Content uploaded by Carsten Merkwirth
Author content
All content in this area was uploaded by Carsten Merkwirth
Content may be subject to copyright.
Content uploaded by Carsten Merkwirth
Author content
All content in this area was uploaded by Carsten Merkwirth
Content may be subject to copyright.
Loss of Prohibitin Membrane Scaffolds Impairs
Mitochondrial Architecture and Leads to Tau
Hyperphosphorylation and Neurodegeneration
Carsten Merkwirth
1,2,3¤
, Paola Martinelli
4.
, Anne Korwitz
1,2,3.
, Michela Morbin
5
, Hella S. Bro
¨nneke
2
,
Sabine D. Jordan
1
, Elena I. Rugarli
2,3,4
, Thomas Langer
1,2,3,6
*
1Institute for Genetics, University of Cologne, Cologne, Germany, 2Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD),
University of Cologne, Cologne, Germany, 3Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany, 4Institute for Zoology, University of
Cologne, Cologne, Germany, 5Neuropathology and Neurology 5, IRCCS Foundation, Neurological Institute Carlo Besta, Milano, Italy, 6Max Planck Institute for Biology of
Aging, Cologne, Germany
Abstract
Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegen-
eration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes
in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds.
In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown
experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the
dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae
morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of
retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific
inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments
and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus,
demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1,
affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A
destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the
appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These
results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability,
mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins.
Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a
dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative
disorders caused by deficiencies in mitochondrial dynamics.
Citation: Merkwirth C, Martinelli P, Korwitz A, Morbin M, Bro
¨nneke HS, et al. (2012) Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and
Leads to Tau Hyperphosphorylation and Neurodegeneration. PLoS Genet 8(11): e1003021. doi:10.1371/journal.pgen.1003021
Editor: Nils-Go
¨ran Larsson, Max Planck Institute for Biology of Aging, Germany
Received May 3, 2012; Accepted August 23, 2012; Published November 8, 2012
Copyright: ß2012 Merkwirth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants of the Deutsche Forschungsgemeinschaft to TL (SFB635, C4) and EIR (RU1653/1-1) and the European Research
Council (AdG No. 233078) to TL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Thomas.Langer@uni-koeln.de
¤ Current address: The Salk Institute for Biological Studies, La Jolla, California, United States of America
.These authors contributed equally to this work.
Introduction
The dynamic behavior of mitochondria that constantly divide
and fuse is pivotal to maintain their pleiotropic activities and their
distribution within cells. Conserved protein machineries in the
outer and inner membrane of mitochondria mediate membrane
fusion events, ensure cristae formation and regulate the interaction
of mitochondria with the endoplasmic reticulum [1–3]. Loss of
mitochondrial fusion leads to neuronal loss in mice, highlighting
the vulnerability of neurons for deficiencies in mitochondrial
dynamics [4–6]. Mutations in the dynamin-like GTPases MFN2
and OPA1, which mediate mitochondrial membrane fusion, cause
neurodegeneration in Charcot-Marie-Tooth disease type 2A and
autosomal dominant optic atrophy, respectively [7–9]. Moreover,
defects in mitochondrial dynamics are associated with multiple
neurodegenerative diseases, including Parkinson’s, Alzheimer’s
(AD) and Huntington’s disease [10–12].
Recent evidence identified prohibitins in the mitochondrial
inner membrane as novel modulators of mitochondrial fusion [13–
15]. Prohibitins comprise a conserved and ubiquitously expressed
protein family [16,17]. Two homologous proteins, prohibitin-1
(PHB1) and prohibitin-2 (PHB2), assemble into large ring
complexes in the inner membrane with putative functions as
protein and lipid scaffolds [18]. The genetic interaction of yeast
PHB1 and PHB2 with genes involved in the mitochondrial
cardiolipin and phosphatidyl ethanolamine metabolism suggests
PLOS Genetics | www.plosgenetics.org 1 November 2012 | Volume 8 | Issue 11 | e1003021
that prohibitin complexes may also affect the lipid distribution in
the inner membrane [19]. Consistently, PHB1 and PHB2 are
homologous to members of the SFPH-family that were found in
association with membrane microdomains in various cellular
membranes [20,21].
Despite emerging evidence for a scaffold function of prohibitins
[16], only limited information is available on the physiological
relevance of a defined spatial organization of the inner membrane
for mitochondrial activities. Loss of prohibitin genes in Caenorhab-
ditis elegans and mice results in embryonic lethality, pointing to
essential functions during embryonic development [22,23].
Knockdown of PHB1 and PHB2 in adult, non-neuronal tissues
of C. elegans influences aging by moderating fat metabolism and
energy production [24]. However, it remained unclear whether
prohibitins affect mitochondrial respiratory activities directly. In
mammalian cells, prohibitins appear to affect mitochondrial
respiration in a cell-type specific manner. While knockdown of
PHB1 impaired complex I activity in endothelial cells [25],
mitochondrial respiratory function was not affected in prohibitin-
deficient mouse embryonic fibroblasts (MEFs) [13]. These studies
identified the processing of OPA1 as the central process regulated
by prohibitins in vitro. The function of OPA1 in mitochondrial
fusion and cristae morphogenesis depends on the presence of both
long and short forms of OPA1, the latter being generated by
proteolytic processing of long forms [26–29]. Loss of PHB2
destabilizes long OPA1 forms and inhibits mitochondrial fusion,
resulting in the fragmentation of the mitochondrial network and
an increased susceptibility of the cells towards apoptotic stimuli
[13,15]. Interestingly, a destabilization of long OPA1 forms has
also been observed in cells lacking m-AAA proteases [30], ATP-
dependent quality control enzymes with regulatory functions
during mitochondrial biogenesis [4], which assemble with
prohibitin complexes in the inner membrane of yeast, mammalian
and plant mitochondria [31,32]. Mutations in m-AAA protease
subunits cause axonal degeneration in spinocerebellar ataxia,
hereditary spastic paraplegia, and a spastic-ataxia neuropathy
syndrome [33–35].
