Age-related hearing loss due to Mitochondrial Apoptosis

Introduction to Mitochondrial age-related disease

Mitochondria play an extremely important part in age-related disease. These diseases are intimately connected to ROS. Superoxide (ROS) is the toxic by-product of the mitochondria high energy electron transport chain and oxidative phosphorylation. ROS increases with age due to pertubations in mitochondrial function.
 

Disease due to mitochondrial perturbations and increased ROS can be divided into three categories of mitochondrial disease:

1. Decrease in production of ATP; example: age-associated cardiomyopathy.

2. ROS signaling that causes apoptosis, (programmed death of cell); example: Parkinsons disease, age-related hearing loss (AHL).

3. ROS signaling that can increase mTOR or decrease NO (Nitric oxide); example: Hypertension

Age-related Hearing loss

This section takes a very close look at paper, "Age-related hearing loss in C57BL/6J mice is mediated by Bak-dependent mitochondrial apoptosis", by Someya, Peter Rabinovitch, 2009. It is very important paper for 3 reasons:

1. The elucidation of etiology of AHL, an extremely common disease.

2. As a prototype for other similar diseases.

3. Introduction of extremely valuable new class of drugs to improve mitochondrial function in aging: Mitochondrial-targeted antioxidants. Two drugs in this class, (Mito-Q, alpha Lipoic acid) are available as supplements.

Introduction

"Age-related hearing loss (AHL), also known as prebycusis, is characterized by an age-dependent decline of auditory function associated with loss of sensory hair cells, spiral ganglion (SG) neurons and stria vascularis cells in the cochlea of inner ear. Hair cells and SG neurons do not regenerate in mammals, and loss of these long-lived cochlear cells leads to permanent hearing impairment. AHL affects more than 40% of people greater than 65 years of age in the United States."

 

"Most inbred mouse strains display some degree of AHL, and the age of onset varies. The C57BL/6J mouse strain which is widely used for aging research; displays the classic pattern of AHL by 12 to months of age. The onset of AHL in this strain begins in the high-frequency region and spreads toward the low frequencies with age; these functional deficits are accompanies by the loss of hair cells and neurons" in the cochlea with age. 

 

The mitochondrial theory of aging postulates that reactive oxygen species (ROS) generated inside the mitochondria causes age related damage. Mitochondrial damage and dysfunction are thought to play a central role in AHL.
 

Apoptosis (programmed cell death) plays a key role in the age-related decline in multiple organs included the cochlea. Mitochondria play a key role in apoptosis. One way in which mitochondria cause apoptosis is through induction of proapoptotic proteins, Bak and Bax. These proteins promote permeability of outer mitochondrial membrane, leading to caspase activation and cell death. 

 

Interestingly, caloric restriction which lowers mTOR activity, slow the progression of both aging and reduces levels of apoptosis in the cochlea and slow the progression of AHL in C57BL mice.

 

This study was about the role of mitochondria and ROS in AHL.

Results

1. Bak Deficiency Reduces Apoptotic 

Cochlear Cell Death and Delays the Onset of AHL.

Bak and Bax are both proteins which promote apoptosis and programmed cell death. C57BL/6J (CBL mouse) is a mouse strain widely used for aging research. They typically display the classic pattern of AHL by 12 to 15 months of age. To investigate contribution of Bak they conducted hearing tests on 5 and 15 month old CBL mice, one group WT mice and the other genetically modified Bak -/-, which would not produce Bak. Auditory brainstem response (ABR) was used to monitor progression of AHL. 

 

The Bak-/- mice and WT were same at 5 months.

 

At 15 months the Bak-/- mice were significantly better than WT old mice in all frequencies. Compared to young mice, the old Bak-/- were surprising same as young mice in middle and high; but did have some loss at low frequencies, although much less hearing loss than old WT mice.
 

The same tests were done with Bax-/- mice; but there was no difference between mice lacking Bax and wild type.
 

These results showed that genetic absence of Bak; but not Bax prevented AHL. 

 

To confirm results they counted hair cells numbers and SG neuron density. The hair cells detect sound and relay their electrical response to SG neurons which transmit signal to auditory cortex. 

 

Bak prevented loss of hairs cells and SG neurons.

 

2. Oxidative Stress Triggers Bak Expression in Primary Cochlear Cell and Bak deficiency Reduces Apoptotic Cell Death.

"To uncover the pathogenic mechanisms that trigger Bak-mediated apoptosis in the cochlea, we conducted in vitro studies to investigate whether primary cochlear cells lacking Bak are resistant to oxidative stress-induced cell death using paraquat (PQ), which damages neurons and cochlear cells by generating ROS, such as superoxide anions." [ASG note: Paraquat is used to induce Parkinsons disease in animal models of PD suggesting a similar mechanism.]

 

Results showed Cochlear cells lacking Bak are resistant to PQ-induced apoptotic cell death. This was true for both cohlear cells and neurons and was confirmed by percent positive TUNEL cells which is marker for apoptosis cell death.

 

3. Mitochondrially Targeted Catalase Suppresses Bak Expresion in the Cochlea, Reduces ROS-Induced DNA damage, and Delays the Onset of AHL.

"To test the hypothesis that mitochondria-derived ROS plays a causal role in AHL, we conducted hearing tests in C57BL/6J transgenic mice to overexpress catalase localized to mitochondria (MCAT)

[See Cardiomyopathy section for discussion of MCAT]

"At 13 months of age the mean ABR hearing thresholds of MCAT transgenic mice were significantly lower than those of age-matched WT mice at all frequencies tested. In agreement with the ABR tests results, middle-aged MCAT mice displayed only minor age-related loss of SG neurons and hair cells. Cell counting also demonstrated that catalase overexpression reduced outer and inner hair cell loss. Theses results indicated the mitochondria-derived ROS plays a causal role in AHL."

 

"To investigate whether damage to nucleic acids isblocked by catalase overexpression, we measured oxidative damage to DNA and RNA in the cochlea of WT and MCAT mice at 13 months of age.
 

(a) We found that oxidative DNA damage, but not RNA damage, was reduced in the cochlea of MCAT mice.

(b). We also found that cochlear oxidative damage, but not RNA damage, increased during aging in WT mice.

(c). Cochlear oxidative DNA damage also increased during aging in BAK -/-, suggesting that oxidative stress acts upstream of Bak. 

(d). Next, we measured relative mRNA levels of the proapoptotic Bcl-2 family members Bak, Bax, Bid,Bim, and the anti-apoptotic Bcl-2 the anti-apoptotic Bcl-2 in the cochlea of WT mice at 5 and 15 months of age. 

Bak: "The mean relative expression of Bak increased with age."

Bcl-2: "The mean relative mRNA expression of Bcl-2 decreased with age.

No significant age-related changes were observed in the relative mRNA levels of Bax, Bid, and Bim with age."

(e) "We found that the mean relative mRNA of Bak (but not Bax) in the cochlea of MCAT transgenic mice was significantly lower than that of WT mice. 

 

Together, theses results provide compelling evidence that enhancing mitochondrial antioxidant defenses reduces oxidative DNA damage, Bak expression and cochlear cell death, and also delays the onset of AHL in mice."
 

4. Supplementation with Mitochondrial Antioxidants Suppresses Bak Expression in the Cochlea and Delays the Onset of AHL. 

They tested the effect of 17 antioxidant compounds on AHL in WT CBL mice. Antioxidants that do not selectively target mitochondria did not delay onset of AHL.

 

"Oral supplementation with the mitochondrial antioxidate alpha-lipoic acid and MitoQ suppress Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL."

 

Mice were fed the supplement from 4 months until 15 months of age. MitoQ had best results followed by alpha lipoic acid (LA).

 

Results showed MitoQ and LA prevented hearing loss, survival of SG neurons and reduced expression of Bak. MitoQ was best in all tests.

Discussion

"Previous studies have shown that the apoptotic protein Bak is up-regulated in the human aging brain, as well as in the hippocampus of patients with Alzheimer disease. Therefore, our finding that mitochondrial oxidative stress triggers Bak-mediated apoptosis in the cochlea may have general implications for aging and cell death in the brain and other sensory organs in mammals. The age-related increase in oxidative stress is likely to account for the increased Bak gene expression in the aged cochlea. 

 

[ASG comment: speculation of role of p53]:

"It is well established that the tumor suppressor and nuclear transcription factor p53 is activated by cell stress and DNA damage and that activation of p53 can trigger apoptosis in a wide range of cell types including neurons. In response to cell stress, p53 rapidly translocates to mitochondria and directly binds to Bak and induces its oligomerization, leading to cytochrome c release. Therefore, we speculate that, in response to increased oxidative DNA damage in the aged cochlea, p53 translocates to mitochondria and activates Bak, leading to Bak-mediated apoptosis and eventually to cochlear cell death."

  

[ASG comment; differentiation between Bak and Bax]:

"It is though that the proapoptotic Bcl-2 family members Bak and Bax function in concert as a gateway to the mitochondrial-mediated cell death pathway, and that they are redundant in function in programmed apoptosis that is required for proper development. However, recent studies suggest that Bak acts upstream of Bax in promoting mitochondrial apoptosis, and that Bak plays the central role in apoptosis in the brain, and also in mitochondrial fragmentation. In the current study, Bak defiency prevented AHL wheras Bax deficiency did not. Levels of Bak mRNA were reduced in the cochleae of MCAT transgenic mice and LA and CQ treated mice, whereas levels of Bax mRNA were not altered. We also note that aging resulted in increased expression of Bak, but not Bax, in the WT cochlea. Thus our data provide evidence that the mitochondrial apoptotic function of Bak is not redundant with that of Bax in the adult cochlea."

 

"In summary, our finding strongly support the hypothesis that mitochondrial ROS plays a role in limiting the mammalian health span and provded evidence that the induction of a Bak-mediated apoptosis program may be a central mechanism linking ROS aging and AHL."

Abstract

Age-related hearing loss (AHL), also know as presbycusis, is a universal feature of mammalian aging and is the most common sensory disorder in the elderly population. The molecular mechanisms underlying AHL are unknown, and currently there is no treatment for the disorder. Here we report that C57BL mice with a deletion of the mitochondrial pro-apoptotic gene Bak exhibited reduced age-related apoptotic cell death of spiral gangion neurons and hair cells in the cochlea, and prevention of AHL. Oxidative stress induces Bak expression in primary cochlear cells, and Bak deficiency prevents apoptotic cell death. Furthermore, a mitochondrial targeted catalase transgene suppresses Bak expression in the cochlea, reduce cochlear cell death,and prevents AHL. 

 

Oral supplementation with the mitochondrial antioxidants alpha-lipoic acid and MITO-Q also suppress Bak expression in the cochlea, reduces cochlear cell death and prevents AHL. 

 

Thus, induction of a Bak-dependent mitochondral apoptosis program in response to oxidative stress is a key mechanism of AHL in C57BL mice. 

 

Significance paper:

A remarkable discussion and proof of role of mitochondrial generated ROS in very common AHL.

A demonstration of mechanism of ROS induced DNA damage inducing Bak which (speculate) increases p53 which causes apoptotic cell death of hair cells and SG neurons.

 

The demonstration that supplements that target ROS in mitochondria, specifically, MitoQ and alpha lipoic acid, can prevent disease process. 

 

A validation of importance of ROS theory of aging.

 

Also note, ROS can signal increased production of mTOR, so lowering ROS can also lower mTOR