Late-onset corticohippocampal neurodepletion attributable to catastrophic failure of oxidative phosphorylation in MILON mice

Article (PDF Available)inThe Journal of Neuroscience : The Official Journal of the Society for Neuroscience 21(20):8082-90 · November 2001with19 Reads
Source: PubMed
Abstract
We generated mitochondrial late-onset neurodegeneration (MILON) mice with postnatal disruption of oxidative phosphorylation in forebrain neurons. They develop normally and display no overt behavioral disturbances or histological changes during the first 5 months of life. The MILON mice display reduced levels of mitochondrial DNA and mitochondrial RNA from 2 and 4 months of age, respectively, and severely respiratory chain-deficient neurons from 4 months of age. Surprisingly, these respiratory chain-deficient neurons are viable for at least 1 month without showing signs of neurodegeneration or major induction of defenses against oxidative stress. Prolonged neuronal respiratory chain deficiency is thus required for the induction of neurodegeneration. Before developing neurological symptoms, MILON mice show increased vulnerability to excitotoxic stress. We observed a markedly enhanced sensitivity to excitotoxic challenge, manifest as an abundance of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactive cells after kainic acid injection, in 4-month-old MILON mice, showing that respiratory chain-deficient neurons are more vulnerable to stress. At approximately 5-5.5 months of age, MILON mice start to show signs of disease, followed by death shortly thereafter. The debut of overt disease in MILON mice coincides with onset of rapidly progressive neurodegeneration and massive cell death in hippocampus and neocortex. This profound neurodegenerative process is manifested as axonal degeneration, gliosis, and abundant TUNEL-positive nuclei. The MILON mouse model provides a novel and powerful tool for additional studies of the role for respiratory chain deficiency in neurodegeneration and aging.

Figures

Figure
Figure
Figure

Full-text (PDF)

Available from: Mats I Ekstrand, Apr 07, 2014
Late-Onset Corticohippocampal Neurodepletion Attributable to
Catastrophic Failure of Oxidative Phosphorylation in MILON Mice
Lene So¨ rensen,
1
Mats Ekstrand,
1
Jose´ P. Silva,
1
Eva Lindqvist,
2
Baoji Xu,
3
Pierre Rustin,
4
Lars Olson,
2
and
Nils-Go¨ ran Larsson
1
1
Departments of Medical Nutrition and Biosciences, Karolinska Institutet, Novum, Huddinge Hospital, S-141 86
Huddinge, Sweden,
2
Department of Neuroscience, Retzius Laboratory, Karolinska Institutet, S-171 77 Stockholm,
Sweden,
3
Department of Physiology, Howard Hughes Medical Institute, University of California, San Francisco, San
Francisco, California 94143, and
4
Unite de Recherches sur les Handicaps Genetiques de l’Enfant, Institut National de la
Sante´ et de la Recherche Me´ dicale U393, Hoˆ pital des Enfants Malades, F-75015 Paris, France
We generated mitochondrial late-onset neurodegeneration
(MILON) mice with postnatal disruption of oxidative phosphor-
ylation in forebrain neurons. They develop normally and display
no overt behavioral disturbances or histological changes during
the first 5 months of life. The MILON mice display reduced
levels of mitochondrial DNA and mitochondrial RNA from 2 and
4 months of age, respectively, and severely respiratory chain-
deficient neurons from 4 months of age. Surprisingly, these
respiratory chain-deficient neurons are viable for at least 1
month without showing signs of neurodegeneration or major
induction of defenses against oxidative stress. Prolonged neu-
ronal respiratory chain deficiency is thus required for the induc-
tion of neurodegeneration. Before developing neurological
symptoms, MILON mice show increased vulnerability to exci-
totoxic stress. We observed a markedly enhanced sensitivity to
excitotoxic challenge, manifest as an abundance of terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling (TUNEL) reactive cells after kainic acid injection, in
4-month-old MILON mice, showing that respiratory chain-
deficient neurons are more vulnerable to stress. At 5–5.5
months of age, MILON mice start to show signs of disease,
followed by death shortly thereafter. The debut of overt disease
in MILON mice coincides with onset of rapidly progressive
neurodegeneration and massive cell death in hippocampus and
neocortex. This profound neurodegenerative process is mani-
fested as axonal degeneration, gliosis, and abundant TUNEL-
positive nuclei. The MILON mouse model provides a novel and
powerful tool for additional studies of the role for respiratory
chain deficiency in neurodegeneration and aging.
Key words: mitochondria; respiratory chain; reactive oxy-
gen species; ROS; neurodegeneration; oxidative stress;
apoptosis; oxidative phosphorylation; OXPHOS; cre-loxP;
Tfam; mitochondrial transcription factor A; MILON; neocor-
tex; hippocampus
Deficient function of the mitochondrial respiratory chain is in-
creasingly recognized as an important cause of neurodegenera-
tion (Larsson and Clayton, 1995; Larsson and Luft, 1999; Wal-
lace, 1999). The respiratory chain is located in the mitochondrial
inner membrane, and it generates most of the cellular ATP
through the process of oxidative phosphorylation. The respira-
tory chain consists of 100 different polypeptides distributed in
five enzyme complexes, which are encoded by either nuclear or
mitochondrial genes. The mitochondrial DNA (mtDNA) encodes
13 subunits of respiratory chain complexes I, III, IV, and V,
whereas nuclear DNA encodes all of the remaining respiratory
chain proteins and all proteins needed for maintenance and
expression of mtDNA. More than 50 point mutations and hun-
dreds of rearrangements of mtDNA, as well as a variety of
nuclear gene mutations, have been found in patients with mito-
chondrial disease (Larsson and Clayton, 1995; Larsson and Luft,
1999; Wallace, 1999). Mitochondrial dysfunction has also been
implicated in the pathogenesis of age-associated neurodegenera-
tive diseases, such as Parkinson’s, Huntington’s, and Alzheimer’s
diseases, as well as in naturally occurring aging (Wallace, 1992).
Somatic mtDNA mutagenesis has been proposed to contribute to
the aging process by creating a vicious circle, in which such
mutations impair the synthesis of respiratory chain subunits and
thus reduce respiratory chain function, leading to increased pro-
duction of reactive oxygen species (ROS), which, in turn, addi-
tionally damages mtDNA.
We developed a system for tissue-specific inactivation of
mtDNA expression in the mouse by conditional knock-out of the
nuclear mitochondrial transcription factor A gene (Tfam), which
encodes a mitochondrial protein necessary for transcription and
replication of mtDNA (Larsson et al., 1998). Previous reports
from our laboratory have established that animals homozygous
for a loxP-flanked Tfam allele (Tfam
loxP
/Tfam
loxP
), which also
harbor a transgene directing tissue-specific expression of bacte-
riophage P1 recombinase (cre), will develop a profound tissue-
specific mtDNA depletion and respiratory chain deficiency
(Wang et al., 1999; Li et al., 2000; Silva et al., 2000). We demon-
Received May 23, 2001; revised July 12, 2001; accepted July 20, 2001.
N.-G.L. was supported by Swedish Medical Research Council Grants 13X-12197
and 13P-12204 and funds from the Karolinska Institutet, Torsten and Ragnar
So¨derbergs Stiftelse, the Human Frontiers Science Program, and the Swedish Foun-
dation for Strategic Research. L.S. was supported by a stipend from the Swedish
Foundation for Strategic Research. P.R. was supported by the Association Fran-
c¸aises contre les Myopathies (AMF). L.O. was supported by grants from the Swedish
Medical Research Council, United States Public Health Service, the AMF, Hed-
lunds Stiftelse, the Human Frontiers Science Program, and funds from the Karo-
linska Institutet. B.X was supported by the Howard Hughes Medical Institute and
National Institute of Neurological Disorders and Stroke Grant P01-16033.
L.S. and M.E. have contributed equally to this work.
Correspondence should be addressed to Nils-Go¨ran Larsson, Department of
Medical Nutrition, Karolinska Institutet, Novum, Huddinge Hospital, S-141 86
Huddinge, Sweden. E-mail: nils-goran.larsson@mednut.ki.se.
Copyright © 2001 Society for Neuroscience 0270-6474/01/218082-09$15.00/0
The Journal of Neuroscience, October 15, 2001, 21(20):8082–8090
Advertisement:
strated recently that decreased mtDNA expression in vivo is
associated with induction of antioxidant defenses and increased
apoptosis in Tfam knock-out mouse hearts (Wang et al., 2001).
There is a connection between respiratory chain deciency, ROS
production, and apoptosis induction (Geromel et al., 2001), but
the molecular pathways are poorly understood. Correlative data
suggest that these processes are linked by unknown pathways
and are involved in the pathophysiology of various types of
age-associated neurodegeneration (Wallace, 1992). We now
investigated this issue further by postnatal disruption of Tfam
in neurons of hippocampus and neocortex to create mitochon-
drial late-onset neurodegeneration (MILON) mice.
MATERIALS AND METHODS
Matings and genotyping of transgenic animals. Tfam
loxP
/Tfam
loxP
mice of
mixed genetic background (Larsson et al., 1998; Wang et al., 1999) were
mated to /CaMKII-cre mice (Xu et al., 2000). Compound heterozygotes
(/Tfam
loxP
, /CaMKII-cre) were identied and backcrossed to the
Tfam
loxP
/Tfam
loxP
strain to generate MILON mice with the genotype
Tfam
loxP
/Tfam
loxP
, /CaMKII-cre. This mating gave rise to normal litter
sizes (mean litter size, 10.3 pups) with genotypes of the expected Men-
delian distribution (of 542 animals, the distribution was as follows:
Tfam
loxP
/Tfam
loxP
, 25.5%; /Tfam
loxP
, /CaMK II-cre, 26.6%; MILON,
23.2%; and /Tfam
loxP
, 24.7%). The Tfam genotype and the cre recom
-
binase gene were identied at weaning by PCR analyses (Larsson et al.,
1998; Wang et al., 1999). Animal studies were approved by the animal
welfare ethics committee and performed in compliance with Swedish law.
Southern, Northern, and Western blot analyses. Southern, Northern, and
Western blot analyses were performed as described previously (Larsson
et al., 1996). Phosphoimaging (BAS 1500; Fujilm, Toyko, Japan), and
appropriate software (Image Gauge v3.41; Fujilm) were used to mea-
sure relative levels of mtDNA and mitochondrial RNA (mtRNA) in
brain samples of neocortex and cerebellum from Tfam
loxP
/Tfam
loxP
;
/Tfam
loxP
, /CaMKII-cre and MILON animals of ages 1, 2, 4, and 6
months (n 4 6). The mtDNA levels were normalized to the nuclear
18S rRNA gene, whereas the complete smear of mtRNA was normalized
to 18S rRNA transcript levels. Southern blots were also probed with Tfam
cDNA to calculate the relative Tfam gene recombination frequency, as
described previously (Wang et al., 1999). For gene expression studies,
RNA from neocortex was extracted and Northern blots were performed
as described previously (Wang et al., 2001). One-sided, unpaired t tests
were used to assess statistical signicance. Western blot analyses of Tfam
protein levels were performed on total protein extracts from neocortex of
MILON mice and Tfam
loxP
/Tfam
loxP
littermate controls at the age of 2, 3,
4, and 5 months as described previously (Larsson et al., 1996).
In situ hybridization. Probes against the mitochondrial transcripts cy-
tochrome c oxidase subunit I (5-TGGGT CCCCT CCTCC AGCGG
GATCA AAGAA AGTTG TGTTT AGGTT GCGG-3) and nicotin-
eamide adenine dinucleotide (NADH) dehydrogenase subunit 4 (5-
CCATT TGAAG TCCTC GGGCC ATGAT TATAG TACGG CTGTG
GATCC GTTCG-3) were 3-end labeled with
35
S and used to detect
mtRNA in 14
m cryostat sections from fresh frozen brains by in situ
hybridization (Dagerlind et al., 1992). A random oligonucleotide probe
was used as a negative control. Hybridizations were performed at 42°C
for 16 18 hr, and sections were rinsed ve times in 1 SSC, dehydrated,
and exposed to photographic emulsion (Kodak NTB2; Eastman Kodak,
Rochester, NY) for 4 d.
Biochemistry and enzyme histochemistr y. The activities of the respira-
tory chain complexes were measured in fresh frozen samples of neocor-
tical and hippocampal tissue from MILON and Tfam
loxP
/Tfam
loxP
control
mice at 2, 4, and 5 months of age (n 4) as described previously (Rustin
et al., 1994). Glutathione peroxidase (Gpx) and mitochondrial superox-
ide dismutase (Sod2) activities were determined as described previously
(Wang et al., 2001). Enzyme histochemical analyses of succinate dehy-
drogenase (SDH) and cytochrome c oxidase (COX) activities were
performed on 14
m cryostat sections of fresh frozen brains of MILON
and control mice as described previously (Wang et al., 1999).
Histochemistry and immunohistochemistr y. MILON and Tfam
loxP
/
Tfam
loxP
littermate controls were perfused with Ca
2
-free Tyrodes
solution, followed by 4% paraformaldehyde with 0.4% picric acid in 0.16
M phosphate buffer. The brains were dissected out, post-xed overnight,
and equilibrated to 10% sucrose containing 0.1% sodium azide. Primary
antibodies used for indirect immunohistochemistry (Ho¨kfelt et al., 1973;
Zetterstro¨m et al., 1994) included polyclonal antibodies against
neurolament-10 (NF-10) (1:100; Sigma, St. Louis, MO), glial brillary
acidic protein 19 (GFAP-19) (1:500; Sigma), von Willebrand factor
(1:300; Dako, Glostrup, Denmark), cleaved caspase 3 (1:100; Cell Sig-
naling Technology, Beverly, MA), cleaved caspase 7 (1:100; Cell Signal-
ing Technology), and nitrotyrosine (1:50; Cell Signaling Technology).
Cryostat sections (14
m) were incubated with primary antibodies over-
night at 4°C, rinsed, and incubated with appropriate FI TC-labeled sec-
ondary antibodies. Sections were analyzed by uorescence microscopy.
Proliferating cell nuclear antigen (PCNA) expression was studied with a
monoclonal antibody (1:50; Dako). Brain sections were permeabilized
with ice-cold ethanol/acetic acid (1:2) and blocked (M.O.M Immunode-
tection kit; Vector Laboratories, Burlingame, CA) before application of
specic antibodies. Peroxidase activity was detected with 3,3-
diaminobenzidine (DAB substrate kit for peroxidase; Vector Laborato-
ries), and the sections were counterstained with methyl green (Dako).
Controls included omitting the primary antibody.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling and DNA ladder gel electrophoresis. Terminal deoxynucleoti-
dyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)
(Apoptag in situ Apoptosis Detection kit; Intergen, Purchase, NY) was
performed according to the instructions of the manufacturer. DNA from
neocortex and hippocampus was extracted, and the DNA ladder assay
performed as described previously (Wang et al., 2001). We also used a kit
(ApoAlert LM-PCR Ladder Assay; Clontech, Palo Alto, CA) for detec-
tion of DNA ladders in samples containing small fractions of apoptotic
cells following the instructions of the manufacturer.
Kainic acid induction of seizures. Kainic acid was dissolved in Ringers
isotonic saline (pH 6) and administered intraperitoneally at a dose of 20
or 30 mg/kg body weight in MILON and control mice at the age of 4
months. Mice were monitored continuously for 23 hr after injection to
determine the onset and level of seizures. Seizure levels were rated
(Sperk et al., 1985): level 1, immobility; level 2, forelimb and/or tail
extension, rigid posture; level 3, repetitive movements, head bobbing;
level 4, rearing; and level 5, rearing and falling. Mice injected with saline
were included as negative controls. Brains were collected 24 hr after
seizure onset.
RESULTS
Forebrain-specific disruption of the Tfam gene
We generated mice with disruption of Tfam in forebrain neurons
by mating Tfam
loxP
/Tfam
loxP
mice (Wang et al., 1999) to mice
heterozygous for a transgene expressing cre recombinase from the
calcium-dependent calmodulin kinase II promoter, /CaMKII-
cre (Xu et al., 2000). The resulting MILON mice (genotype
Tfam
loxP
/Tfam
loxP
, /CaMKII-cre) displayed a highly tissue-
specic pattern of Tfam knock-out as determined by PCR (Fig.
1a) and Southern blot analyses (data not shown). The knock-out
(Tfam
) allele was present in neocortex but, with the exception of
testis, not in other tissues, including cerebellum (Fig. 1a). The
CaMKII-cre transgenic mouse used in this study has been shown
previously to express cre recombinase from postnatal day 14
(P14), and maximal recombination of loxP-anked alleles was
observed at P29 in neocortex, hippocampus, and some other
regions (Xu et al., 2000). The CaMKII-cre transgene causes re-
combination in 50% of neocortical and hippocampal neurons
harboring a loxP-anked allele (Xu et al., 2000). Consistent with
these observations, we found that the levels of Tfam
alleles were
equal in 1-month-old (relative level, 24 3%) and 5-month-old
(25 2%) presymptomatic MILON mice, demonstrating that
maximal Tfam recombination was obtained by 1 month after
birth. Tfam protein levels were clearly reduced in neocortex of
MILON mice from 2 months of age and throughout life as shown
by Western blot analysis (Fig. 1b). The MILON mice appeared
normal and showed no overt behavioral abnormalities until the
age of 5 6 months, when they started to display signs of deteri-
orating physical condition progressing rapidly until death within
So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons J. Neurosci., October 15, 2001, 21(20):80828090 8083
12 weeks. There was thus a surprisingly long time span of 4 5
months between the completed Tfam recombination and obvious
signs of disease in the MILON mice.
Reduced mtDNA expression in neocortex and
hippocampus of MILON mice
There was an 40% reduction of mtDNA copy number and
mtRNA levels in neocortex at 2 and 4 months of age, respectively,
and onward (Fig. 1c,d). Furthermore, in situ hybridizations
showed a drastic decrease in mtRNA levels in neocortex and
hippocampus, especially in the CA1 and CA3 regions (Franklin
and Paxinos, 1997) at age 4 months (Fig. 2b). The CA2 region of
hippocampus and the polymorphic layer of the dentate gyrus
displayed no evident reduction of mtRNA levels. Analyses of
respiratory chain enzyme activities in neocortical samples dem-
onstrated decreased activities of NADH dehydrogenase (complex
I) and COX (complex IV), which both contain critical mtDNA-
encoded subunits, in 4- and 5-month-old MILON mice (Fig. 2a).
The activity of SDH (complex II), which is exclusively nucleus
encoded, remained unchanged in MILON mice (Fig. 2a). Severe
respiratory chain deciency in individual cells was demonstrated
by enzyme histochemistry to determine COX and SDH activities
on brain tissue sections. Hippocampus, piriform cortex, and
amygdala in 4-month-old MILON mice contained many COX-
decient cells. In hippocampus, the CA3 region was most affected
with massive amounts of COX-decient cells, but also CA1 and
the dentate gyrus contained numerous COX-decient cells (Fig.
2b). A large number of COX-decient cells were also seen
throughout neocortex and were particularly abundant in cingulum
cortex of 4-month-old MILON mice (data not shown).
It should be emphasized that the CaMK II-cre transgene causes
recombination of loxP-anked alleles in neurons but not in the
other cell types of neocortex and hippocampus (Xu et al., 2000).
It is thus likely that the moderate reduction of respiratory chain
function (Fig. 2a) in tissue extracts of neocortex, containing many
different cell types, is explained by the profound COX deciency
present in individual Tfam knock-out neurons (Fig. 2b).
Corticohippocampal nerve cell loss and gliosis in
MILON mice
Next, we investigated the neuropathological consequences of re-
spiratory chain deciency in forebrain neurons. Early-stage (age
55.5 months) and end-stage (age 5.5 6 months) symptomatic
MILON mice displayed a progressive marked nerve cell loss in
neocortex and hippocampus (Fig. 3). There was substantial de-
generation in neocortex and a severe disruption of cortical orga-
nization in end-stage MILON mice as determined by cresyl violet
staining (Fig. 3). Early-stage symptomatic MILON mice dis-
played a substantial nerve cell loss, as well as a cellular inltration
indicative of an inammatory response in the CA1 region of
hippocampus (Fig. 3). This was further supported by the obser-
vation of macrophages, including characteristic gitter cells. Other
hippocampal areas appeared normal in these animals. In end-
stage animals, the pyramidal cell layer of the medial part of CA1
was completely absent, CA2 was intact, and CA3 only slightly
affected. Although the granule cell layer of the dentate gyrus was
present, many of the nuclei appeared condensed. Such pyknotic
nuclei, which could be consistent with apoptosis, were found in all
affected areas of the hippocampal formation.
We used the cell cycle S-phase marker PCNA to detect divid-
ing cells. Numerous PCNA-positive cells were found in hip-
pocampus, especially in early-stage symptomatic MILON ani-
mals, and in neocortex (data not shown). The majority of these
dividing cells in hippocampus were glia or inammatory response
cells, as judged from morphological appearance. Some labeled
cells were found clustered in the subgranular zone of the dentate
gyrus, indicative of increased neurogenesis.
Immunohistochemistry further supported the presence of neu-
rodegeneration and an inammatory response in end-stage
MILON mice (Fig. 4). Neurolament (NF-10) immunohisto-
chemistry showed extensive axonal degeneration, as evidenced by
increased axonal beading and fragmentation in both early- and
end-stage symptomatic MILON mice. Axonal degeneration was
unevenly distributed in neocortex, being more pronounced ante-
Figure 1. Characterization of Tfam re-
combination, Tfam protein expression,
mtDNA copy number, and mtRNA lev-
els in MILON mice and controls. a,
PCR analysis to determine the tissue-
specic recombination pattern in a
2-month-old MILON mouse. The
Tfam knock-out allele (Tfam
)isonly
present in neocortex and testis. b,
Western blot analyses of cortical
protein extracts from MILON (M;
Tfam
loxP
/Tfam
loxP
, /CaMK II-cre) and
control (C; Tfam
loxP
/Tfam
loxP
) animals.
Tfam protein is detected with a poly-
clonal anti-mouse Tfam antibody,
whereas a monoclonal anti-actin anti-
body is used as a loading control. The
Tfam protein levels are notably de-
creased in the MILON mice from 2
months of age. c, Results from phos-
phoimager quantication of Southern
blots to determine mtDNA copy number in neocortex of control (C; black bars), heterozygous knock-out (H; /Tfam
loxP
, /CaMK II-cre; gray bars), and
MILON (M; white bars) mice. All values are normalized to the nuclear 18S rRNA gene and depicted as the percentage of the mean value of age-matched
controls (n 4 6 pairs of MILON and control mice). *p 0.05; **p 0.01. The levels of mtDNA are signicantly decreased in MILON mice from
2 months of age. d, Results from phosphoimager quantication of Northern blots to determine mtRNA levels in neocortex of control (C; black bars),
heterozygous knock-out (H; gray bars), and MILON (M; white bars) mice. All values are normalized to 18S rRNA and depicted as the percentage of the
mean value of age-matched controls (n 4 6 pairs of MILON and control mice). **p 0.01; ***p 0.001. The levels of mtRNA are signicantly
decreased in MILON mice from 4 months of age.
8084 J. Neurosci., October 15, 2001, 21(20):80828090 So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons
riorly and also increasing with the progression of disease. Early-
stage animals showed signs of axonal degeneration in hippocam-
pus, as well as neocortex. In end-stage animals having lost the
medial pyramidal cell layer of CA1, axonal degeneration was no
longer noted in this area of hippocampus, but there was neuro-
lament accumulation in some granular cell somata of the den-
tate gyrus. This was probably the result of a loss of axonal
connections to CA3.
We demonstrated the presence of gliosis by using GFAP anti-
bodies to detect reactive astrocytes (Fig. 4). Neocortex did not
contain any signicant amount of reactive astrocytes, but there
was a large increase of GFAP-immunoreactive cells in corpus
callosum, with many astrocytes having processes directed into the
degenerating neocortex. In all hippocampal regions, we observed
massive gliosis, corresponding fully to the increased cellularity
noted with cresyl violet staining. There was no difference in the
distribution of reactive astrocytes between early symptomatic and
end-stage MILON mice. However, the amount of gliosis was
clearly larger in end-stage MILON mice. Pathological vascular-
ization, as indicated by large abnormal vessels, particularly in
neocortical areas, could also be seen using antibodies against the
von Willebrand factor (data not shown). None of the different
neuropathological changes described above were present in 2-, 3-,
or 4-month-old MILON mice, in heterozygous knock-out ani-
mals, or in controls.
To assess the magnitude of cell death at different time points,
we performed TUNEL. No TUNEL-positive nuclei could be
detected in 2-, 3-, or 4-month-old presymptomatic MILON mice,
further supporting that Tfam knock-out neurons are viable for
several months. In early- and end-stage symptomatic animals (age
56 months), TUNEL-positive nuclei were seen in all areas
suffering from cell loss, as demonstrated by cresyl violet staining
(Fig. 3). Moreover, TUNEL reactivity was often observed before
any cell loss was evident using a cresyl violet stain. TUNEL in
neocortex was limited to the outer layers, corresponding well to
the areas most depleted of mtRNA, as seen with in situ hybrid-
Figure 2. Biochemical and histochemical
assessment of respiratory chain function
and in situ hybridization to detect
mtRNA. a, Enzyme activities of respira-
tory chain complexes I, II, and IV in neo-
cortex from 2-, 4-, and 5-month-old con-
trol (C; black bars) andMILON (M; white
bars) mice. Values are depicted as the
percentage of the mean value of age-
matched controls. **p 0.01. The activity
of complexes I and IV are signicantly
decreased in MILON mice from 4 months
of age. b, Top row, In situ hybridization to
detect the mtDNA-encoded COXI tran-
script (mtRNA) in brain from a 4-month-
old presymptomatic MILON mouse and a
littermate control. Scale bars, 1 mm. NCX,
Neocortex; DG, dentate gyrus. The levels
of mtRNA are decreased in neocortex and
hippocampus in MILON mice. Bottom
row, Enzyme histochemical double stain-
ing for COX and SDH activities. Cells
with C OX and SDH activity appear
brown, whereas cells with decient COX
activity appear blue. Scale bars, 0.2 mm.
So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons J. Neurosci., October 15, 2001, 21(20):80828090 8085
ization. The rst areas of hippocampus to display TUNEL-
positive cells were CA1 and to some extent also CA3. Later,
when medial CA1 was completely lost and CA3 was slightly
affected, most TUNEL-positive cells were seen in the granule
cell layer of the dentate gyrus. Because TUNEL may detect
necrotic as well as apoptotic cell death, we used complemen-
tary methods to detect apoptosis. DNA gel electrophoresis of
cortical and hippocampal samples from symptomatic 5- to
6-month-old MILON mice yielded a smear, and a DNA ladder
was faintly visible. By using a sensitive PCR assay, we detected
DNA ladders in cortical and hippocampal samples of MILON
mice at the age of 5 6 months but not in MILON mice at the
age of 4 months or in control mice (Fig. 5d). Northern Blot
analysis did not show any increase in transcripts encoding the
proapoptotic Bax or anti-apoptotic Bcl-xL proteins in 2-, 4-,
and 5-month-old mutants (Fig. 5a,b). We could not detect
activated caspase 3 or 7 by immunohistochemistry of brain
sections from MILON mice at different ages (data not shown).
Northern blot analyses showed normal levels of transcripts for
glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (Fig. 5a),
suggesting that upregulation of glycolysis does not occur in
MILON mice.
MILON mice display a low induction of
antioxidant defenses
We next evaluated whether respiratory chain deciency in neu-
rons affect ROS production and ROS defense mechanisms.
Northern blot analyses of RNA samples from neocortex of
MILON mice showed moderately increased Gpx transcript levels
at 5 months of age and a tendency toward increased Sod2 tran-
script levels at 2, 4, and 5 months of age (Fig. 5a, b). We also
measured the Sod2 and Gpx enzyme activities at different ages
Figure 3. Characterization of cell loss in neocortex and hippocampus of a 5-month-old control mouse, a 5-month-old early symptomatic MILON mouse,
and a 5.5-month-old end-stage MILON mouse stained with cresyl violet or TUNEL. NCX, Neocortex; DG, dentate gyrus The arrowheads indicate cell
loss in the CA1 and CA3 regions. The boxes are close-ups of indicated areas. Scale bars, 0.5 mm. There is cell loss and cell inltration in the CA1 area
of hippocampus of early symptomatic MILON mice. There is extensive cell loss and degenerative changes in neocortex and in the CA1, CA3, and dentate
gyrus areas of the hippocampal formation of end-stage MILON mice.
8086 J. Neurosci., October 15, 2001, 21(20):80828090 So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons
and found no signicant differences between MILON and control
mice (Fig. 5c).
Western blot analysis and immunohistochemistry to detect
protein nitrosylation, another marker for oxidative stress induced
by the conversion of superoxide and nitric oxide to peroxynitrite,
did not show any difference between 4-to 6-month-old mutant and
control animals (data not shown). The activity of the nucleus-
encoded respiratory chain complex II, an ironsulfur group-
containing enzyme readily impaired by oxidative stress, was un-
changed in the mutants (Fig. 2). These results suggest
unexpectedly low levels of ROS production in the MILON mice.
Excitotoxic stress induces marked neuronal cell death
in MILON mice
Surprisingly, there were no signs of neurodegeneration, as deter-
mined by cresyl violet, TUNEL, GFAP, NF-10, and PCNA
labeling of brain sections (Figs. 3, 4), in 4-month-old MILON
mice with widespread and profound respiratory chain deciency
in forebrain neurons (Fig. 2). We therefore challenged MILON
mice with kainic acid injections to determine whether they would
be more susceptible to stress-induced neuronal death.
We injected 16 mice (MILON, n 8; controls, n 8) with 20
mg/kg kainic acid at the age of 4 months and observed that three
MILON and three control mice developed level 4 or 5 seizures,
whereas the remaining mice developed level 2 seizures. Next, we
injected 13 mice (MILON, n 6; controls, n 7) with 30 mg/kg
kainic acid at the age of 4 months and observed that six MILON
and six control mice developed level 35 seizures, whereas no
seizures were induced in one of the control animals. There was
thus no signicant difference in the probability to develop high-
level (grade 35) seizures after kainic acid injections between
MILON and control mice at the age of 4 months in response to
20 or 30 mg/kg kainic acid.
Next, we investigated the neuropathological consequences of
seizures by harvesting brains 24 hr after kainic acid injection and
performing TUNEL staining. There was no difference in the low
amount of TUNEL-positive cells that could be seen in hippocam-
Figure 4. Characterization of neurodegenerative
changes and gliosis in a MILON and a control
mouse at the age of 5.5 months. Neurolaments were
detected with a polyclonal antibody against NF-10,
and astrocytes were detected with a polyclonal anti-
body against GFAP. Antibody reactivity was visual-
ized with immunouorescence. CC, Corpus callo-
sum; DG, dentate gyrus. Scale bars, 0.25 mm. NF-10
immunouorescence shows extensive axonal bead-
ing in neocortex and accumulation of neurolaments
in granular cell somata of dentate gyrus in MILON
mice. GFAP immunouorescence shows extensive
gliosis in corpus callosum and hippocampus.
So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons J. Neurosci., October 15, 2001, 21(20):80828090 8087
pus of MILON (n 2) and control (n 2) mice with level 2
seizures. MILON mice (n 2) injected with saline displayed no
TUNEL-positive cells in hippocampus. However, MILON mice
(n 5) with grade 35 seizures displayed larger numbers of
TUNEL-positive cells in the CA3 region of hippocampus than
did controls (n 5) (Fig. 6). There was no overlap between the
ve MILON specimens with abundant TUNEL-reactive cells in
the CA3 area of hippocampus and the ve control specimens with
much less TUNEL reactivity, when the amount of TUNEL-
reactive cell proles in hippocampus was rated by two indepen-
dent observers using a semiquantitative scale on coded slides.
DISCUSSION
Neurodegenerative diseases in man typically become manifest
late in life. There are few animal models that replicate the
late-onset and progressive features of this major group of human
CNS diseases. Here, we characterize the MILON mice, who are
apparently healthy well into adulthood, at which time they de-
velop progressive lethal corticohippocampal neurodegeneration.
Before the development of overt neurological symptoms, the
MILON mice are considerably more vulnerable to excitotoxic
challenge. We also characterized some of the mechanisms leading
to nerve cell death in the MILON mice and found that neurons
are able to survive for at least 1 month after shutting off oxidative
phosphorylation and that nerve cell death is preceded by only
minimal activation of defense mechanisms against reactive oxy-
gen species.
Figure 5. Gene expression proles, en-
zyme activities, and DNA ladder gel
analysis in MILON and control mice of
different ages. a, Results from phospho-
imager quantication of Northern blots
to determine mitochondrial Sod2, Gpx,
Bax, Bcl-xL, Gapdh, and mtRNA tran-
script levels in neocortex of control (C;
black bars) and MILON (M; white bars)
mice at the age of 2, 4, and 5 months.
Transcript levels were normalized to
18S rRNA and are presented as the
percentage of the mean value of age-
matched controls. **p 0.01; ***p
0.001. b, Northern blot analysis of tran-
script levels in neocortex from controls
(C) and MILON (M ) mice at different
ages. The same blot was reprobed to
detect different transcripts as indicated
in the panel. c, Sod2 and Gpx enzyme
activities in neocortex from controls (C;
black bars) and MILON (M; white bars)
mice at different ages. Values are pre-
sented as the percentage of the mean
enzyme activity of age-matched con-
trols. d, PCR DNA ladder gel assay of
DNA prepared from neocortex of
MILON (M ) and control ( C) mice at
different ages. DNA fragmentation is
present in neocortex of 5- to 6-month-
old symptomatic MILON mice, as indi-
cated by arrowheads. A staurosporine-
treated human osteosarcoma cell line
143B was used as a positive control.
Figure 6. TUNEL analysis of neuronal cell death 24 hr after injection of
kainic acid in MILON and control mice at the age of 4 months. Scale bars,
0.2 mm. MILON mice consistently display more TUNEL-labeled cells in
the CA3 area compared with controls with similar seizure activity.
8088 J. Neurosci., October 15, 2001, 21(20):80828090 So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons
Human mitochondrial disorders have an overall incidence (1:
10,000) comparable with other common genetic forms of neuro-
degeneration (Chinnery et al., 2000; Darin et al., 2001). Point
mutations or deletions affecting tRNA genes of human mtDNA
will impair mitochondrial translation and result in the same type
of respiratory chain deciency affecting multiple complexes as in
the MILON mice. Affected patients mainly have encephalomyo-
pathic syndromes and display mosaic tissue-specic patterns of
respiratory chain deciency that, at least partly, are determined
by the distribution of mutated mtDNA in different cell types
(Larsson and Clayton, 1995). We reported previously that cell
type-specic disruption of Tfam in mouse heart (Wang et al.,
1999; Li et al., 2000) and pancreatic
-cells (Silva et al., 2000)
faithfully mimics phenotypes found in humans with syndromes
caused by mtDNA deletions (mtDNA). Similar to the MILON
mouse, neuropathological ndings in patients with mitochondrial
disorders include nerve cell loss, gliosis, and white matter changes
(Leigh, 1951; Oldfors et al., 1990; Oldfors et al., 1995). There is
typically a time lag between the occurrence of respiratory chain
deciency and onset of mitochondrial neurodegeneration in hu-
man patients. In some cases, this can be attributed to hetero-
plasmy, i.e., a mixture of normal and mutated mtDNA, with
increasing levels of mutated mtDNA with time in affected organs
(Larsson et al., 1990). However, this time lag is also observed in
mitochondrial neurodegeneration syndromes attributable to nu-
clear mutations, such as Friedreichs ataxia (Rotig et al., 1997), in
which temporal differences in gene dosage is not an issue. Similar
to humans, prolonged respiratory chain deciency is also re-
quired for the induction of neurodegeneration in MILON mice
and therefore probably reects an inherent property of neurons
to withstand long periods of respiratory chain deciency. Inter-
estingly, MILON mice exhibited marked neuronal cell death in
response to exogenous stress; similarly, patients with Leigh syn-
drome and other mitochondrial encephalomyopathies may ex-
hibit profound clinical deterioration in response to moderate
stress, e.g., viral infections.
Aging is associated with increased levels of mtDNA, attrib-
utable to somatic mutagenesis, and some studies have shown a
decline of respiratory chain function with increasing age (Wal-
lace, 1992). There are always multiple forms of mtDNA in old
subjects with high levels in postmitotic tissues, such as brain and
skeletal muscle. An uneven distribution of mtDNA with clonal
expansions within single cells has been reported in several studies
(Oldfors et al., 1993; Brierley et al., 1998; Cottrell et al., 2000). A
recent enzyme histochemical study of brains from old subjects
demonstrated an age-related increase of respiratory chain-
decient pyramidal neurons in hippocampus (Cottrell et al.,
2001), further supporting the concept of uneven distribution of
age-associated mtDNA mutations in postmitotic cells. It is likely
that presumptive functional impairment and neuropathological
changes caused by mtDNA will be determined by the fraction of
respiratory chain-decient neurons. It is also possible that af-
fected neurons may impair the function of many respiratory-
competent neurons through transneuronal degeneration mecha-
nisms. The generation of chimeras between MILON and control
mice should enable us to establish threshold fractions of respira-
tory chain-decient cortical neurons needed to induce severe
neurodegeneration and thus shed light on the role of the increas-
ing numbers of COX-decient neurons during aging.
We reported previously that disruption of Tfam in mouse
cardiomyocytes causes dilated cardiomyopathy with heart con-
duction blocks (Wang et al., 1999; Li et al., 2000). Interestingly,
these severely respiratory chain-decient hearts display signs of
apoptosis, as manifested by moderate increase of TUNEL-
reactive cells, expression of activated caspase 3 and 7, increased
levels of transcripts for Bax and Bcl-xL, and DNA fragmentation
on gels (Wang et al., 2001). We observed much more abundant
TUNEL-reactive cells in the MILON mice than in the mitochon-
drial cardiomyopathy mice, but, surprisingly, activated caspase 3
or 7 was not detected by immunohistochemical assays, the levels
of transcripts for Bax and Bcl-xL were not changed, and DNA
fragmentation was only observed by using a sensitive PCR assay
in symptomatic MILON mice. These ndings suggest that the
pathways leading to cell death may be different in respiratory
chain-decient cardiomyocytes and neurons. It is known that
differences in intracellular ATP levels are of importance for the
execution of apoptosis (Leist et al., 1997). Consistent with this
hypothesis, we found massive upregulation of transcripts encod-
ing the glycolytic enzyme Gapdh in respiratory chain-decient
cardiomyocytes (Wang et al., 2001) but not in MILON mice
neurons. Future studies of MILON mice, including pharmaco-
logical treatments as well as breeding to other transgenic mouse
strains, should help elucidate molecular pathways leading to neu-
ronal cell death. The MILON mice should thus help unravel the
molecular mechanisms leading to death of nerve cells with de-
fective mitochondrial function and model CNS aspects of mito-
chondrial diseases.
REFERENCES
Brierley EJ, Johnson MA, Lightowlers RN, James OF, Turnbull DM
(1998) Role of mitochondrial DNA mutations in human aging: impli-
cations for the central nervous system and muscle. Ann Neurol
43:217223.
Chinnery PF, Johnson MA, Wardell TM, Singh-Kler R, Hayes C, Brown
DT, Taylor RW, Bindoff LA, Turnbull DM (2000) The epidemiology
of pathogenic mitochondrial DNA mutations. Ann Neurol 48:188 193.
Cottrell DA, Ince PG, Blakely EL, Johnson MA, Chinnery PF, Hanna M,
Turnbull DM (2000) Neuropathological and histochemical changes in
a multiple mitochondrial DNA deletion disorder. J Neuropathol Exp
Neurol 59:621 627.
Cottrell DA, Blakely EL, Johnson MA, Ince PG, Borthwick GM, Turn-
bull DM (2001) Cytochrome c oxidase decient cells accumulate in
the hippocampus and choroid plexus with age. Neurobiol Aging
22:265272.
Dagerlind A, Friberg K, Bean AJ, Ho¨kfelt T (1992) Sensitive mRNA
detection using unxed tissue: combined radioactive and non-
radioactive in situ hybridization histochemistry. Histochemistry
98:39 49.
Darin N, Oldfors A, Moslemi AR, Holme E, Tulinius M (2001) The
incidence of mitochondrial encephalomyopathies in childhood: clinical
features and morphological, biochemical, and DNA abnormalities. Ann
Neurol 49: 377383.
Franklin KBJ, Paxinos G (1997) The mouse brain. San Diego:
Academic.
Geromel V, Kadhom N, Cebalos-Picot I, Ouari O, Polidori A, Munnich
A, Ro¨tig A, Rustin P (2001) Superoxide-induced massive apoptosis in
cultured skin broblasts harboring the neurogenic ataxia retinitis pig-
mentosa (NARP) mutation in the ATPase-6 gene of the mitochondrial
DNA. Hum Mol Genet 10:12211228.
Ho¨kfelt T, Fuxe K, Goldstein M, Joh TH (1973) Immunohistochemical
localization of three catecholamine synthesizing enzymes: aspects on
methodology. Histochemie 33:231254.
Larsson NG, Clayton DA (1995) Molecular genetic aspects of human
mitochondrial disorders. Annu Rev Genet 29:151178.
Larsson NG, L uft R (1999) Revolution in mitochondrial medicine.
FEBS Lett 455:199 202.
Larsson NG, Holme E, Kristiansson B, Oldfors A, Tulinius M (1990)
Progressive increase of the mutated mitochondrial DNA fraction in
Kearns-Sayre syndrome. Pediatr Res 28:131136.
Larsson NG, Garman JD, Oldfors A, Barsh GS, Clayton DA (1996) A
single mouse gene encodes the mitochondrial transcription factor A
and a testis-specic nuclear HMG-box protein. Nat Genet 13:296302.
Larsson NG, Wang J, Wilhelmsson H, Oldfors A, Rustin P, Lewandoski
M, Barsh GS, Clayton DA (1998) Mitochondrial transcription factor
A is necessary for mtDNA maintenance and embryogenesis in mice.
Nat Genet 18:231236.
So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons J. Neurosci., October 15, 2001, 21(20):80828090 8089
Leigh D (1951) Subacute necrotizing encephalomyopathy in an infant.
J Neurol Neurosurg Psychiatry 14:216 221.
Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P (1997) Intracel-
lular adenosine triphosphate (ATP) concentration: a switch in the
decision between apoptosis and necrosis. J Exp Med 185:14811486.
Li H, Wang J, Wilhelmsson H, Hansson A, Thoren P, Duffy J, Rustin P,
Larsson NG (2000) Genetic modication of survival in tissue-specic
knockout mice with mitochondrial cardiomyopathy. Proc Natl Acad Sci
USA 97:34673472.
Oldfors A, Fyhr I-M, Holme E, Larsson N-G, Tulinius M (1990) Neu-
ropathology in Kearns-Sayre syndrome. Acta Neuropathol 80:541546.
Oldfors A, Larsson N-G, Lindberg C, Holme E (1993) Mitochondrial
DNA deletions in inclusion body myositis. Brain 116:325336.
Oldfors A, Holme E, T ulinius M, Larsson NG (1995) Tissue distribution
and disease manifestations of the tRNA(Lys) A-G(8344) mitochon-
drial DNA mutation in a case of myoclonus epilepsy and ragged red
bres. Acta Neuropathol 90:328333.
Rotig A, de Lonlay P, Chretien D, Foury F, Koenig M, Sidi D, Munnich
A, Rustin P (1997) Aconitase and mitochondrial iron-sulphur protein
deciency in Friedreich ataxia. Nat Genet 17:215217.
Rustin P, Chretien D, Bourgeron T, Gerard B, Rotig A, Saudubray JM,
Munnich A (1994) Biochemical and molecular investigations in respi-
ratory chain deciencies. Clin Chim Acta 228:3551.
Silva JP, Kohler M, Graff C, Oldfors A, Magnuson MA, Berggren PO,
Larsson NG (2000) Impaired insulin secretion and beta-cell loss in
tissue-specic knockout mice with mitochondrial diabetes. Nat Genet
26:336340.
Sperk G, Lassmann H, Baran H, Seitelberger F, Hornykiewicz O (1985)
Kainic acid-induced seizures: dose-relationship of behavioural, neuro-
chemical and histopathological changes. Brain Res 338:289295.
Wallace DC (1992) Mitochondrial genetics: a paradigm for aging and
degenerative diseases? Science 256:628 632.
Wallace DC (1999) Mitochondrial diseases in man and mouse. Science
283:14821488.
Wang J, Wilhelmsson H, Graff C, Li H, Oldfors A, Rustin P, Bru¨ning JC,
Kahn CR, Clayton DA, Barsh GS, Thoren P, Larsson NG (1999)
Dilated cardiomyopathy and atrioventricular conduction blocks in-
duced by heart-specic inactivation of mitochondrial DNA gene ex-
pression. Nat Genet 21:133137.
Wang J, Silva JP, Gustafsson CM, Rustin P, Larsson NG (2001) In-
creased in vivo apoptosis in cells lacking mitochondrial DNA gene
expression. Proc Natl Acad Sci USA 98:4038 4043.
Xu B, Zang K, Ruff NL, Zhang YA, McConnell SK, Stryker MP,
Reichardt LF (2000) Cortical degeneration in the absence of neuro-
trophin signaling: dendritic retraction and neuronal loss after removal
of the receptor TrkB. Neuron 26:233245.
Zetterstro¨m RH, Simon A, Giacobini MM, Eriksson U, Olson L (1994)
Localization of cellular retinoid-binding proteins suggests specic roles
for retinoids in the adult central nervous system. Neuroscience 62:899
918.
8090 J. Neurosci., October 15, 2001, 21(20):80828090 So¨ rensen et al. Respiratory Chain Deciency in Mouse Cortical Neurons
    • "However, animals were still alive and had to be sacrificed only a few weeks later due to general weakness and respiratory failure (Wredenberg, personal communication ). An even more impressive gap of at least 1 month between the total absence of RC subunits and death of the animals was observed in the brain cortex after TFAM knockout in cortical neurons (Sorensen et al. 2001). Finally, we have shown previously that epidermal stem cells and their descendants can even hyperproliferate and form a well-functioning epidermis without any mtDNA and consequently without RC (Baris et al. 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Due to its epidemiological dimensions, there are tremendous efforts to understand the ultimate pathways that lead from modern Western lifestyles to the development of insulin resistance and, finally, overt type 2 diabetes (T2DM), which is often accompanied by nonalcoholic fatty liver disease (NAFLD). The insulin-resistant liver is intimately involved in T2DM, since it importantly contributes to high circulating blood glucose levels due to the unsuppressed release of glucose, even in the fasted state. There is a large body of literature on the “involvement” of mitochondrial dysfunction in the liver in the development of T2DM. However, it is unclear if mitochondrial dysfunction causes hepatic insulin resistance, thereby truly contributing to the development of T2DM and NAFLD, or if it is just a consequence. Also, the term mitochondrial dysfunction has been used in a very uncritical way. Finally, there seems to be a continuum of mitochondrial changes during the development of NAFLD, from the initial benign steatosis to nonalcoholic steatohepatitis (NASH). In this chapter, we summarize the current knowledge on mitochondrial functions and their failure and critically review the existing literature on these processes in the liver during the development of T2DM and NASH.
    Full-text · Chapter · Nov 2015 · PLoS ONE
    • "We believe that this reduction in mitochondrial amounts exerts an impact in the axonopathy exhibited in X-ALD for the reasons above mentioned. Indeed, deletion of TFAM is characterized by decrease of both mtDNA and OXPHOS activity, correlated to axonal degeneration and gliosis [106]. To compensate for the defective mitochondrial biogenesis, we sought to stimulate the PGC-1α/PPARγ axis with an agonist of PPARγ, thus treating the X-ALD mice with the thiazolidinedione pioglitazone, widely used as antidiabetic drug [107] . "
    [Show abstract] [Hide abstract] ABSTRACT: Peroxisomal and mitochondrial malfunction, which are highly intertwined through redox regulation, in combination with defective proteostasis, are hallmarks of the most prevalent multifactorial neurodegenerative diseases- including Alzheimer's (AD) and Parkinson's disease (PD) - and of the aging process, and are also found in inherited conditions. Here we review the interplay between oxidative stress and axonal degeneration, taking as groundwork recent findings on pathomechanisms of the peroxisomal neurometabolic disease adrenoleukodystrophy (X-ALD). We explore the impact of chronic redox imbalance caused by the excess of very long-chain fatty acids (VLCFA) on mitochondrial respiration and biogenesis, and discuss how this impairs protein quality control mechanisms essential for neural cell survival, such as the proteasome and autophagy systems. As consequence, prime molecular targets in the pathogenetic cascade emerge, such as the SIRT1/PGC-1α axis of mitochondrial biogenesis, and the inhibitor of autophagy mTOR. Thus, we propose that mitochondria-targeted antioxidants; mitochondrial biogenesis boosters such as the antidiabetic pioglitazone and the SIRT1 ligand resveratrol; and the autophagy activator temsirolimus, a derivative of the mTOR inhibitor rapamycin, hold promise as disease-modifying therapies for X-ALD. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jun 2015
    • "The primary objective of our work was to generate a model of partial mtDNA depletion by genetically silencing mitochondrial transcription factor A, TFAM. Indeed, tissue specific [19, 36, 37] and global knockdown [38] of the TFAM gene in mouse animal models has proven an effective means of depleting mtDNA levels. After transcriptionally silencing the TFAM gene in MIN6 cells by.80%, we achieved a 40% reduction in mtDNA levels, which was comparable to the degree of mtDNA depletion seen in aged human islets [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes.
    Full-text · Article · Dec 2014
Show more

Recommended publications

Discover more