These results prompted us to assess in vivo the role of prohibitins
in neurons, which contain high levels of prohibitins and are
particularly vulnerable to disturbances in mitochondrial dynamics.
Using conditional gene ablation in mice, we demonstrate that a
post-natal loss of PHB2 in the forebrain triggers massive
neurodegeneration which is associated with the accumulation of
aberrant mitochondria and hyperphosphorylation of the microtu-
bule-associated protein tau.
Results
Forebrain-specific PHB2-deficient mice
Previous experiments using a genetic loss-of-function approach to
uncover physiological functions of PHB2 revealed an early
embryonic lethality phenotype in mice [13,23]. To circumvent
gene ablation during embryogenesis, conditional Phb2 mice
(Phb2
fl/fl
) were bred to mice expressing the Cre recombinase
under control of the postnatally expressed CaMKIIapromoter
(CaMKIIa-Cre) [36] resulting in neuron-specific PHB2-deficient
mice (Phb2
fl/fl;CaMKIIa-Cre
; hereafter referred to as Phb2
NKO
mice).
This mouse line shows a defined and restricted recombination
pattern and a progressive increase in recombination efficiency
after completed neuronal development [36]. Histological exam-
inations of brains derived from CaMKIIa-Cre mice crossed to
ROSA26-LacZ reporter mice revealed selective Cre-mediated
recombination in forebrain regions including the cortex, striatum
and hippocampus, to a minor extent in hypothalamic regions, but
not in hind- and midbrain regions like the cerebellum (Figure S1)
[37]. To demonstrate efficient depletion of Phb2,in-situ hybrid-
ization against the endogenous Phb2 mRNA was performed.
Notably, Phb2 mRNA was virtually depleted in hippocampal
neurons of 8-week-old Phb2
NKO
mice (Figure 1A). Consistently,
immunoblotting of tissue lysates prepared from various brain
compartments of mice of different age revealed maximal
depletion of PHB2 in Cre-expressing tissues at 14-weeks, but
not in the cerebellum where Cre recombinase is not expressed
(Figure 1B). Notably, PHB2 depletion was accompanied by
efficient loss of its assembly partner PHB1 (Figure 1B). This
observation is consistent with previous findings in cultured MEFs
[13] and demonstrates that prohibitin subunits are functionally
interdependent in neurons in vivo.
Homozygous Phb2
NKO
mice were born at expected mendelian
ratios, showed normal fertility and were anatomically indistin-
guishable from their WT littermates. From 12 to 14 weeks of age,
however, Phb2
NKO
mice progressively developed aging-related
phenotypes, including weight loss, cachexia and kyphosis
(Figure 1C, 1D; Figure S2). Furthermore, Phb2
NKO
mice, but
not control littermates, showed an excessive pathological groom-
ing behavior characterized by facial hair loss and self-inflicted
facial lesions (Figure 1C). An extensive analysis of behavioral and
cognitive abilities in early-stage 8-week-old Phb2
NKO
animals
revealed decreased hippocampus-dependent learning abilities and
memory formation (Figure S3), and an impairment of innate fear
behavior and motor coordination (Figure S4) (for details, see Text
S1). The phenotypes of Phb2
NKO
animals deteriorated with age
and led to premature death of Phb2
NKO
mice starting at the age of
14 weeks (Figure 1E). The maximal lifespan of Phb2
NKO
mice was
22 weeks only. Survival was not affected in homozygous Phb2
fl/fl
or heterozygous Phb2
fl/WT;CaMKIIa-Cre
(Phb2
HET
) mice (Figure 1E).
We therefore conclude that PHB2 in the forebrain is essential for
postnatal mouse survival.
Author Summary
Mitochondria are the major site of cellular ATP production
and are essential for the survival of neurons. High ATP
levels are required to sustain neuronal activities and axonal
transport of macromolecules and organelles. The func-
tional integrity of mitochondria depends on fusion and
fission of their membranes, which maintain a dynamic
mitochondrial network in cells. Interference with these
processes causes neurodegenerative disorders that are
characterized by axonal degeneration of distinct neurons.
However, how an impaired fusion affects mitochondrial
activities and neuronal survival remains poorly understood.
Here, we have addressed this question by analyzing
forebrain-specific knockout mice lacking prohibitins. Pro-
hibitin complexes form membrane scaffolds in the inner
membrane, which we now show are required for
mitochondrial fusion, ultrastructure, and genome stability
in neurons. Loss of prohibitins triggers extensive neuro-
degeneration associated with behavioral and cognitive
deficiencies. Surprisingly, we observe hyperphosphoryla-
tion and filament formation of the microtubule-associated
protein tau, reminiscent of a large group of neurodegen-
erative disorders termed tauopathies. Our findings, there-
fore, not only provide new insight into how defects in
mitochondrial fusion affect neuronal survival, but also
point to an intimate relationship of deficiencies in
mitochondrial dynamics and tau pathologies.
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 2 November 2012 | Volume 8 | Issue 11 | e1003021
Figure 1.
CaMKIIa-Cre
-mediated inactivation of the mouse
Phb2
gene in forebrain neurons. (A) In-situ hybridization of Phb2 mRNA in the
hippocampus of 8-week-old Phb2
NKO
and Phb2
fl/fl
control mice. Scale bar: 500 mm. (B) Immunoblot analysis of tissue lysates generated from the
indicated brain regions of Phb2
NKO
(KO) and Phb2
fl/fl
(WT) control mice of different age using PHB1- and PHB2-specific antibodies. Ponceau S (PoS)
staining was used to monitor equal gel loading. Cortex (CO), striatum (ST), hippocampus (HC), cerebellum (CB). (C) Representative photographs of 20-
week-old Phb2
NKO
mice of the indicated genotypes showing lordokyphosis (left panel) and excessive pathological grooming (right panel). White
arrows indicate regions of self-inflicted open skin lesions. (D) Body weight analysis of Phb2
NKO
and Phb2
fl/fl
control animals. n = 20. ***P,0.001. Error
bars indicate SEM. (E) Kaplan-Meier survival plot of Phb2
NKO
(n = 30) and control animals (Phb2
fl/fl
(n = 59), Phb2
HET
(n = 19)). P,0.0001.
doi:10.1371/journal.pgen.1003021.g001
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 3 November 2012 | Volume 8 | Issue 11 | e1003021
Progressive forebrain atrophy and neuronal loss in
Phb2
NKO
mice
To investigate the underlying defects at the cellular level, we
analyzed gross brain morphology of Phb2
NKO
and control brains.
Phb2
NKO
brains were indistinguishable from controls in size, weight
and gross morphology at 14 weeks of age (Figure 2A). In contrast,
at the age of 20 weeks we observed a massive atrophy of Phb2
NKO
forebrains, which was accompanied by a severe total brain weight
loss (Figure 2A). Histological examinations of Phb2
NKO
brains
further supported the progressive nature and severity of the
phenotypes. Nissl stainings and semithin sections from Phb2
NKO
animals revealed that the region most prominently affected was
the hippocampus, which undergoes progressive degeneration over
time, culminating in the almost complete loss of neurons in both
the dentate gyrus (DG) and cornu ammonis (CA) regions at 20
weeks of age (Figure 2B, Figure S5A). At this age, cortical neurons
in all layers also appeared affected in Phb2
NKO
mice, showing
shrinkage of the cell body and loss of processes (Figure S5B). Since
the hippocampal region appeared to be a preferential target in the
absence of PHB2, we analyzed this area in more detail. Neuronal
loss was accompanied by a progressive development of astrogliosis,
as demonstrated by increased GFAP reactivity already observable
at 6 weeks of age in the DG (Figure 2B). At this age, a significant
fraction of DG neurons in Phb2
NKO
mice appeared vacuolated and
neuronal loss was already apparent (Figure 2D, 2E). At 14 weeks,
the DG consisted of only one neuronal layer, with more than 50%
of residual neurons showing degenerative features (Figure 2C–2E).
Remarkably, while DG neurons were markedly reduced in
number already at 14 weeks (Figure 2E), neurons in the CA1
region were less affected and neuronal loss became apparent only
in 20-week-old Phb2
NKO
mice (Figure 2F, Figure S6). TUNEL
staining of the hippocampal DG regions revealed few positive
neuronal cell bodies, suggesting that neuronal loss in Phb2
NKO
brains is at least partially caused by apoptosis (Figure S5). We
therefore conclude that PHB2 is generally required for neuronal
survival in vivo. However, the time-course and severity of neuronal
degeneration show regional differences.
Loss of prohibitins affects the structural integrity and
distribution of mitochondria in neurons
To define whether the depletion of PHB2 affects mitochondrial
ultrastructure in neurons at early stages of the pathological
process, we analyzed the DG of young Phb2
NKO
mice by
transmission electron microscopy. DG neurons of 6-week-old
Phb2
fl/fl
control mice contained mitochondria with a normal
appearance characterized by lamellar-shaped cristae inside dou-
ble-membrane layered organelles (Figure 3A). In contrast, several
neurons in the DG of Phb2
NKO
mice contained mitochondria with
almost complete absence of lamellar cristae (Figure 3A). Moreover,
in some cases these mitochondria appeared moderately swollen.
These ultrastructural features account for the appearance of
vacuolated neurons observed in semithin sections (Figure 2C). The
number of neurons containing mitochondria with defective
ultrastructure was further enhanced in 14-week-old animals
confirming the progressive nature of this pathology (not shown).
To further investigate whether lack of PHB2 affects the
mitochondrial network in neurons in a cell-autonomous manner,
we isolated primary hippocampal neurons from conditional E18.5
Phb2
fl/fl
and Phb2
fl/WT
embryos and infected them with lentiviruses
expressing nuclear-targeted Cre recombinase to genetically
inactivate Phb2 in vitro. The mitochondrial network in these
neurons was visualized by the simultaneous infection with
lentiviral particles encoding a mitochondrially targeted EGFP
(Su9-EGFP). Tubular mitochondria were present in the cell body
and along the neurites in Phb2
fl/fl
(Figure 3B; a, a9) and Phb2
fl/WT
neurons, which were infected with Cre-expressing lentiviruses
(NLS-Cre::Phb2
fl/WT
) (Figure 3C). In contrast, mitochondria were
greatly fragmented and clustered in perinuclear regions of .70%
of infected Phb2
fl/fl
neurons (NLS-Cre::Phb2
fl/fl
) (Figure 3B; b, b9).
We further evaluated the mitochondrial distribution in Phb2-
depleted neurons and determined the total number of mitochon-
dria protruding into the neurites. Strikingly, neurites of Cre-
infected Phb2
fl/fl
neurons contained fewer mitochondria when
compared to controls consistent with the perinuclear clustering of
fragmented mitochondria after acute loss of prohibitins
(Figure 3D).
Different isoforms of the dynamin-like GTPase OPA1 with
seemingly varying activities exist, which are expressed in a tissue-
specific manner in mice [38]. The expression of OPA1 isoform 1
predominates in the central nervous system giving rise to bands b
(L-OPA1) and, upon proteolytic processing, to band e (S-OPA1)
[38]. To examine whether depletion of PHB2 affects the
accumulation of OPA1 in neuronal tissue in vivo, we analyzed
Phb2
NKO
and control forebrain lysates by immunoblotting with
OPA1-specific antibodies. The loss of prohibitins was accompa-
nied by the selective loss of the L-OPA1 isoform b in the
hippocampus (Figure 3E), cortex and striatum but not in the
cerebellum (Figure S7). These alterations occurred in a time-
dependent manner simultaneous with the depletion of prohibitins
and were already detected at 10 weeks of age. This does not reflect
a general impairment of the biogenesis of mitochondrial inner
membrane proteins, as various subunits of respiratory chain
complexes accumulated at similar levels in the brain of Phb2
NKO
and control animals (Figure 3E; Figure S7). Overall, these data
demonstrate that neuronal PHB2 ensures stabilization of L-OPA1
and the maintenance of the mitochondrial network and ultra-
structure in vivo.
Tau hyperphosphorylation in PHB2-deficient neurons
Surprisingly, ultrastructural examination of hippocampi of 14-
week-old Phb2
NKO
mice revealed the accumulation of straight
tubular structures in unmyelinated neuronal processes. These
filamentous structures measure about 12–20 nm in diameter
(mean 20.8 nm60.323; range 9.9–25.72 nm) and are reminiscent
of inclusions composed of aberrantly phosphorylated species of the
microtubule-associated protein tau. Although morphologically
distinct from paired helical filaments (PHF), they are similar to
those found in ‘classical’ intracytoplasmic inclusions of tau-positive
astrocytes and neurons, which are observed in several neurode-
generative conditions such as frontotemporal dementia and other
tauopathies (Figure 4A) [39].
To explore a role for Phb2 in tau phosphorylation, hippocampal
tissue sections were immunostained with AT-8 antibodies, which
selectively recognize phosphorylated species of tau (phospho-
Ser202 and phospho-Thr205). Intraneuronal inclusions were
detected in the DG but not in other hippocampal regions of
Phb2
NKO
mice as early as at 6 weeks but not in control littermates,
and accumulated in both cell body and neurites (Figure 4B). We
substantiated these observations by immunoblotting using phos-
pho-tau specific AT-8 antiserum (Figure 4C). Several hyperpho-
sphorylated tau species selectively accumulated in hippocampal
lysates from 14-week-old Phb2
NKO
mice, but not in lysates from
control mice (Figure 4C).
Several kinases have been implicated in tau phosphorylation
both in vitro and in vivo [40,41]. We therefore assessed the activation
status of candidate kinases by immunoblotting of hippocampal
extracts of Phb2
NKO
mice. Phosphorylated, active forms of the
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 4 November 2012 | Volume 8 | Issue 11 | e1003021
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 5 November 2012 | Volume 8 | Issue 11 | e1003021
extracellular signal-regulated MAP kinases ERK1/2 and of the c-
Jun N-terminal kinase JNK were detected specifically in Phb2
NKO
mice (Figure 4C). In contrast, the b-form of glycogen synthase
kinase (GSK3), another putative major tau kinase, was robustly
inactivated by phosphorylation at Ser position 9 (Figure 4D).
Concomitantly, this was accompanied by the parallel activation of
the upstream kinase AKT suggesting that the AKT-GSK3 axis
might not be causative for the increased tau pathology in Phb2
NKO
mice (Figure 4D). Similarly, cyclin-dependent kinase 5 (CDK5)
apparently does not contribute to tau hyperphosphorylation in
Phb2
NKO
mice as we did not detect proteolytic conversion of its
substrate p35 to p25 in Phb2-deficient hippocampal lysates
(Figure 4D).
Taken together, we conclude from these experiments that
deletion of Phb2 activates MAP kinases leading to tau hyperpho-
sphorylation and the deposition of aberrant filamentous structures
in hippocampal neurons.
Late-onset mitochondrial dysfunction and selective
mtDNA loss in Phb2
NKO
tissues
Mitochondrial dysfunction is an early phenomenon in many
human tauopathies [42,43]. To examine whether compromised
mitochondrial respiratory function might be the underlying defect
causing tau pathology and neurodegeneration in Phb2
NKO
mice, we
monitored respiratory activities in situ and in isolated PHB2-
deficient brain mitochondria. Enzymatic COX/SDH stainings on
whole brain cryosections of 6-week-old Phb2
NKO
brains did not
provide evidence for the presence of respiratory deficient cells
(Figure S8). Consistently, substrate-driven respiration was not
affected in mitochondria that had been isolated from hippocampal
tissues of 12-week-old Phb2
NKO
mice (Figure 5A). Consistently, we
obtained no evidence for increased ROS production and oxidative
damage in 14-week-old Phb2
NKO
mice (Figure S9).
While not apparent in young mice, OXPHOS activities
declined with age and were decreased significantly in 18-week-
old Phb2
NKO
mice (Figure 5B). Mitochondria isolated from
hippocampi of these mice were generally able to consume oxygen,
as the basal mitochondrial respiration in the presence of pyruvate
was similar in 18-week-old Phb2
NKO
and control mitochondria.
However, respiration rates in PHB2-deficient mitochondria
decreased significantly in the presence of saturating concentrations
of ADP to maximally stimulate respiration, indicating that
coupling is impaired in mitochondria depleted of PHB2.
Moreover, enzymatic activities of complex I (monitored in the
presence of glutamate and malate), complex II (in the presence of
succinate) and of complex IV [in the presence of TMPD
(N,N,N9,N9-Tetramethyl-1,4-phenylendiamine)] were significantly
reduced in mitochondria isolated from 18-week-old Phb2
NKO
mice
suggesting that respiratory activities in hippocampal tissues
progressively deteriorate over time in the absence of PHB2
(Figure 5B).
The broad functional impairment of respiratory complexes in
aged PHB2-deficient mice could be explained by a loss of the
mitochondrial genome (mtDNA), which encodes essential respira-
tory chain subunits. We therefore determined mtDNA levels by
quantitative real-time PCR analysis of mtDNA isolated from
several neuronal tissues of Phb2
NKO
and control mice. Strikingly,
mtDNA levels relative to nuclear DNA deteriorated in a
progressive manner in the hippocampus and striatum but not in
the cerebellum of Phb2
NKO
mice (Figure 5C, 5D, Figure S10). In
20-week-old Phb2
NKO
animals, relative mtDNA levels were
reduced to 30% of controls in the hippocampus, providing a
rationale for the decreased respiratory activities in these mice. It is
noteworthy that mtDNA levels were not affected in cortical PHB2-
deficient mitochondria (Figure S10), pointing to neuronal-specific
differences in the mechanisms that stabilize mtDNA.
In conclusion, these experiments demonstrate that PHB2 is
required for the maintenance of mtDNA in neuronal mitochon-
dria. The loss of PHB2 in the forebrain leads to a progressive
destabilization of mtDNA and ultimately to an impaired
respiratory function. However, respiratory deficiencies become
apparent at significantly later stages than tau phosphorylation
suggesting that they are not the primary cause for the tau
pathology in PHB2-deficient mice.
Discussion
Our analysis of Phb2
NKO
mice unravelled essential functions of
prohibitins for the survival of adult neurons in vivo. Impaired
OPA1 processing and hyperphosphorylation of tau manifest early
during this degeneration process. Our observations therefore
establish the requirement of prohibitins for mitochondrial fusion
and ultrastructure in neurons and provide a novel model for tau
pathologies induced by mitochondrial dysfunctions.
Prohibitins are required for neuronal survival
We observe massive degeneration of PHB2-deficient neurons
in the forebrain. Neurons expressing Cre recombinase are lost
or severely affected in Phb2
NKO
mice, demonstrating a general
requirement of prohibitins for neuronal survival in vivo.TUNEL
stainings of DG neurons suggest that apoptosis contributes to
neuronal loss but other types of cell death cannot be excluded.
Consistently, depletion of prohibitins was found to facilitate
apoptosis in different cell types in vitro [13,44,45]. It is
noteworthy that the susceptibility towards apoptosis appears to
vary between different cell types [13,44,45]. Similarly, the loss
of PHB2 in Phb2
NKO
mice leads to faster death of DG neurons
when compared to CA1 neurons, pointing to neuron-specific
differences.
The loss of hippocampal neurons in Phb2
NKO
mice is associated
with anxiolytic behavior and deficiencies in memory function and
in learning abilities. Moreover, Phb2
NKO
mice develop progressive
cachexia and kyphosis. In view of massive neuronal loss in the
hippocampal region of Phb2
NKO
mice, it appears likely that
reduced food intake causes these phenotypes. As Phb2 might only
be partially deleted in the hypothalamic region of Phb2
NKO
mice
Figure 2. Progressive astrogliosis and loss of hippocampal neurons in
Phb2
NKO
mice. (A) Representative photographs of brains isolated
from 14- or 20-week-old Phb2
NKO
and Phb2
fl/fl
control mice (upper panel). Brain weights of Phb2
NKO
and Phb2
fl/fl
control animals were monitored at
the indicated time points (lower panel). n = 5 per genotype and time point, *P,0.05; **P,0.01; ***P,0.001. Error bars indicate SEM. (B) Nissl staining
of coronal sections across the hippocampal region from Phb2
NKO
and Phb2
fl/fl
control brains of the indicated age (left panel). Immunohistochemistry
using GFAP antibody reveals progressive astrogliosis in the hippocampus of Phb2
NKO
mice (right panel). Scale bars: 400 mm. (C) Coronal semithin
sections of the hippocampal DG from 6-week (upper panel) or 14-week-old (lower panel) Phb2
NKO
and Phb2
fl/fl
control mice. Black vertical bars show
the thickness of the neuronal layers. White scale bar: 40 mm. (D) Quantification of neurons with degenerative features and vacuolization in the DG of
Phb2
NKO
and Phb2
fl/fl
control mice of the indicated age. Data are expressed as percentage of total cells counted. At least 200 cells were scored per
section; in case of the Phb2
NKO
at 14 weeks all residual neurons were scored. Error bars indicate SEM (n= 3). (E) Number of DG and (F) CA1 neurons in
6- and 14-week-old Phb2
NKO
mice. Error bars indicate SEM (n = 3).
doi:10.1371/journal.pgen.1003021.g002
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 6 November 2012 | Volume 8 | Issue 11 | e1003021
Figure 3. Defective mitochondrial morphogenesis and ultrastructure in
Phb2
-deficient neurons
in vivo
.(A) Transmission electron
microscopy analysis of the mitochondrial ultrastructure in DG neurons of 6-week-old Phb2
NKO
and Phb2
fl/fl
control mice. The enlargements show the
double membrane of the mitochondrion and the emergence of one crista. Scale bar: 400 nm. (B) Fragmentation and perinuclear clustering of PHB2-
deficient neuronal mitochondria. Primary hippocampal neurons isolated from E18.5 Phb2
fl/fl
embryos were infected with lentiviruses expressing
mitochondrially targeted EGFP and Cre recombinase (NLS-Cre) as indicated. Fixed samples were immunostained with antibodies directed against GFP
and neuronal bIII-tubulin followed by DAPI staining. a9,b9are magnifications of the boxed insets shown in a, b. Scale bars: 10 mm. (C) Quantification
of mitochondrial morphology in PHB2-deficient and control primary hippocampal neurons. Cells were infected with lentiviruses expressing Cre
recombinase when indicated and processed as described in (B). Cells containing tubular (white bars) or fragmented mitochondria (red bars) were
classified. .200 cells were scored in three independent experiments. ***P,0.001. Error bars indicate SEM. (D) Quantification of mitochondria per
neurites in PHB2-deficient primary hippocampal neurons. Phb2
fl/fl
neurons were infected with lentiviruses expressing Cre recombinase when
indicated and processed as described in (B). .30 cells were scored in three independent experiments. **P,0.01. Error bars indicate SEM. (E)
Immunoblot analysis of hippocampal tissue lysates from Phb2
NKO
(KO) and Phb2
fl/fl
(WT) control mice of the indicated age. Lysates were analyzed by
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 7 November 2012 | Volume 8 | Issue 11 | e1003021
[36], it remains to be determined how the loss of PHB2 in the
forebrain causes these phenotypes, which are reminiscent of other
mouse lines harboring dysfunctional mitochondria [46,47].
Regardless, they are likely the consequence of the massive
neuronal loss in Phb2
NKO
mice rather than reflecting specific
functions of prohibitins in the forebrain.
SDS-PAGE and immunoblotting using the indicated antibodies. Antibodies directed against VDAC and the 70 kDa subunit of complex II were used to
monitor equal gel loading. b/e: long/short OPA1 isoforms.
doi:10.1371/journal.pgen.1003021.g003
Figure 4. Tau hyperphosphorylation and filaments in
Phb2
NKO
mice. (A) Transmission electron microscopy analysis of hippocampal tissue
from 14-week-old Phb2
NKO
mice revealed the presence of straight filamentous tubules in neuronal unmyelinated processes reminiscent of tau
filaments. Scale bars: 1.5 mm (left panel); 1 mm (right panel). (B) Immunohistochemistry using anti-AT8 antibody detecting hyperphosphorylated tau
specifically on hippocampal tissue sections from 6-week-old Phb2
NKO
mice. Hyperphosphorylated tau accumulated both in the cell body (arrow head)
and in dendrites (arrow) of DG neurons. The lower panel illustrates magnifications of the boxed insets depicted in the upper panel. Scale bars:
100 mm (upper panel), 50 mm (lower panel). (C) Immunoblot analysis of tau hyperphosphorylation and associated signalling molecules. Hippocampal
tissue lysates from individual 14-week-old Phb2
NKO
and Phb2
fl/fl
control mice were analyzed by SDS-PAGE and immunoblotting using the indicated
antibodies. b-actin was used as loading control. (D) Immunoblot analysis of signalling components that have been linked functionally to tau
hyperphosphorylation. Hippocampal lysates were analyzed as in (C) using the indicated antibodies. b-actin was used as loading control.
doi:10.1371/journal.pgen.1003021.g004
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 8 November 2012 | Volume 8 | Issue 11 | e1003021
Prohibitins as mitochondrial membrane scaffolds in
neurons
Ring complexes formed of multiple PHB1 and PHB2 subunits
act as scaffolds in the inner membrane affecting the spatial
organization of membrane proteins and lipids [16,48]. Previous
studies in proliferating cells in vitro revealed that prohibitin
complexes ensure the accumulation of L-OPA1 within mitochon-
dria [13]. We now extend these findings to adult neurons in vivo
and establish an essential role of prohibitins for the maintenance of
mitochondrial ultrastructure. Destabilization of L-OPA1 in the
absence of PHB2 likely inhibits fusion and ongoing fission events
lead to the fragmentation of the mitochondrial network in
hippocampal neurons. Moreover, we demonstrate that prohibitin
scaffolds are required to maintain the mitochondrial genome,
which is progressively lost in neurons lacking PHB2 and likely
explains respiratory deficiencies that occur in aged PHB2-deficient
neurons. Notably, mtDNA is absent in fusion-incompetent
mitochondria in MFN2-deficient fibroblasts [5], indicating that
Figure 5. Late-onset mtDNA loss and respiratory dysfunction in
Phb2
NKO
mice. (A) and (B) Oxygen consumption of mitochondria isolated
from the hippocampus of (A) 12-week-old or (B) 18-week-old Phb2
NKO
and Phb2
fl/fl
control mice in the presence of specific substrates for individual
respective respiratory chain complexes. Pyr, pyruvate; ADP, adenosine diphosphate; glu, glutamate; mal, malate; succ, succinate; TMPD, N,N,N9,N9-
tetramethyl-1,4-phenylendiamine. n = 5. Error bars represent SEM. ***P,0.001. (C) and (D) Relative levels of mtDNA in the hippocampus (D) and the
cerebellum (E) of Phb2
NKO
and Phb2
fl/fl
control mice. Total DNA was extracted from brain subregions of mice of the indicated age and genotype and
analyzed by quantitative real-time PCR analysis using primers specific for mtDNA and nuclear DNA. Data represent average of at least three
independent experiments, each sample assayed in quadruples. mtDNA, mitochondrial DNA. Error bars represent SEM. *P,0.05, ***P,0.001.
doi:10.1371/journal.pgen.1003021.g005
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 9 November 2012 | Volume 8 | Issue 11 | e1003021
mitochondrial fusion or the protein machinery involved is required
to maintain mtDNA. It is therefore conceivable that neurons
lacking PHB2 lose mtDNA because mitochondrial fusion is
inhibited. Alternatively, PHB2 acting as a membrane scaffold
may directly affect the stability of mitochondrial nucleoids in
neurons. Prohibitins have been identified as peripheral compo-
nents of mitochondrial nucleoids and were found to maintain their
organization and stability at least in some cell lines in vitro [14,49].
In yeast, depletion of prohibitins in combination with components
affecting the accumulation of phosphatidyl ethanolamine in
mitochondrial membranes induces the loss of the mitochondrial
genome [19,50], supporting a critical role of the membrane
environment for the maintenance of mtDNA.
Taken together, our observations demonstrate that neuronal
survival in vivo critically depends on prohibitin scaffolds in the
inner membrane and identify the processing of OPA1 and the
stability of the mitochondrial genome as processes within
mitochondria, whose perturbation leads to neurodegeneration in
the absence of prohibitins.
Loss of PHB2 causes tau hyperphosphorylation and
neurodegeneration
Our findings also provide insight into the cellular mechanisms
through which a dysfunction of mitochondria leads to neurode-
generation. The observation of impaired OPA1 processing and
defective mitochondrial ultrastructure preceding massive neuronal
loss in Phb2
NKO
mice supports emerging evidence that neurons are
particularly susceptible to perturbations in mitochondrial dynam-
ics. Studies on the cerebellum of MFN2-deficient mice revealed
electron transport deficiencies of Purkinje cells prior to neuronal
death, which are consistent with the lack of mtDNA nucleoids
observed in fibroblasts [5]. The dependence of mtDNA stability
and respiratory activity on mitochondrial fusion provides an
elegant mechanism to explain neuronal loss in MFN2-deficient
mice [5]. However, while the lack of PHB2 destabilizes mtDNA in
the hippocampus and striatum, respiratory deficiencies manifest
only in aged Phb2
NKO
mice, indicating that alternative mechanisms
lead to neurodegeneration in this model.
The analysis of mitochondrial morphology in PHB2-deficient
hippocampal neurons suggests that deficiencies in mitochondrial
distribution may trigger neuronal loss. Fragmented mitochondria
accumulate in the perinuclear region of hippocampal neurons
lacking PHB2 in vitro and are depleted from neurites. The
surprising observation of tau hyperphosphorylation and aggrega-
tion provides a possible explanation for the altered distribution of
mitochondria in PHB2-deficient neurons. Consistent with an
important role for neurodegeneration, we detected tau phosphor-
ylated at AT-8 sites already in 6-week-old Phb2
NKO
mice, i.e. before
neuronal loss becomes apparent. Tau is predominantly present in
axons, where it binds and stabilizes microtubules and regulates
axonal transport processes [43,51,52]. Hyperphosphorylated
forms of tau were found to detach from microtubules, accumulate
in the soma and are prone to aggregation. Consistently,
phosphorylated tau was found to interfere with the binding of
kinesin motors to mitochondria and distinct vesicles affecting
cargo-selective anterograde transport in cultured neurons [52].
Moreover, phosphorylation of tau at AT-8 sites was recently found
to modulate mitochondrial movement in cortical neurons [53]. It
is therefore conceivable that tau hyperphosphorylation in the
absence of PHB2 causes mitochondrial transport deficiencies
triggering progressive neuronal loss in Phb2
NKO
mice.
Hyperphosphorylation of tau has been observed in AD brains
[54]. Stress-activated kinases like JNK and ERK1/2 have been
implicated in the hyperphosphorylation of tau during AD. In fact,
fibrillar Abcan induce ERK activation, abnormal phosphoryla-
tion of Tau, and progressive neurodegeneration [55]. In addition,
JNK-related kinases are activated in AD brains and are associated
with the development of amyloid plaques [56]. However, despite
extensive studies on tau hyperphosphorylation, the complexity of
kinases and phosphatases involved has precluded to define its
pathogenic role for AD until now [57].
Regardless, the discovery of tau hyperphosphorylation and
filament formation upon loss of PHB2 sheds new light on the
possible role of mitochondria in neurodegeneration in AD and
related disorders. While mitochondrial dysfunction has been
recognized as a prominent, early event in a number of tauopathies
including AD [51], it remained open whether mitochondrial
defects are of direct pathogenic relevance or secondary to other
cellular deficiencies. Our analysis of Phb2
NKO
mice provides first
genetic evidence that a dysfunction of mitochondria can trigger
tau hyperphosphorylation and aggregation. We detected phos-
phorylated tau in PHB2-deficient hippocampal neurons lacking
apparent respiratory defects or evidence for oxidative damage
strongly suggesting that other mechanisms induce tau pathologies
in this model. Perturbations in mitochondrial dynamics and
ultrastructure that occur early in Phb2
NKO
mice and may interfere
with axonal trafficking are attractive candidates. Our findings
therefore raise the possibility that tau pathologies might be
associated with other neurodegenerative disorders caused by
deficiencies in mitochondrial dynamics. Studies along these lines
may turn out to be of relevance for tauopathies as well.
Materials and Methods
Histology and immunohistochemistry
Animals were anesthetized with avertin and perfused intracar-
dially with 4% paraformaldehyde in PBS. Brain were removed,
post-fixed overnight with 4% paraformaldehyde in PBS and
conserved in 0.12 M phosphate buffer. Immunohistochemistry
and immunofluorescence were performed on 30 mm sagittal
vibratome sections, as previously described [58]. Anti-GFAP
antibodies were purchased by NeoMarkers (Fremont, CA, USA).
Anti-4-HNE antibodies were purchased from Abcam (Cambridge,
UK). Immunohistochemistry with anti-AT-8 (Thermo Fisher
Scientific,Walthman, MA, USA) was performed with Vector
M.O.M. Immunodetection kit (Vector Lab, Burlingame, CA,
USA) according to the manufacturer’s protocol. For TUNEL
assays, tissues were frozen on liquid nitrogen vapour for 5 s after
fixation and then conserved in liquid nitrogen. TUNEL assays
were performed on 20 mm thick coronal frozen sections with
ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit
(Chemicon International Temecula, CA) according to the
manufacturer’s protocol. All immunohistochemical and immuno-
fluorescence analyses were performed on at least three mice per
genotype.
Neuropathology and ultrastructural analysis
Age-matched Phb2
NKO
and control mice (n = 3 for each
genotype) were anesthetized intraperitoneally with avertin and
perfused with 2% glutaraldehyde in PBS. Brains were removed
and postfixed in 0.12 M phosphate buffer/2% glutaraldehyde.
After treatment with osmium tetroxide, brains were embedded in
Epon (Fluka, Buchs SG, Switzerland). Semithin (1 mm) coronal
sections were cut from hippocampus and cerebral cortex. To
quantify the number of DG neurons with degenerative features,
we performed morphometry on semithin sections by scoring the
percentage of DG neurons with abnormal morphology and
vacuoles in the cytoplasm, and by counting the number of
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 10 November 2012 | Volume 8 | Issue 11 | e1003021
neurons in the DG and CA1 areas (n = 3 per genotype).
Morphometric analyses were performed blinded to the mouse
genotype. For ultrastructural analyses, blocks of tissue were
selected for electron microscopy after light microscopy examina-
tion of semithin sections. Ultrathin sections (70 nm) were cut,
collected on 200 mesh copper grids (Electron Microscopy
Sciences, Hatfield, PA, USA) and stained with uranium acetate
(Plano GMBH, Wetzlar, Germany) and lead citrate (Electron
Microscopy Sciences).
RNA in situ hybridization
To obtain specific probes for in situ hybridization, the coding
sequence of the mouse Phb2 (nucleotides 1–900) cDNA was PCR-
amplified from mouse liver cDNA, subcloned and used as
templates to transcribe either sense or antisense digoxygenin-
labeled riboprobes using the DIG RNA labeling kit (Roche).
Vibratome sections were permeabilized with proteinase K (10 mg/
ml) for 10 min. In situ hybridization was performed essentially as
described previously [59].
Enzyme activity staining of brain cryosections
Frozen brain cryosections were thawed and incubated in COX
staining solution (DAB, cytochrome c, sucrose, catalase, phosphate
buffer pH 7.4), SDH staining solution (succinic acid, phosphate
buffer pH 7.4) or both in a humid chamber for 15 min at 37uC.
Slides were washed three times with water for 5 min. For
dehydration samples were incubated in increasing concentrations
of ethanol: 90% EtOH for 1 min, 95% EtOH for 1 min and 100%
EtOH for 1 min. Subsequently, the sections were washed two
times in xylol for 2 min each and finally mounted in mounting
medium.
Primary neuronal cultures
Mouse primary hippocampal neurons were isolated from E18.5
embryos (Phb2
fl/fl
and Phb2
fl/wt
) and grown on coverslips for 7 DIV
before transduction with lentiviral vectors. Detailed experimental
procedures are found in the supplement.
Supporting Information
Figure S1 Spatially restricted Cre-recombination in mice
expressing Cre recombinase under the control of the CaMKIIa
promoter. b-galactosidase activity staining of parasagittal (a, d) and
coronal sections (b, c) of CaMKIIa-Cre/ROSA26-lacZ reporter
brains revealed spatially-restricted Cre recombination in the
cortex (CO), the striatum (ST), the hippocampus (HC) and the
hypothalamus (d). Maximal recombination efficiency was observed
in the hippocampus, in which all neuronal compartments [cornu
ammonis (CA), dentate gyrus (DG)] showed strong b-galactosidase
staining. CB = cerebellum. Scale bars: 1 mm (a, b); 0,5 mm (c, d).
(PDF)
Figure S2 Whole-body CT scans of Phb2
NKO
mice. (A) and (B)
Representative Micro-CT scans of 21-week-old (A) male and (B)
female Phb2
NKO
and Phb2
HET
control mice. Phb2
NKO
mice
displayed a strong curvature of the spinal column (lordokyphosis)
and reduction of body size and mass.
(PDF)
Figure S3 Impaired learning and memory abilities of Phb2
NKO
mice. (A) Escape latencies of 8-week-old Phb2
NKO
(n = 12) and
Phb2
fl/fl
control mice (n = 13) were examined with the Morris
water maze hidden platform paradigm during a 5-day training
period. ***P,0.001. Error bars indicate SEM. (B) Swim path
comparisons of 8-week-old Phb2
NKO
(n = 12) and Phb2
fl/fl
(n = 13)
control mice assessed during the training phase in the Morris
water maze on five consecutive days. The total distance travelled
in four trials per training day is indicated. *P,0.05; **P,0.01;
***P,0.001. Error bars indicate SEM. (C) Swimming times of 8-
week-old Phb2
NKO
(n = 12) and Phb2
fl/fl
control mice (n = 13) spent
in each quadrant in the probe trial on day 5. The dotted line
indicates the chance level (25%). ***P,0.001. Error bars indicate
SEM. (D) Representative path tracings of 8-week-old Phb2
NKO
and
Phb2
fl/fl
control mice during the probe trial on day 5. The coloured
quadrant indicates the target region after removal of the platform.
(E) Swim path comparisons of Phb2
NKO
mice and Phb2
fl/fl
controls
assessed during the probe trial in the Morris water maze on day 5.
Values are expressed as the total distance travelled during 60 s of
the probe trial. ***P,0.001. Error bars indicate SEM. (F) Swim
velocities of 8-week-old Phb2
NKO
(n = 12) and Phb2
fl/fl
(n = 13)
control mice assessed during the probe trial in the Morris water
maze on day 5. The total distance travelled per 60 sec during the
probe trial is indicated. Error bars indicate SEM.
(PDF)
Figure S4 Reduced anxiety and loss of motor coordination in
Phb2
NKO
mice. (A) Elevated zero maze analysis of 8-week-old
Phb2
NKO
(n = 12) and Phb2
fl/fl
control mice (n = 13). Values are
expressed as percentage of time spent in either open or closed
areas of the maze. **P,0.01. Error bars indicate SEM. (B) Total
distance of Phb2
NKO
(n = 12) and Phb2
fl/fl
control mice (n = 13)
travelled in the elevated zero maze (EZM). **P,0.01. Error bars
indicate SEM. (C) Open field test of 8-week-old Phb2
NKO
(n = 12)
and Phb2
fl/fl
control mice (n = 13). Values are expressed as
percentage of time spent in the center of the open field.
***P,0.001. Error bars indicate SEM. (D) Vertical locomotion
of 8-week-old Phb2
NKO
(n = 12) and Phb2
fl/fl
(n = 13) control mice
assessed from total rearing events during a 5-minute test phase in
the open field paradigm. ***P,0.001. Error bars indicate SEM.
(E) