potential. Uncoupling mitochondria

with FCCP (carbonyl cyanidep-trifluoromethoxyphenylhydrazone),

completely eliminated the increase in conductance recorded dur-

ing and after nerve stimulation. The acute changes in mitochondrial

membrane conductance were also correlated with synaptic plasticity,

because FCCP application also eliminated short term potentiation of

the synapse following nerve stimulation.

The possible candidate channels that could be activated on

mitochondrial membranes during high frequency activity of the

synapse include the channels known to be most abundant in the

outer membrane of adult mitochondria in healthy resting neurons

such as VDAC (51). The opening of VDAC is most likely very tightly

regulated. Kinnally and Tedeschi (54) have pointed out that there

are several hundred VDAC channels in a patch that has a diameter

of 0.5 m, assuming a random distribution of channels. If even one

channel were open, the patch resistance of a resting mitochondrial

membrane would be 1.7 giga-ohms (G) for a channel with a

conductance of 650 pS, yet studies have demonstrated the ability

to obtain patch resistances of up to 10 G(11, 54). Regulation of

VDAC may influence important functions of the synapse such as

learning and memory, because knock out mice lacking two of thethree known mammalian isoforms of VDAC display abnormalities

consistent with the absence of long term potentiation, the elec-

trophysiological correlate of learning found in hippocampal slice

recordings (55). Regulation of VDAC opening influences the flux

of ATP and other metabolites across the outer mitochondrial mem-

brane (56) and, therefore, could be the conduit for the synchronous

release of ATP or calcium during high frequency synaptic events.

The opening of VDAC is also modulated by the presence of NADH

on the outside of the outer membrane (5759), suggesting that

the metabolic state of the neuron might determine whether VDAC

remains closed or opens.

In the squid presynaptic terminal, the activation of mitochon-

drial channel activity during synaptic transmission is calcium sensi-tive. Although the recordings were most likely obtained on outer

mitochondrial membranes, the only known calcium-sensitive (as

opposed to calcium conducting) channel is an inner membrane

channel that is activated by elevated calcium concentrations within

the mitochondrial matrix. Thus, Ca2+-dependent responses during

synaptic stimulation could represent opening of an inner membrane

channel whose activity might be linked to the opening of VDAC

in the outer membrane (60). A channel spanning two membranes

could permit the efflux of calcium (as well as ATP and other ions

and metabolites) from the matrix into the cytosol during synaptic

potentiation (9, 10).

BCL-xL Is Expressed inAdult Nervous System

Another important set of proteins expressed in the mitochondrial

outer membrane that could be regulated during synaptic events is

that of the BCL-2 family. The properties of BCL-xL and other BCL-2

family members position them to regulate the processes of synaptic

transmission, synaptic plasticity, and synaptic development. BCL-xL

is highly expressed in the mammalian nervous system both during

development and in adults (6164), and is localized at least par-

tially to mitochondria (65).During synaptic development, levels of

Review


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Volume 6, Issue 4

BCL-xL rise in the brain (66)with a similar time course to that thatgoverns the increase in size of presynaptic vesicle clusters (67, 68).

In adult brain, only a few BCL-2 proteins continue to be expressed

at high levels including the pro-apoptotic protein BID and the anti-

apoptotic protein BCL-xL (66). BCL-2 family proteins both cause

and prevent cell death, but their precise mechanism of action is still

incompletely understood. Properties of these molecules have been

widely studied in the hopes of increasing understanding of the com-

plex set of cellular behaviors that occurs during cell death. Some of

the characteristics of the molecules that have been uncovered have

shed light on their possible function in the synapse. BCL-2 proteins

may either protect the synapse from untimely elimination or con-

tribute to its elimination either during development of redundant

synapses or in pathological states such as ischemia and neurode-

generative diseases. Several of the known properties of BCL-xL may

contribute to mitochondrial function in the synapse.

BCL-xL Regulates Apoptosis

Programmed cell death (or apoptosis) plays an important role in

the development and throughout the life of many organ systems,

including the nervous system (69).In the nervous system, damaged

cells or cells not destined for the adult animal are removed (70).

Failure of the death program can lead to growth and proliferation of

cancer cells, whereas untimely onset of cell death leads to degenera-

tive changes such as found in Alzheimer Disease, and amyotrophic

lateral sclerosis (71). In addition, during some pathological insults

to the brain such as ischemia or trauma, some cells die immediately,

whereas others meet their demise by turning on a programmeddeath pathway(72, 73).

BCL-2 family proteins regulate the permeabilization of mito-

chondrial membranes, release of cytochrome c, and eventual activa-

tion of caspases, enzymes that support the breakdown of cellular

components (7478). Although it is widely held that anti-apoptotic

proteins protect against cell death, and pro-apoptotic molecules kill

cells, it is now also firmly acknowledged that anti-apoptotic proteins

such as BCL-2 and BCL-xL can be transformed into pro-apoptotic

molecules by activation of endogenous proteases (7982). In addi-

tion, some pro-apoptotic molecules serve important pro-survival

functions in neurons and in the synapse (83, 84). In their classical

role, however, anti-apoptotic molecules such as BCL-xL regulateand prevent cell death in several ways, including binding to pro-

apoptotic molecules (85), thereby preventing the effects of the pro-

apoptotic molecules on mitochondrial membrane permeability to

cytochrome c and other pro-death factors (8688); increasing the

conductance of the outer mitochondrial membrane to metabolites

(89); and possibly by directly altering the efficiency of mitochondrial

metabolism (90, 91).

In the synapse, the role of both anti-and pro-apoptotic proteins

is emerging. Evidence is accumulating that mitochondrial ion chan-

nel activity of the BCL-2 family proteins can strengthen or eliminate

a synapse during plasticity or degeneration without causing the death

of the cell soma (9295). Therefore, in addition to their role in con-

trolling cell death, BCL-2 family proteins regulate aspects of synaptic

physiology even when cell death is not occurring (83, 93, 94).

BCL-xL Is an Ion Channel That

Regulates Conductance of the

Mitochondrial Outer Membrane

BCL-2 family proteins conduct ions when reconstituted into artificial

lipid bilayers (86, 9698). The three-dimensional structure of BCL-

xL consists of two central hydrophobic helices surrounded by five

amphipathic helices (99).The structure is similar to that of pore-

forming bacterial toxins. In lipid vesicles or planar lipid bilayers, the

induction of ion channel activity by BCL-xL is related to its known

ability to target to, and insert into, lipid membranes. In these arti-

ficial membranes, the channel is cation selective at neutral pH, anddisplays multiple conductances, with a prominent conductance

of 276 pS, and several smaller conductance levels. Some of the

small conductance channels appear to display typical single chan-

nel behavior, whereas the larger conductances have more complex

behavior, indicating that multiple proteins could influence the activ-

ity of, or constitute, the channel.

A key feature of the ion channel activity of BCL-xL is that it can

induce metabolite exchange across mitochondrial membranes (89,

100). In particular, it performs this function in mitochondria from

cells that have been exposed to apoptotic stimuli, such as growth

factor deprivation. In this pathological setting, BCL-xL appears to

protect cells from death by maintaining VDAC in its open configura-

tion despite the pro-apoptotic effect of an early loss of permeabilityto metabolic substrates.

A surprising dichotomy of the effects of anti-apoptotic molecules

is that they enhance the release of ATP and phosphocreatine from

mitochondria (101), but prevent the release of cytochrome c(87), and

large fluorescent moieties from artificial lipid vesicles (86). How this

works is not completely understood, but one possibility is that bind-

ing of BCL-xL and BCL-2 to pro-apoptotic molecules may alter the

death promoting functions of the pro-apoptotic molecules (85, 88,

102). Therefore, both the channel activity of BCL-xL and its ability

to alter the activities of pro-death molecules through protein-protein

interactions may comprise the anti-apoptotic functions of BCL-xL.

BCL-xL Produces Mitochondrial Ion Channel

Activity within the Presynaptic Terminal

Whether the ion channel activity of BCL-xL might participate in

synaptic plasticity and development apart from its role in protection

from cell death is now being explored. As a preview to experiments in

mammalian neurons, we have studied the effects of BCL-xL on syn-

aptic plasticity in the squid giant presynaptic terminal, a high fidelity,

exctitatory, axo-axonal synapse that is critical for the animals escape

behavior(93). Squid stellate ganglia are immunoreactive by light

microscopy for BCL-xL in the large presynaptic terminal fingers. As is



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typical for mitochondrial staining in large neurons and axons (7, 8),

staining is found throughout the axoplasm, but the density of immu-

noreactivity is greatest close to the plasma membrane, particularly

that apposed to the postsynaptic axon. At higher power, striations

in BCL-xL staining likely correspond to the location of the spine-like

postsynaptic structures that represent contact points between the pre-

synaptic terminal and the postsynaptic axon (103, 104). Observation

at still higher power reveals a punctate cytoplasmic pattern that co-

localizes BCL-xL with a mitochondria-specific dye.

Further evidence for the mitochondrial localization of BCL-xL

in squid was obtained by preparation of a purified mitochondrial

fraction from the stellate ganglion (105). Immunoblot analysis of

this fraction revealed the presence of squid BCL-xL that co-migrated

with recombinant human BCL-xL. Also detected in these fractions

was the mitochondrial outer membrane protein VDAC1.

Full-length recombinant human BCL-xL protein produces char-acteristic channel activity with multiple conductances when applied

by patch pipette to mitochondrial patches within the living presyn-

aptic terminal (93). Unitary openings of the channel correspond to

conductances between 100 pS and 760 pS, and a series of rapid

voltage steps to successive potentials reveals current-voltage relations

that are linear or very slightly outwardly rectifying.

BCL-xL Enhances Synaptic Transmission

Because BCL-xL induces mitochondrial ion channel activity and

induces a change in mitochondrial membrane conductance within

the squid presynaptic terminal, BCL-xL might influence the release

of calcium or metabolites into the cytosol that could in turnregulate synaptic responses. In support of this hypothesis, injec-

tion of recombinant BCL-xL protein into the presynaptic terminal

enhances the rate of rise of postsynaptic responses, resulting in

an earlier latency for evoked action potentials in the postsynaptic

cell as compared to the latency recorded in control synapses (93).

Interestingly, injected BCL-xL protein produced potentiation of

synaptic transmitter release in both healthy synapses and in those in

which transmission had run down (i.e., decreased) to the point that

the postsynaptic potential no longer triggered postsynaptic action

potentials. Under these conditions, injection of BCL-xL protein into

the terminal enhanced the amplitude of the postsynaptic potential,

restored suprathreshold responses, and effectively brought the syn-apse back to life.

The time course of enhanced postsynaptic responses after

injection of BCL-xL is longer than the changes produced by opening

of mitochondrial ion channels during short-term synaptic plasticity.

Enhancement of transmission lasts as long as forty-five minutes in

some cells, with an average peak response of twenty minutes after

injection, suggesting that perhaps endogenous BCL-xL participates

in longer lasting changes in synaptic function. The initial record-

ings of mitochondrial ion channels during synaptic transmission in

response to tetanic stimulation (11) suggested that the activation

of a calcium-dependent conductance of the outer mitochondrial

membrane regulates short term synaptic changes. Although that

conductance is clearly activated during normal physiological behav-

iors of the synapse, its identity is not clear. Activity of BCL-xL could

contribute to such changes in permeability of the outer membrane.

BCL-xL Enhances Recovery

from Synaptic Depression

In addition to its ability to stimulate neurotransmitter release in an

infrequently active synapse (0.03 Hz), injection into the synapse of

recombinant BCL-xL protein also enhanced transmitter release from

presynaptic terminals stimulated at 2 Hz, a higher frequency that

normally produces a significant degree of synaptic depression (21).

This finding suggested that, just as calcium buffering by mitochon-

dria alters the recovery from depression in the MNTB (20), BCL-xL

may counteract the effects of synaptic depression on the readilyreleasable pool (93).

Experiments to test the role of BCL-xL in management of ves-

icle pools demonstrated that recovery of vesicle pools after synaptic

depression is, indeed, regulated by BCL-xL. Different stimulus para-

digms were employed in order to study the effects of BCL-xL on the

kinetics of different vesicle pools. In the first paradigm, stimulation

was carried out at 2 Hz before and after the tetanus (Figure 3A). As

reported previously(21),during this basal, high rate of stimulation,

synaptic depression occurs as the readily releasable pool is depleted.

After the depletion, a more reluctantly releasable set of vesicles is

usedthat of the recycling pool. During the recovery phase follow-

ing the administration of a tetanus given against the background of

continuous 2 Hz stimulation, vesicles do not re-populate all pools,but re-populate only the recycling pool. The time course of the

recovery of the recycling pool is rapid, and is not affected by previ-

ous injection of BCL-xL.

In the second paradigm, recovery from tetanic stimulation

was measured during very infrequent basal stimulation (Figure

3B). Under these conditions, full recovery of all pools occurs, as

evidenced by the ability of the synapse to release as fully after the

tetanus as it does during the control period at the beginning of the

experiment. Nevertheless, the time course of recovery of synaptic

responses following the tetanus is slower than it is at 2 Hz, sug-

gesting that, when all the pools are re-populated, the most readily

releasablethe first pool to be released at the onset of stimula-tionre-populates quite slowly(17, 18).The amount of recovery to

this pool measured within thirty seconds after the end of the admin-

istered tetanus, however, was significantly enhanced by BCL-xL

injection when compared to recovery measured in controls. Thus,

although synaptic depression during a tetanus is unaffected by BCL-

xL, a slow component of the time course of recovery of the total

vesicle pool is sensitive to the actions of BCL-xL, and the pool that

is affected may be the most readily releasable pool. BCL-xL therefore

appears to enhance the ability of a subset of neurotransmitter-con-

taining vesicles to become available for release.

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10/15 2August 2006

Volume 6, Issue 4

BCL-xL Effects on Synaptic Transmission

Do Not Require Calcium Buffering

Calcium release from mitochondria is known to participate in syn-

aptic plasticity; specifically, re-release of calcium from mitochondria

following the initial buffering of calcium entering the presynaptic

terminal is responsible for the long tail of residual calcium that

causes posttetanic potentiation at many synapses (Figure 1) (9, 10,

11). At the squid giant synapse in physiological solutions, how-

ever, the calcium that enters the terminal during repeated action

potentials produces strong synaptic depression, thought to result

from depletion of synaptic vesicles (21). Thus, it is unlikely that the

enhancement of transmission by BCL-xL, particularly at higher stim-

ulus frequencies (e.g. 2 Hz), results from further elevation of calcium

levels alone in the presynaptic terminal.

To examine the potential role of mitochondrial calcium flux in

the enhancement of synaptic transmission during BCL-xL injection,

neurons were treated with ruthenium red, an agent that is taken up

by neurons and inhibits uptake of calcium into mitochondria (106,

107). Ruthenium red blocks short term synaptic potentiation that

is dependent upon mitochondrial calcium handling in the synapse.

Even under these experimental conditions, however, BCL-xL poten-

tiates transmitter release, suggesting that the actions of BCL-xL in

squid presynaptic terminal do not require calcium uptake by mito-

chondria, and further suggesting that BCL-xL might regulate the

local production or release of ATP.


Post

Pre

Recycling Pool

Reserve Pool


A

B

Readily releasable poolRecycling pool

Reserve pool

Time (min)

PSP

(mV/ms)

Control

50 Hz 50 Hz

BCL-xL injection

BCL-xL

Figure 3. BCL-xL enhances recovery of vesicles to the readily releasable pool. A. During 2 Hz stimulation of the squid synapse, the readily releasable pool

remains depleted, and vesicles recycle and re-release from the recycling pool. B. After BCL-xL protein injection into the presynaptic terminal, the postsynaptic

potential is enhanced, but after a tetanus, there is no effect on recovery of the recycling or reserve pools. C. Between stimuli at 0.033 Hz, full recovery of all

pools occurs. D. Injection of BCL-xL protein into the presynaptic terminal speeds recovery of the readily releasable pool of neurotransmitter. See text for details.


Recycling pool

Reserve pool

Time (min)

PSP

(mV/ms)Control

50 HzBCL-xL injection

BCL-xL

Post

Pre

Recycling Pool

Reserve Pool


C

D


11/15218

BCL-xL Controls Mitochondrial Bioenergetics

Mitochondria require substrates such as the end products of glycoly-

sis in order to carry out oxidation. Oxidation of substrates hyperpo-

larizes the mitochondrial membrane potential for the purpose of ATP

production. In growing or proliferating cells, growth factors induce

cells to increase nutrient uptake from the environment in order to

supply the proper amount of substrate for mitochondrial metabolism

(91, 108). Nutrients provide energy sources and building blocks for

cell growth (91). In the setting of growth factor withdrawal, signals

within the cell are activated that can lead to a decrease in the abil-

ity of cells to use glycolytic or oxidative substrates. The decline in

substrate use eventually causes mitochondrial membrane depolariza-

tion. The delicate balance between pro- and anti-apoptotic BCL-2

family proteins appears necessary for the regulation of mitochondrial

metabolism at times of deprivation and controls the onset of theeventual release of pro-apoptogenic factors such as cytochrome c into

the cytosol (76). Although overexpression of anti-apoptotic BCL-2 pro-

teins such as BCL-xL protects cells from death, in cells that express

BCL-xL at normal physiological levels, growth factor withdrawal and

metabolic decline can still cause pro-apoptotic proteins to override

the protective effects of BCL-xL (91). Whether BCL-2 family proteins

participate directly in changes in mitochondrial metabolism in healthy

cells is being explored.

Limitation of nutrient stores or oxygen causes the decline in

ATP/ADP ratio in the cell cytoplasm. Evidence suggests that, in this

setting, BCL-xL acts downstream of metabolic changes in the cell

to increase the release of ATP into the cytososl (91). When cells

deprived of growth factors were made to overexpress BCL-xL veryearly in apoptosis, their ability to condense the mitochondrial matrix

in response to ADP could be restored, suggesting that, within twelve

hours of growth factor deprivation in the absence of BCL-xL, the

cause of the change in cellular metabolism is the reversible inability of

mitochondria to translocate ADP and ATP across the outer membrane

(101). BCL-xL can reverse the pathological situation by activating the

opening of VDAC (89). If, despite the protective actions of BCL-xL,

the apoptotic program progresses, the eventual release of cytochrome

c will occur, and indeed may mark the time of irreversibility of the

apoptotic event.

Effects of BCL-xL on Synaptic Transmission

Mimicked by Synaptic Perfusion of ATP

If BCL-xL regulates the flux of metabolites across the outer mitochon-

drial membrane (89, 100), then this property may enhance neuro-

transmission in the physiological setting. The evidence to support this

hypothesis came from studies of the effect of ATP injection into the

synapse on the degree of synaptic responses (23, 93).Direct microin-

jection of ATP into the synapse produced a similar degree and time

course of enhancement of synaptic transmission as did the effects of

BCL-xL injection (93), and, in fact, occluded the effects of injection of

BCL-xL, suggesting that the two agents acted via the same mechanism.

Pro-apoptotic Proteolytic Cleavage Fragment of

BCL-xL Causes Synaptic Decline

Growth factor or oxygen withdrawal causes a decline in ATP/ADP

ratio in the cell (101). Therefore, it may follow that processes that

use a lot of energy such as synaptic vesicle recycling and membrane

pumps that maintain ionic homeostasis within the cell are at risk.

Mitochondria from growth-factor deprived cells have lost their abil-

ity to condense their matrix in response to ADP, and this sign of

dysfunction is accompanied by a loss of ability to make ATP during

respiration (101).

After the changes in mitochondrial respiration occur, if nutrient

or substrate deprivation continues, then apoptotic events at the cell

soma may become irreversible. If this occurs in a neuronal synapse,

that synapse could be marked for elimination. At this time, a set of

changes occurs in the mitochondrial outer membrane that negativelyaffects synaptic function (75, 105, 109, 110). Under pro-apoptotic

conditions, BCL-2 family proteins activate large channel activity that

participates in the release of cytochrome c, either in the absence

of any change to the properties of the inner membrane (112) or, as

may occur during ischemia, accompanying induction of permeability

transition of the inner mitochondrial membrane (75).

In the squid synapse, the effects of hypoxia serve as a model to

study the role of BCL-xL in neuronal injury(94, 105). The presyn-

aptic terminal is very sensitive to hypoxia, which attenuates synaptic

transmission over 1030 minutes (94). Patch clamp recordings of

mitochondrial membrane channel activity during hypoxia revealed

large conductance activity not found frequently in controls. The

channel activity was larger than that induced by pipette-mediatedapplication of BCL-xL protein.

Injurious stimuli such as hypoxia promote the N-terminal pro-

teolytic cleavage of BCL-xL to form the killer protein N-BCL-xL,

which induces cell death and cytochrome c release (79, 80, 81).The

large conductance channel activity recorded in the outer mitochon-

drial membranes of hypoxic synaptic terminals therefore could be

a result of activity of proteolytically altered BCL-xL that has formed

a new kind of channel activity in the outer mitochondrial mem-

branes. In support of this, when recombinant N BCL-xL protein

was added to the patch pipette during mitochondrial recordings

within the synapse or to recordings of isolated mammalian brain

mitochondria (111) large conductance channels were induced inmitochondrial outer membranes (105, 111). In addition, the appear-

ance of the hypoxia-induced channel in squid could be prevented

by pre-treatment of the synapse with zVAD-fmk, a pan-caspase/cal-

pain inhibitor that prevents the cleavage of BCL-xL. Immunoblots

confirmed loss of the BCL-xL protein during hypoxia, and although

antibodies against the proteolytic cleavage fragment N BCL-xL

did not function in squid, in mammalian brain, neurons that had

undergone ischemic injury manifested a high level ofN BCL-xL in

mitochondria (111). Appearance of the channel associated with N

BCL-xL during hypoxia most likely arose from specific proteolysis of

BCL-xL and not from general injury, because levels of VDAC were

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12/15 2August 2006

Volume 6, Issue 4

preserved in both zVAD-treated and untreated hypoxic synapses,

whereas only in the hypoxic synapses treated with zVAD were levels

of BCL-xL preserved (94).

N BCL-xL produces loss of membrane potential and cyto-

chrome c release from mammalian mitochondria (80, 113). When

N BCL-xL protein was injected into the squid presynaptic termi-

nal, it caused a marked decrease in synaptic responses, the opposite

effect of that observed with full-length BCL-xL, even though both

variants of recombinant protein produce channel activity when

added to the pipette during recordings of mitochondria inside

the synapse. The time course of rundown of synaptic responses

matched that of hypoxia, suggesting a correlation between the two

types of synaptic decline (114). In addition, the data suggested that

large mitochondrial channel activity such as that recorded in the

setting of hypoxia or with recombinant N 76 BCL-xL could cause

the synaptic decline, whereas the smaller conductance changesproduced by full length BCL-xL produce synaptic potentiation (84,

114). A further understanding of how the different channel activities

produce differential effects on mitochondrial physiology and how

those effects, in turn, alter synaptic responses is needed.

VDAC participates in large conductancemitochondrial membrane activity

VDAC is a relatively non-selective channel that is believed to be the

major conductance pathway for metabolites such as ATP, ADP, and

creatine phosphate across the mitochondrial outer membrane (51,

89, 100). Although BCL-xL causes channel activity in artificial lipid

membranes, whether it does so in mitochondrial membranes, orwhether it produces all its effects though its biophysical interactions

with VDAC is still partly in question. To address this issue more

fully, we have taken advantage of the evidence that NADH reduces

the conductance of VDAC in mitochondrial membranes (57, 59) but

has no effect on the conductance ofN BCL-xL in artificial lipid

vesicles (105). Therefore, if BCL-xL produces its effects solely by

interacting with VDAC, we would be able to inhibit those effects by

application of NADH to mitochondrial membranes and to the syn-

apse itself. Indeed, the activity of recombinant N BCL-xL is attenu-

ated by application of NADH to patches of mitochondria inside

the synapse, and both the channel activity produced by hypoxia on

mitochondrial membranes and the decline in synaptic responsesproduced by hypoxia were inhibited by application of NADH to the

patches or injection of NADH into the presynaptic terminal during

synaptic transmission (94, 111). The findings suggest that, during

hypoxic-ischemic injury, the activity ofN BCL-xL is produced by

its interaction with VDAC, further supporting a metabolic role for

the channel activity in cell death in injured neurons.

Conclusions and Future Directions

We have painted a picture of BCL-xL as an important regulator of

events inside the synapse. It actions position BCL-xL to play an

important role in protecting synapses from a decline in function

in the setting of injurious stimuli. Not only may BCL-xL serve as a

protector, however, it can also become biochemically altered rapidly

inside the synapse, and thereby hasten synaptic decline. The models

advanced thus far suggest that the two opposite actions of BCL-xL

could help balance synaptic function between under-and overactiv-

ity, to protect against both synaptic degeneration and excitotoxic

death. Furthermore, a protein so integrally related to mitochondrial

metabolism inside the synapse could serve as sensor of synaptic

activity, to provide for acute and long term changes in the metabolic

properties of the synapse necessary for the changes in synaptic

efficacy that underlie memory and learning. Amounts of BCL-xL

increase during periods of synaptogenesis in mammalian brain (66),

thus, in addition to its actions on increasing the availability of ATP

acutely for synaptic transmission, BCL-xL may play an important

role in axonogenesis and synaptogenesis, for example, by alteringthe local production of ATP at the synapse during the formation of

new vesicle pools, or in targeting and docking mitochondria to syn-

aptic sites during the process of neuronal maturation (Figure 4).

BCL-xL is expressed in neurons that are rapidly increasing

in size and complexity. The events that occur during neuronal

development require not only protein synthesis, but also an ever

increasing supply of ATP for energy dependent processes of a neu-


BCL-xL

BCL-xL

BCL-xL

Dendrites

AxonStage 3 Axonal differentiation

Stage 1 Lamellipodia form

Stage 2 Neurons developshort processes

Stage 4 Synaptophysinrestricted to axon

After Stage 4 Synaptophysinstaining becomes punctate

Neurotrophin receptor

Neurotrophin

Undifferentiated neuron

X

Figure 4. Stages of neuronal growth possibly associated with BCL-xL

expression. In the absence of BCL-xL, some neuronal processes and syn-

apses may fail to form or function normally.


13/15220

ron of increased size and complexity (Figure 4). If, during neuronal

development, BCL-xL increases the efficiency of production of ATP,

then it could strongly influence the ability of a neuron to develop to

the point of being able to perform the critical functions of rapid and

intense release of neurotransmitter that are characteristic of synapses

in the adult nervous system. Without crucial changes in mitochon-

drial morphology, metabolism and targeting, synapses may not form,

mature, or display plasticity, because of the absence of local, care-

fully regulated availability of metabolites. doi:10.1124/mi.6.4.7

AcknowledgmentsThe author thanks J.M. Hardwick for comments and discussion,

L.K. Kaczmarek for comments, discussion, and assistance with fig-

ure preparation, and J. Eisen for help with manuscript preparation.

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