Mitochondrial dysfunction in hearing loss

Nathan Fischel-Ghodsiana, Richard D. Kopkeb,*, Xianxi Gec

aDepartment of Pediatrics, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USAbHough Ear Institute, Oklahoma City, OK, USA

cDepartment of Defense Spatial Orientation Center, Department of Otolaryngology, Naval Medical Center, San Diego, CA, USA

Received 9 December 2003; accepted 12 July 2004

Abstract

Mitochondrial pathology plays an important role in both inherited and acquired hearing loss. Inherited mitochondrial DNA

mutations have been implicated in both syndromic and non-syndromic hearing loss, as well as in predisposition to

aminoglycoside ototoxicity. Acquired mitochondrial dysfunction in the absence of mitochondrial DNA mutations has also

been proposed as playing an important role in noise-induced and toxin-induced hearing loss. Presbycusis, the hearing loss

associated with aging, may be caused by mitochondrial dysfunction resulting from the accumulation of acquired

mitochondrial DNA mutations and other factors. The pathophysiological mechanisms and clinical implications of these

findings are discussed.

q 2004 Published by Elsevier B.V. on behalf of Mitochondria Research Society.

Keywords: Hearing loss; Presbycusis, ototoxicity; Noise induced hearing loss; Mitochondrial dysfunction

1. Introduction

Mitochondrial pathology plays an important role in

both inherited and acquired hearing loss. Inherited

mitochondrial DNA mutations have been implicated

in both syndromic and non-syndromic sensorineural

hearing loss (Jacobs, 1997; Fischel-Ghodsian, 1998,

1999). Acquired mitochondrial dysfunction in the

absence of mitochondrial DNA mutations has also

been proposed as playing an important role in noise-

induced hearing loss (NIHL), and toxin-induced

1567-7249/$ - see front matter q 2004 Published by Elsevier B.V. on be

doi:10.1016/j.mito.2004.07.040

* Corresponding author. Tel.: C1 405 943 1716; fax: C1 405 947

6226.

E-mail address: rickkopke@swbell.net (R.D. Kopke).

hearing loss (Kopke et al., 1999, 2000, 2002). The

mechanisms of mitochondrial dysfunction are being

studied, and in NIHL seem to involve glutamate

toxicity, glutathione depletion, increased reactive

oxygen species (ROS) and oxidative stress. Presby-

cusis, the hearing loss associated with aging, may lead

to mitochondrial dysfunction through both the

accumulation of mitochondrial DNA mutation, and

other factors (Seidman et al., 2000). The purpose of

this paper is to review some of the latest information

linking inherited and acquired mitochondrial patho-

logies to a variety of etiologies of sensorineural

deafness. The clinical relevance of these findings will

be discussed, in particular with respect to preventive

and therapeutic interventions.

Mitochondrion 4 (2004) 675–694

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N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694676

2. Brief review of cochlear function

The function of the cochlea is to convert an

acoustic waveform into an electrochemical stimulus

transmitted to the central nervous system. The inner

hair cell (IHC) function is primarily as a sensory

receptor. Movement of the hair cell (HC) stereocilia

opens transduction ion channels allowing entry of KC

and CaCC, generating a transduction current. The

transduction current then activates voltage sensitive

calcium channels along the IHC lateral wall and base

as well as CaCC-activated KC channels. The end

result is release of the neurotransmitter glutamate at

the HC base (Raphael and Altschuler, 2003).

Glutamate binds to the afferent nerve terminals that

surround the base of the HC, resulting in an action

potential being propagated down the afferent nerve

fibers. The role of the outer hair cell (OHC) is to

provide the amplification of acoustic signal by

elongation and contraction of the OHC that results

from acoustically driven depolarization and hyper-

polarization of the cell augmenting the displacement

of the basilar membrane. The major functions of

mitochondria in HCs are to provide adenosine

triphosphate (ATP) formation by oxidative phos-

phorylation, modulation of intracellular calcium

concentration, and the regulation of apoptotic cell

death. The number of mitochondria is related to the

metabolic activity of the cell. Mitochondrial density

Table 1

Mitochondrial mutations and hearing impairment

Hearing impairment Mutations identified Inherited

Syndromic

Syst. neuromuscular Deletions, A3243G,. Rare

DiabetesCdeafness A3243G-tRNAleu(UUR) Yes

Deletion/rearrangement Yes

A8296G-tRNAlys Yes

T14709C in the tRNAglu Yes

PPKCdeafness A7445G-non-coding Yes

Non-syndromica A1555G-12S rRNA Yes

A7445G-non-coding Yes

Cins7472-tRNAser(UCN) Yes

T7511C-tRNAser(UCN) Yes

Ototoxic A1555G-12S rRNA Yes

C1494T-12SrRNA Yes

DT961Cn-12SrRNA Yes

Presbycusis Random Not know

a A pathogenic role has been proposed, but not been established, for the

appears higher in the basal turn of the cochlea and in the

infra-nuclear region of the HC, suggesting a higher

metabolic activity in the basal turn, and a greater energy

requirement in the region close to nerve endings.

(Kopke et al., 2003) The stria vascularis contributes the

metabolic energy necessary to maintain a positive

endocochlear potential. Enzymes, specifically Na/K

ATPase, utilize ATP generated by the mitochondria

from cells of the stria and spiral ligament, to pump NaC

and KC ions against their concentration gradients.

3. Inherited mitochondrial hearing loss

Inherited hearing loss can be due to both hetero-

plasmic and homoplasmic mitochondrial mutations.

These data have recently been reviewed (Fischel-

Ghodsian, 2002, 2003) and are summarized with the

inclusion of the most recent data in Table 1.

3.1. Mitochondrial mutations and syndromic

hearing loss

Systemic neuromuscular syndromes due to hetero-

plasmic mitochondrial DNA mutations, such as Kearns–

Sayre syndrome, mitochondrial encephalomyopathy,

lactic acidosis, and stroke-like episodes (MELAS), and

mitochondrial encephalomyopathy with ragged red

fibers, frequently have hearing loss as one of their

Acquired Homoplasmy Heteroplasmy

Usually No Yes

Possible No Yes

Not observed No Yes

Not known No Yes

Not observed No Yes

Not observed Yes Minimal

Not observed Yes Minimal

Not observed Yes Minimal

Not observed Nearly Yes

Not observed Nearly Yes

Not observed Yes Minimal

Not observed Yes No

Possible Yes Multiplasmy

n Yes No Yes

T7510C and G7444A sequence changes (see text).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 677

clinical signs (Schon et al., 1997; Chomyn, 1998; Sue

et al., 1998).

In 1992 several families with diabetes mellitus and

sensorineural hearing loss were described, and

surprisingly were found to have inherited the hetero-

plasmic A3243G mutation in the gene for tRNAleu

(UUR) the very same mutation associated with the

systemic MELAS syndrome (Reardon et al., 1992;

van den Ouweland et al., 1992). In none of these cases

were other neurological symptoms present. One

family had instead of the 3243 mutation a hetero-

plasmic large deletion/insertion event (Ballinger

et al., 1992), and more recently the heteroplasmic

point mutations T14709C in the tRNAglu gene and

A8296G in the tRNAlys gene were also found to be

associated with maternally inherited diabetes and

deafness (Vialettes et al., 1997; Kameoka et al.,

1998). This association between diabetes mellitus,

hearing loss, and mitochondrial mutations has been

confirmed in population studies of diabetic patients

(Oka et al., 1993; Alcolado et al., 1994; Kadowaki

et al., 1994; Katagiri et al., 1994; Sepehrnia et al.,

1995; Newkirk et al., 1997; Rigoli et al., 1997;

Guillausseau et al., 2001). Kadowaki et al. (1994), for

example, found the 3243 mutation in 2–6% of diabetic

patients in Japan, and in three out of five patients with

diabetes and deafness. Twenty-seven of their 44

patients with diabetes and the 3243 mutation had

hearing loss. The hearing loss is sensorineural and

usually develops only after the onset of diabetes. In

addition, diabetes mellitus, diabetes insipidus, optic

atrophy and deafness have been well described as the

Wolfram syndrome, a usually autosomal recessive

condition (Cremers et al., 1977), but which may also

occur as a consequence of mitochondrial deletions

(Rotig et al., 1993; Bu and Rotter, 1993).

Some of the mutations described below in the non-

syndromic section have been found associated

occasionally with other symptoms. Most prominently,

the A7445G in the tRNAser gene mutation was initially

described as a non-syndromic deafness mutation, but

was subsequently found to be also associated with the

skin condition, palmoplantar keratoderma (PPK) in at

least some of the cases (Reid et al., 1994a; Fischel-

Ghodsian et al., 1995; Sevior et al., 1998). Another

mutation in the tRNAser gene has also been found in

one family to be associated with ataxia and myoclonus

(Tiranti et al., 1995). The most common non-syndromic

mutation, the A1555G mutation in the 12S rRNA gene

has been described in one family with Parkinson, in

another with a constellation of spinal and pigmentary

disturbances, and in one case of a woman with a

restrictive cardiomyopathy (Shoffner, 1999; Nye et al.,

2000; Santorelli et al., 1999). It remains, however, not

unlikely that these associations occurred by chance and

are not causally related. Similarly, the homoplasmic

mitochondrial sequence change A5568G in the

tRNAtrp gene was proposed as pathogenic for a family

with hearing loss and hypopigmentation, but the

evidence for pathogenicity is speculative at this time

(Hutchin et al., 2001).

3.2. Mitochondrial mutations and non-syndromic

hearing loss

The first mutation associated with non-syndromic

deafness was identified in an Arab–Israeli pedigree,

when the striking pattern of transmission only through

mothers was noted (Jaber et al., 1992; Prezant et al.,

1993). Most of the deaf family members had onset of

severe to profound sensorineural hearing loss during

infancy, but a minority of family members had onset

during childhood or even adulthood (Braverman et al.,

1996). The homoplasmic A1555G mutation in

the mitochondrial 12S ribosomal RNA gene was

identified as the pathogenic mutation (Prezant et al.,

1993). Initially, in all additional pedigrees and

individual patients with the same A1555G mutation,

the hearing loss occurred only after aminoglycoside

exposure (Hutchin et al., 1993; Fischel-Ghodsian et al.,

1993, 1997; Matthijs et al., 1996; Pandya et al., 1997;

Gardner et al., 1997). Predisposing mutations for

aminoglycoside ototoxicity will be discussed more

generally in Section 4.3. below. However, subsequently

a significant number of pedigrees were described in

Spain, with family members who went deaf with and

without aminoglycosides (El-Schahawi et al., 1997;

Estivill et al., 1998). The age of onset of hearing loss in

the Spanish families was rarely congenital, which is

different from the Arab–Israeli pedigree. In particular,

the study by Estivill et al. is remarkable for two reasons,

both of which indicate a higher than previously

expected frequency of this mutation. First, it describes

19 families with the A1555G mutation out of a total of

70 families with sensorineural hearing loss collected.

Even if the selection of families led to a bias toward

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694678

families with multiple affected individuals, and even

when only the individuals without aminoglycoside

exposure are considered, this represents an unexpect-

edly high frequency of familial sensorineural hearing

loss due to the A1555G mutation. Second, the fact that

the mutation was identified on different haplotypes, a

finding supported by the study of Torroni et al. (1999)

indicates that this mutation exists in other populations

as well, and may not be rare. Similarly, in Mongolia the

mutation appears to be common, although it is not clear

to what extent this is a selection bias due to

aminoglycoside exposure (Pandya et al., 2001). How-

ever, unpublished results from screening of hearing-

impaired populations in other parts of the world seem so

far to indicate a very low frequency of the A1555G

mutation. In the United States, newborn screening of

1173 anonymized random dried blood spot cards

revealed a single case of the A1555G mutation (Tang

et al., 2002). Despite that, families with the A1555G and

non-syndromic non-ototoxic hearing loss have been

described in different places of the world (Usami et al.,

1997; Casano et al., 1998; Bu et al., 2000; Feldmann

et al., 2001; Mingroni-Netto et al., 2001).

Another close to homoplasmic inherited mutation

leading to hearing loss is the A7445G mutation.

It was first described in a family from Scotland,

and confirmed and established in two unrelated

pedigrees from New Zealand and Japan (Reid et al.,

1994a; Fischel-Ghodsian et al., 1995; Sevior et al.,

1998). In the New Zealand and Japanese pedigrees, a

mild form of the skin condition palmoplantar

keratoderma also segregates in the maternal line

(Sevior et al., 1998). Interestingly, the penetrance of

this mutation for hearing loss in the Scottish pedigree

is quite low, while in the New Zealand and Japanese

pedigrees is very high. Thus, in similarity to the Arab–

Israeli pedigree, the mitochondrial mutation by itself

does not appear to be sufficient to cause hearing loss,

but requires additional genetic or environmental

factors, which seem to be rare in the Scottish pedigree

and common in the New Zealand and Japanese

pedigrees. The difference in penetrance in this

situation appears to be due to a difference in

mitochondrial haplotype. In the New Zealand pedi-

gree, complete sequencing of the mitochondrial DNA

revealed three additional sequence changes in com-

plex I protein genes, two of which have been also

labeled as secondary Leber’s hereditary optic

neuroretinopathy mutations (Fischel-Ghodsian et al.,

1995). Since these or similar sequence changes are not

present in the Scottish pedigree (Reid et al., 1994b),

mitochondrial haplotype appears to account for the

differences in penetrance in this case.

A third mitochondrial mutation, a cytosine inser-

tion at position 7472 in the tRNAser (UCN) gene, was

identified in one large Dutch family (Verhoeven et al.,

1999). The same mutation had been previously

described in a Sicilian family with hearing loss,

some of the family members having also other

neurological symptoms, such as ataxia and myoclonus

(Tiranti et al., 1995). In the Dutch family, the hearing

loss is sensorineural progressive with onset in early

adulthood. Most of the individuals over 30 years of

age were deaf, indicating that the penetrance in this

family is high. The mutation is heteroplasmic,

although most individuals have over 90% of abnormal

mitochondrial chromosomes in the tissues examined.

More recently, a large African American pedigree

with maternal inheritance and non-syndromic hearing

loss has been identified (Friedman et al., 1999), and

shown to have a close to homoplasmic T7511C

mutation in the tRNAser (UCN) gene (Sue et al.,

1999). The same mutation was subsequently found in

a Japanese and two French families (Ishikawa et al.,

2001; Feldmann et al., 2001). Also, a British family

with a T7510C mutation, and non-syndromic deafness

was described, although the mitochondrial chromo-

some was not fully evaluated (Hutchin et al., 2000).

Lastly, the G7444A substitution has been described in

deaf individuals with and without the A1555G

mutation, but its pathogenicity has not been estab-

lished (Pandya et al., 2001).

3.3. Pathophysiology of hearing loss due

to mitochondrial DNA mutations

Mitochondrial DNA is essential for normal ATP

production in nearly every cell of the body, and thus it

is not unexpected that hearing loss occurs in systemic

neuromuscular disorders due to mitochondrial DNA

mutations. Similarly, in syndromic hearing loss with

heteroplasmic mitochondrial DNA mutations, it is

possible to speculate that the distribution of abnormal

mitochondrial DNA molecules is responsible for the

phenotype. For the homoplasmic mitochondrial DNA

mutations associated with non-syndromic hearing

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 679

loss, at a first glance, it is possible to speculate that

mitochondrial mutations interfere with energy pro-

duction, that the cochlea is highly dependent on

sufficient energy production, and that insufficient

energy production leads to degeneration of cochlear

cells. However, the cochlea is not the most energy-

dependent organ in the body, and in the systemic

neuromuscular disorders, the extraocular muscles

appear to be the most energy-sensitive cells, and

hearing loss is certainly not the most prominent

clinical sign. Thus, in order to understand the

pathophysiological pathways leading from the mito-

chondrial mutations to hearing loss, two major

biological questions need to be answered: Why does

the same mutation cause severe hearing loss in some

family members but not in others (phenotypic

expression), and why is the ear the only organ affected

(tissue specificity)?

Study of the mitochondrial mutations leading to

hearing loss has led to three possible precipitating

factors modulating phenotypic expression, and it is

likely that a combination of them also play a

significant role in the phenotypic expression of

acquired mitochondrial disorders. The first such factor

involves environmental agents, and aminoglycosides

are the prime example as a triggering event in the case

of the 1555 mutation. It is not unlikely that other, as

yet unrecognized environmental factors, could play

similar, but perhaps less dramatic, roles. Diet and

drugs affecting oxygen radical formation and break-

down come to mind. The second factor involves the

mitochondrial haplotype, and, as noted above, the

7445 mutation provides a dramatic example of that

effect. The third factor involves nuclear genes. The

Arab–Israeli pedigree and some of the Spanish and

Italian pedigrees are good examples of the role of

nuclear genes. For example, the entire Arab–Israeli

family lives in the similar environmental surroundings

of a small Arab village in Israel, and all maternal

relatives share the same mitochondrial haplotype.

Biochemical differences between lymphoblastoid cell

lines of hearing and deaf family members with the

identical mitochondrial chromosomes provide direct

support for the role of nuclear factors (Guan et al.,

1996). An extensive genome wide search has led to

the conclusion that this nuclear effect is unlikely to be

due to the effect of a single nuclear locus but involves

a number of modifier genes (Bykhovskaya et al.,

1998, 2000). The chromosomal location of one of

these modifier genes has been identified, and linkage

disequilibrium has been obtained in families from

varied ethnic backgrounds (Bykhovskaya et al., 2000,

2001).

Additional nuclear-encoded putative modifier

genes have been identified using a candidate gene

approach (Bykhovskaya et al., 2003). Thus, the model

that emerges for explaining penetrance is a threshold

model, where a combination of environmental,

mitochondrial and nuclear factors can push a cell

over a threshold, with dramatic clinical differences on

either side of this threshold.

The second major biological question relates to

tissue specificity: If a homoplasmic mutation affects

oxidative phosphorylation (the only known function

of the human mitochondrial chromosome and an

essential process in every nucleated cell of the human

body), it is unclear how the clinical defect remains

confined to the cochlea, rather than affecting every

tissue. We propose that cochlea-specific isoforms or

splice-variants involved in mitochondrial RNA pro-

cessing or translation interact abnormally with the

mutated rRNA, tRNA, or polycistronic mRNA, and

lead to qualitative or quantitative changes in the

protein products. Different processing of mitochon-

drial RNA and protein, leading to tissue specific

defects or functions have been described. Several

examples of tissue specificity in oxidative phos-

phorylation and of tissue specific secondary functions

of mitochondrial RNAs exist. Tissue-specific subunits

for oxidative phosphorylation have been described

(Arnaudo et al., 1992). Even more relevant is the case

report of a 22-year-old patient who died from

respiratory failure due to a mitochondrial myopathy.

It was shown that the causative mutation in the

mitochondrial tRNALeu (UUR) gene causes a RNA

processing defect in skeletal muscle but not in the

patient’s fibroblasts (Bindoff et al., 1993), raising the

possibility of a skeletal muscle specific mitochondrial

RNA processing gene. Examples of secondary func-

tion include the mitochondrial large ribosomal RNA

gene in Drosophila melanogaster, which in addition

to being involved in mitochondrial translation, can

also be processed in a few cells for export into the

cytoplasm where it induces pole cell formation in

embryos, a key event in the determination of the germ

line (Kobayashi et al., 1993). Similarly, in the mouse

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694680

the ND1 protein can be processed in two different

ways, part of it being presented on the cell membrane

with a minor histocompatibility protein (Wang et al.,

1991).

It can be hoped that the identification of the nuclear

modifier genes through genetic positional cloning or

candidate gene testing will shed light on the

pathophysiological pathways leading from the mito-

chondrial mutation to hearing impairment, and

provide targets for prevention and therapy. In

addition, the recent identification of the first mouse

model of a naturally occurring pathogenic mitochon-

drial DNA mutation provides a ready experimental

model to dissect the complex genetic factors and

interactions (Johnson et al., 2001). In that model, a

mitochondrial DNA mutation in the tRNA-Arg gene

worsens hearing impairment when combined with the

nuclear-encoded Ahl gene locus on mouse chromo-

some 10, which has been described as a major gene

for age-related hearing loss in mice (Johnson et al.,

2000, 2001). It is possible that the pathways to hearing

loss are similar in mice and humans, and thus the

human homologues of all nuclear genes and/or

pathways identified in mice will become candidates

for testing in humans.

4. Acquired mitochondrial hearing loss

4.1. Noise-induced hearing loss

4.1.1. Mechanisms for mitochondrial injury

secondary to noise

4.1.1.1. Glutamate excitotoxicity. In a manner

analogous to CNS excitotoxicity, acoustic over-

exposure may initiate mitochondrial damage as the

result of glutamate excitotoxicity (Vercesi et al.,

1997; Castilho et al., 1998, 1999). Glutamate

excitotoxicity is triggered primarily by massive

Ca2C influx arising from over stimulation of the N-

methyl-D-aspartate (NMDA) subtype of glutamate

receptors. Mitochondrial damage results from cal-

cium overload in the mitochondria (Pereira and

Oliveira, 2000). Calcium overload disrupts mito-

chondrial cristae and internal membranes (Chandra-

sekaran et al., 2002). Mitochondria are primary

targets for excitotoxicity evidenced by confocal

imaging of intracellular Ca2C and mitochondrial

membrane potential (Schinder et al., 1996). Gluta-

mate excitotoxicity in neurons involves essentially

two components. The first component marked by

acute neuronal swelling depends on the uptake of

extracellular NaC and ClK by the cell that causes

plasma membrane depolarization and Ca2C channel

opening. This triggers the second component, which

is marked by delayed neuronal degeneration. The

massive influx of extracellular Ca2C, together with

any Ca2C release triggered from intracellular stores,

increases cytosolic-free Ca2C and initiates a cascade-

like effect leading to cell death. Excitotoxicity,

induced by an excessive release of glutamate by

the IHC, causes afferent nerve ending swelling with a

disruption of the postsynaptic structures. After

repetitive noise stimulation, excitotoxicity may

develop metabolic events triggered by the entry of

Ca2C, which leads to neuronal death in the spiral

ganglion (Puel, 1995; Pujol et al., 1995).

4.1.1.2. Oxidative stress. Oxidative stress associated

with glutamate excitotoxicity, mitochondrial respir-

ation and other events that increase ROS, also

causes mitochondrial damage. In early stages of

injury, oxidative stress may affect the mitochondria

and cause auditory functional impairment prior to

HC death. Early elevation of cochlear ROS was

observed following noise exposure (Ohlemiller et

al., 1999). Dilated mitochondria were observed in

the afferent nerve endings after auditory over

stimulation (Omata and Schatzle, 1984). Initially,

ROS production could reduce mitochondrial respir-

ation making mitochondria vulnerable targets of

ROS (Lenaz et al., 1998; Poderoso et al., 2000).

The increasing ROS production is expected to

deplete cellular antioxidant defenses, leading to a

general enhancement of oxidative stress and

radical-mediated injury throughout the cell (Ciani

et al., 1996).

4.1.1.3. Glutathione depletion. Glutathione (GSH) in

mitochondrion is an important antioxidant defense. A

small fraction of the total cellular pool of GSH is

sequestered in mitochondria by the action of a carrier

that transports GSH from cytosol to the mitochon-

drial matrix. Depletion of mitochondrial GSH

renders the cell more susceptible to oxidative stress

(Fernandez-Checa et al., 1998). Buthionine

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 681

sulfoximine (BSO) was used to block GSH synthesis.

The GSH-depressed ears showed more vulnerability

to noise (Henderson et al., 1999). GSH was

diminished in HCs post noise exposure (Kopke et

al., 1999)

4.1.2. Indirect evidence for a prominent role

of mitochondrial injury in NIHL

Indirect evidence published in the literature

indicates that the mitochondria play a prominent

role in NIHL. Hyde and Rubel (1995) evaluated the

role of mitochondrial biogenesis in HC survival after

injury by inhibiting mitochondrial protein synthesis

with chloramphenicol. Addition of chloramphenicol

to noise exposure significantly increased HC loss by

80% demonstrating that mitochondrial biogenesis is

involved in cellular responses to injury. Kopke et al.

(2002) studied the use of acetyl-L-carnitine, carba-

mathione and D-methionine as protective agents in a

chinchilla model of NIHL. Acetyl-L-carnitine

(ALCAR) was chosen because of its capacity to

enhance mitochondrial bioenergetics and repair in

the face of oxidative stress. ALCAR serves as a

precursor for acetyl-CoA, a mitochondrial energy

substrate, and restores a key mitochondrial lipid,

cardiolipin, in oxidatively injured cells, further

restoring mitochondrial integrity. Permanent

threshold shifts and HC loss were significantly

reduced in animals pre-treated with these metabolites

(Figs. 1 and 2). These data suggest that oxidative

stress, glutamate excitotoxicity, impaired mitochon-

drial function, and GSH depletion, play a role in

cochlear injury induced by noise. Similar results

were noted when pre-treated with N-acetylcysteine

(NAC) to bolster cochlear antioxidant defenses

(Kopke et al., 2000).

HC mitochondria are damaged after noise

exposure. Kopke et al. (2003) reported localized

destruction of mitochondrial cristae in cochlear HCs

2 h after noise exposure. Three weeks post-noise

exposure, cristae exhibited shortening, blurring and

dissolution. Mitochondria showed extreme swelling

and vacuolization. The external and internal mem-

branes were destroyed, and numerous mitochondria

were ruptured. Mitochondrial damage was very

limited in the apical turn, apparent in the middle

turn and severe in the basal turn. These authors also

reported that HC mitochondrial injury was reduced

with ALCAR treatment (Kopke et al., 2004).

Compared to saline treated, noise-exposed animals

where most of the mitochondria were damaged in the

area of noise injury to the cochlea, ALCAR-treated

animals demonstrated normal appearing mitochondria

(Fig. 3). The volume density of normal-appearing

mitochondria decreased both in IHC and OHC after

noise exposure in saline-treated animals (P!0.01)

but was preserved with ALCAR treatment (P!0.01)

three weeks post noise exposure (Fig. 4).

Noise injury is associated with release of

cytochrome c and activation of caspases, and

induction of programmed cell death (PCD) leading

to the loss of OHC. Hu et al. (2002) reported

involvement of the apoptotic pathway in the

progression of OHC death in the chinchilla cochlea

following noise exposure. OHC apoptosis developed

asymmetrically toward the apical and basal parts of

the cochleae following the noise exposure. Two days

after the noise exposure, there was still active OHC

pathology with condensed and fragmented nuclei in

the basal part of the cochleae. Caspase-3 activation,

an intracellular marker for apoptosis, showed a

spatial agreement with the apoptotic nuclei (Fig. 5).

Nicotera et al. (2003) further reported that intense

noise exposure causes OHC death primarily through

apoptosis. The authors investigated the apoptotic

signal pathways after noise exposure by examining

the activity of each of three caspases, including

caspase-3, -8, or -9 with carboxyfluorescein-labeled

fluoromethyl ketone (FMK)-peptide inhibitors. The

cochleae were further examined for cytochrome c

release from mitochondria by immunohistology and

for DNA degradation by the TUNEL method. Noise

exposure triggered the activation of caspase-8 and -9

leading to activation of caspase-3. Caspase activation

occurred only in the apoptotic OHCs and not in the

necrotic OHCs (Fig. 6). These results indicate that

multiple signaling pathways leading to caspase-3

activation take place simultaneously in the apoptotic

OHCs. Noise exposure also caused the release of

cytochrome c from mitochondria. In contrast to

activation of caspases, the release of cytochrome c

took place in both apoptotic and necrotic OHCs

(Fig. 7). Moreover, the release of cytochrome c in

a subpopulation of OHCs took place early in the cell

death process, prior to any outward signs of necrosis

or apoptosis.

Fig. 1. (Right) (A) Auditory threshold shifts for saline-treated and ALCAR-treated animals. Mean threshold shifts plotted for treatment groups

(saline-noise and ALCAR-noise treatment), time [1 h (or 0 week), or 1, 2, 3 weeks post noise] by test frequency for 2, 4, 6 and 8 kHz. Noise

exposure was six continuous hours of 105 dB SPL octave band noise centered at 4 kHz. Initial threshold shifts (week 0) were from 65 to 97 dB

for both treatment groups over the test frequencies. There was an overall treatment effect for the ALCAR-noise group compared with the saline-

noise group (P!0.001) for all test frequencies beginning at week one. Error bars are GSEM. Sample (n) size is 12 ears (six animals) for this

and B and C data. (B) Outer hair cell cytocochleogram data. Depicted are meansGSE (SE, shaded area) cytocochleograms for outer hair cells

(OHCs) of noise-exposed animals pre-treated with either ALCAR (solid line) or saline (dotted line). The Y-axis is mean percent missing OHCs.

The lower X-axis represents percent distance from the cochlear apex, and the upper X-axis is the associated frequency range of the cochleae in

kHz. Very little OHC loss occurred in the ALCAR-protected cochleae (less than 10%), whereas there is substantial OHC loss (P!0.001) in the

saline-noise-exposed group (about 60–70% for the 4–10 kHz region). (C) Inner hair cell cytocochleogram data. Illustrated are the mean inner

hair cell (IHC) cytocochleograms with missing IHC percentages on the Y-axis as a function of the measured percent distance from the cochlear

apex (axis are the same as in B). ALCAR treatment (solid line) afforded significant protection (P!0.01) of IHCs (3% or less loss verses 10–30%

in the saline-treated animals). (Left) Postulated cell death pathway from mitochondrial injury: cytochrome c release to activation of caspases

leading to apoptosis of the sensory cells. An injury (noise) generates oxidative stress (ROS and free radicals), which can activate a stress kinase

cell death pathway (c-JNK and c-Jun) triggering BIM and damaging both the inner and outer mitochondrial membranes. Membrane damage

(pores) allows cytochrome c release into the cytoplasm to interact with APAF-1 converting inactive caspase-9 to an activated form. These in

turn activate different downstream effector caspases to degrade membrane lipids, proteins and nuclear DNA resulting in apoptotic cell death.

Bcl-2 and Bclxl act to inhibit pore formation (anti-apoptotic) while BIM (pro-apoptotic) acts to inhibit the mitochondrial membrane stabilizing

action of Bcl-2 and Bclxl. Bax acts directly (pro-apoptotic) to form pores in the mitochondrial membranes. (Kopke et al., 2002, used with

permission).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694682

Fig. 2. Photomicrographs of surface preparations of cochleae from noise-exposed animals. The low (far left) and high power micrographs

(middle) from the 6 kHz cochlear regions show sodium succinate staining of noise-exposed animals treated with saline (controls), acetyl-L-

carnitine (ALCAR), carbamathione or D-methionine (MET). Saline controls exhibit almost total loss of viable (stained) OHCs with scattered

IHC loss. In contrast, the MET- and ALCAR-treated animals show three complete OHCs rows. Carbamathione-treated animals show an

intermediate level of OHC loss. Enhanced high power surface preparations (right panels) depict stained tissue from a similar region of noise-

exposed animals but given only saline (lower) or pre-treated with N-acetylcysteine (NAC) in the top panel. NAC dramatically protected the

IHCs and especially the OHCs compared to the saline noise-exposed animals. (Kopke et al., 2002, used with permission).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 683

4.2. Cisplatin ototoxicity

As with noise, the cancer chemotherapy agent,

cisplatin, may induce oxidative stress. Cisplatin, a

relatively strong ototoxin, will damage HCs (particu-

larly OHCs), the stria vascularis (Tsukasaki et al.,

2000), and auditory neurons (Zheng and Gao, 1996),

making cisplatin-induced deafness the dose-limiting

side effect of this otherwise useful drug (Blakley et al.,

2002). In vitro studies have indicated that exposure of

cochlear neuroepithelium to cisplatin leads to the

production of ROS and depletion of HC GSH

followed by HC death and that these processes can

be prevented by a variety of antioxidant compounds

(Kopke et al., 1997; Feghali et al., 2001). Theories

regarding the genesis of this cisplatin-induced oxi-

dative stress include DNA damage induced by

cisplatin (Saito et al., 1997), interference with the

glutathione antioxidant defense system (Rybak et al.,

1995), or increases in lipid peroxidation (Teranishi

et al., 2001). The consequences of this oxidative stress

can include HC loss and permanent deafness.

Devarajan et al. (2002) examined the mechanisms

of auditory HC death induced by cisplatin in an

immortalized OHC line from mice. They noted

involvement of both cell death receptor and mitochon-

drial-based cell death apoptotic mechanisms. Cisplatin

induced truncation of Bid and upregulation of p53

Fig. 3. Saline-treated noise exposed animals demonstrated that almost all of the mitochondria were damaged in the area of noise injury to the

cochlea. ALCAR-treated animals, in contrast, demonstrated normal appearing mitochondria. (A) Normal mitochondria (no noise exposure), (B)

damaged mitochondria (noise exposure, saline control), (C) normal appearing mitochondria (noise exposure, ALCAR-treated).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694684

coincident with the activation of caspase-8. Further-

more, cisplatin induced mitochondrial membrane

permeability transition and loss of cytochrome c

from the mitochondria into the cytosol along with

activation of Bax. Caspase-8 activation peaked at 6-h

post cisplatin exposure, and this was followed later by

caspase-9 activation. The result of these processes was

dose- and duration-dependent apoptosis of the HCs in

culture. These findings suggest an induction of

apoptosis caused by cisplatin involving mitochondrial

and possibly cell death receptor pathways. Wang et al.

(2003a) have recently made pertinent and interesting

observations about the mechanisms of cisplatin-

induced cochlear injury in vivo. Using a locally

applied thiol protective agent, they were able to show

the prevention of cisplatin-induced mitochondrial

damage, cytochrome c release, DNA fragmentation

and apoptotic or necrotic HC death. Besides preventing

the morphologic and biochemical changes caused by

cisplatin, they were also able to preserve hearing

function.

Fig. 4. Mitochondrial volume density of normal-appearing mito-

chondria decreased both in IHC (A) and OHC (B) after noise

exposure in saline treated animals (P!0.01) and were preserved

with ALCAR treatment compared to noise saline (P!0.01) 3 weeks

post noise exposure.

4.3. Aminoglycoside ototoxicity

Aminoglycoside antibiotics are well known oto-

toxins. Three mitochondrial DNA mutations confer an

increased susceptibility to aminoglycoside antibiotics.

These mutations have recently been reviewed

(Fischel-Ghodsian, 2004), and include the A1555G,

the DT961Cn, and the C1494T mutations in the 12S

rRNA gene. While these known predisposing

mutations account for a significant proportion of

cases, most cases are probably sporadic and related to

receiving a generally toxic dose of the drug. As with

cochlear damage due to noise and cisplatin,

Fig. 5. Depicted are images of double-labeled hair cell nuclei with propidium iodide (PI) and caspase-3 substrate. Two fluorescence images

showing either caspase-3 activity or PI-labeled nuclei were captured simultaneously by confocal microscopy. To facilitate analysis, the image of

PI-labeled nuclei (A) is illustrated separately from that double-labeled with a caspase-3 probe (B). Both apoptotic (arrows) and necrotic

(arrowheads) OHCs coexist in the damaged area. Note that only OHCs with condensed nuclei are stained positive for caspase-3 activity

(arrows), whereas the OHCs with swelling nuclei (arrowheads) lack caspase-3 staining. OHC, outer hair cell. IHC, inner hair cell (Hu et al.,

2002, used with permission).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 685

permanent hearing loss related to the destruction of

initially OHCs, and then IHCs, by aminoglycosides, is

associated with the induction of oxidative stress. A

gentamicin–iron complex is felt to be responsible

for the generation of ROS in cellular systems (Priuska

and Schacht, 1995; Sha and Schacht, 1999a,b) and a

gentamicin-induced increase of ROS in cochlear

explants has been measured (Clerici et al., 1996). In

addition, variation in antioxidant levels including

GSH can modulate the cochlear damage caused by

aminoglycosides (Lautermann et al., 1995). Toxic

levels of ROS can in turn lead to HC death through

apoptosis (Nakagawa et al., 1998; Forge and Fradis,

1985) or necrosis, and mitochondria appear to play an

important role in these processes.

An early indication that mitochondria play a role in

gentamicin-induced HC injury was the report that

inhibition of mitochondrial homeostatic mechanisms

accentuated gentamicin-induced auditory HC death

(Hyde and Rubel, 1995). Gentamicin was shown to

cause cytochrome c release and apoptosis-inducing

factor by inducing mitochondrial membrane per-

meability transition in liver mitochondria (Mather

and Rottenberg, 2001). Cytochrome c is released from

vestibular HC mitochondria after gentamicin

exposure in vivo (Nakagawa and Yamane, 1999).

All of these data are consistent with earlier obser-

vations that gentamicin can decrease inner ear

mitochondrial respiration (Sato et al., 1969, Ann.

Otol. Rhinol) and cause degeneration of mitochondria

in cochlear HCs of guinea pigs (Wersall et al., 1973).

Dehne et al. (2002) reported that lower concen-

trations of gentamicin induced HC death through

a process of apoptosis. Preceding the cell death

Fig. 6. This image shows double labeling for caspase-3 activity (green) and hair cell nuclei (red) with PI of organ of Corti 2 days after noise

exposure. In the left side of the image there is a large lesion containing apoptotic nuclei that are shrunken and irregular in shape with increased

fluorescence for PI and caspase-3 activity. Whereas on the image’s right side nearly all of the OHC nuclei have disappeared, but there is still a

trace of caspase-3 staining in this area. Scale bar 15 mm (Nicotera et al., 2003, used with permission).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694686

there was a consistent nearly universal loss of

mitochondrial membrane potential noted in the HCs.

Cyclosporin A was able to protect the tissue from

gentamicin toxicity implicating involvement of the

mitochondrial permeability pore in the pathologic

process. Dehne et al. suggested that in their in vitro

model, the major apoptotic pathway involved the

ROS-mediated induction of mitochondrial membrane

permeability transition. However, others have

described receptor-mediated processes leading to the

activation of the Jun kinase pathway of apoptosis

(Pirvola et al., 2000). The sum of the data accumu-

lated by these reports confirms the notion that

mitochondrial injury caused by gentamicin-induced

oxidative stress plays a prominent role in aminoglyco-

side ototoxicity.

4.4. Solvents, asphyxiants, and acrylonitrile

A variety of workplace chemicals are potentially

ototoxic if exposures are above certain levels. These

include solvents such as trichloroethylene, xylene,

styrene, hexane, carbon disulfide, toluene, as well as

carbon monoxide, and a variety of heavy metals

(Rybak, 1992). It has been estimated by NIOSH that

some 9.8 million workers are regularly exposed to

such organic solvents (NIOSH, USDHHS, PHS, CDC,

1987). Of concern is the ability of many of these

toxins to potentiate the ototoxicity of noise. Sub-toxic

doses of toxins and noise when experienced together

may cause hearing loss that would not be seen with

either agent alone, or the hearing loss associated with

a noise exposure may be worse than expected if there

is a concomitant exposure even to ‘safe’ levels of

toxin. For example, in animal studies, Fechter et al.

(2000, 2002) have shown that the asphyxiants carbon

monoxide (CO) and hydrogen cyanide (HCN) can

potentiate NIHL. Model-based calculations extrapo-

lated from their data suggested that the potentiation of

noise damage could be seen with current permissible

levels of these two asphyxiants (Fechter et al., 2000,

2002). Additionally, toluene has been reported to

enhance cochlear noise injury in rat (Johnson et al.,

1988, 1990; Lataye and Campo, 1997) and human

(Morata et al., 1993). Another solvent, styrene, has

also been reported to potentiate NIHL (Lataye et al.,

2000; Campo et al., 2001).

As suggested by Fechter (1995) additive effects of

combined noise and toxin damage may stem from

the possibility that the noise and toxin may each

damage a different part of the auditory pathway (i.e.

hexane damages the central auditory pathway while

Fig. 7. The larger panel shows the immunohistological labeling of cytochrome c in normal cochlea without noise exposure. Propidium iodide

(PI) labels OHC nuclei red and the weak green fluorescent ovals are cytochrome c, which is located in mitochondria along the inner plasma

membrane surface. Scale bar 6 mm. (A) Immediately after noise exposure, small points of fluorescence form around an apoptotic nucleus

(arrow) mark release of mitochondrial cytochrome c into the cytosol compared to a lack of fluorescence in adjacent viable OHCs (weak

staining). (B) Punctate fluorescence also appears around a swollen nucleus (arrow), indicating cytochrome c release also from necrotic cells (the

arrowhead indicates a condensed nucleus) [the punctate fluorescence surrounding this nucleus was eliminated to show the fluorescence of the

necrotic cell directly above. The anatomic relationship between the released cytochrome c and swollen nuclei was clearly illustrated by images

that were used to generate image B being reprocessed to form a side-view image (C)]. The released cytochrome c is localized in the HC having

the swollen nucleus and not in the neighboring OHC nuclei (* in B and C). (D) Cytochrome c release also takes place in an OHC having

relatively normal nucleus (arrow). Scale bar 4 mm (Nicotera et al., 2003, used with permission).

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 687

noise damages the cochlea). Noise may enhance the

toxicity of some chemicals by increasing cochlear

blood flow thus enhancing the exposure of the toxin to

the cochlear tissue, or noise damage might render the

cochlea unable to exclude toxins that could have been

excluded from the cochlea without injury, or the

enhanced activity of the cells undergoing acoustic

stimulation might make them more susceptible to a

toxin (Fechter, 1995). In addition, the fact that some

of these toxins potentiate the cochlear damage

associated with noise exposure suggests the possi-

bility that noise and the toxins share common injury

mechanisms. In fact, evidence is accumulating that a

common theme for the toxin noise interaction is

oxidative stress in some cases (Fechter et al., 1997,

2003; Rao et al., 2001). Fechter et al. (1997) reported

that the free radical scavengers PBN or allopurinol

prevented hearing loss caused by carbon monoxide

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694688

suggesting that free radical generation plays an

important role in CO cochlear injury. Additional

work has demonstrated that another free radical

scavenger, POBN, was protective against the com-

bined toxicity of noise and CO (Rao et al., 2001).

These authors also found evidence for the formation

of free radical spin adduct using electron paramag-

netic resonance spectroscopy. Acrylonitrile (ACN), a

common potential manufacturing toxin, potentiated

NIHL in Long–Evans rats and was found to lower

cochlear GSH levels during the time course of the

noise exposure (Fechter et al., 2003).

As stated previously in this review, oxidative

stress, the generation of free radicals and the depletion

of GSH, can all injure mitochondria enhancing further

ROS production, depleting cellular energy, and

eventuating in cell death. However, many of these

toxins may also primarily injure mitochondria. It may

then be possible that toxins potentiate cochlear noise

damage because they both injure this important

organelle. For example, a metabolite of ACN is

hydrogen cyanide HCN. HCN can interfere with

electron flow through the respiratory chain in

mitochondria by inhibiting cytochrome c oxidase

(Fechter et al., 2002, 2003; Jiang et al., 1998). It has

also been postulated that solvents like toluene may

cause brain toxicity by damaging mitochondrial

membranes and interfering in that way with oxidative

phosphorylation, in addition to altering mitochondrial

membrane permeability directly (Garbe and Yukawa,

2001). Thus it can be seen that there is accumulating

evidence that a number of industrial ototoxicants

damage the ear or potentiate NIHL through oxidative

stress mechanisms. More work needs to be done, but it

appears that cochlear mitochondria are a plausible

target of injury by these toxins primarily or seconda-

rily through oxidative stress caused by the toxins.

4.5. Presbycusis

Oxidative damage has been implicated to be a

major factor in the decline in physiologic function that

occurs during the aging process. Decreased activity of

electron transport chain complexes and increased

release of ROS from the mitochondria with age

suggest that alterations in mitochondrial function

occur with age as a consequence of increased

oxidative damage (Van Remmen and Richardson,

2001; Staecker et al., 2001). Alterations in mitochon-

drial turnover with age could also contribute to an

increase in the number of dysfunctional mitochondria.

Mutations and deletions within the mtDNA occur with

increasing frequency in age and presbycusis (Fischel--

Ghodsian et al., 1997; Seidman et al., 2000). When

enough mtDNA damage accrues, the cell becomes

bioenergetically deficient. This mechanism is the

basis of the mitochondrial clock theory of aging, also

known as the membrane hypothesis of aging (Seid-

man et al., 2000).

It is proposed that treatment with antioxidants or

dietary restriction can attenuate age-related hearing

loss. Nutritional compounds have been identified that

enhance mitochondrial function and reverse several

age-related processes. Seidman (2000) provide evi-

dence that long-term treatment with compounds such

as vitamin E, vitamin C, melatonin and lazaroid that

block or scavenge reactive oxygen metabolites

attenuate age-related hearing loss and reduce the

impact of associated deleterious changes at the

molecular level. The antioxidant-treated subjects

had improved auditory sensitivities, and a trend

toward fewer mtDNA deletions. Compounds that

upregulate mitochondrial biogenesis in an aging

model may improve hearing and reduce some of the

effects of aging. Seidman et al. (2000) describe

the effects of two mitochondrial metabolites, alpha-

lipoic acid and ALCAR, on the prevention of age-

related hearing loss. This effect may be related to the

mitochondrial metabolite’s ability to protect from and

repair age-induced cochlear mtDNA damage. Further

study is warranted.

5. Therapeutic implications

(a) Prevention of aminoglycoside ototoxicity. The

ototoxicity associated with the inherited mitochon-

drial mutations is preventable through a combination

of taking family histories and molecular screening. In

the future, the elucidation of the genetic factors

predisposing to aminoglycoside ototoxicity will result

in the development of non-toxic aminoglycoside

analogs or in treatment strategies that prevent

irreversible cochlear damage.

(b) Antioxidant approach. Oxidative damage has

been implicated to be a major factor in the aging,

N. Fischel-Ghodsian et al. / Mitochondrion 4 (2004) 675–694 689

toxin, and NIHL. Using clinically available antiox-

idant compounds such as NAC may reduce hearing

loss.

(c) GSH replenishment. NIHL might be character-

ized as a cochlear-reduced glutathione (GSH)

deficiency state; therefore, strategies to enhance

cochlear GSH levels may reduce NIHL. A GSH

repletion drug such as NAC, methionine, or a-lipoic

acid may be helpful.

(d) Decrease glutamate excitotoxicity. Glutamate

excitotoxicity can be reduced by antagonizing the

action of cochlear-methyl-D-aspartate (NMDA)

receptors using carbamathione or other agents such

as memantine and MK801 (Bienkowski et al., 2000).

(e) Enhance mitochondrial biogenesis. NIHL may

be caused by a defect in mitochondrial bioenergetics

and biogenesis. Therefore, sensorineural hearing loss

may be reduced by acetyl-L carnitine (ALCAR), an

endogenous mitochondrial membrane compound that

helps maintain mitochondrial bioenergetics and bio-

genesis in the face of oxidative stress.

(f) Cell death inhibition. Noise exposure is known

to result in oxidative stress, which contributes to both

the apoptotic and necrotic HC death. Blocking cell

death pathway activation may be a useful strategy to

reduce hearing loss (Pirvola et al., 2000; Hu et al.,

2002; Wang et al., 2003b; Salvi et al., 1998).

6. Summary and conclusions

An increasing body of evidence supports that

mitochondrial dysfunction plays an important role in

both inherited and acquired hearing loss. Mitochon-

drial dysfunction is enhanced by glutamate ototoxi-

city, increasing ROS, oxidative stress and glutathione

depletion. Preventive and therapeutic strategies to

prevent or reduce hearing loss due to mitochondrial

dysfunction are feasible and are currently being

studied in humans.

7. Disclaimer

The views expressed in this article are those of the

authors and do not reflect the official policy or

position of the Department of the Navy, Department

of Defense, or the United States Government.

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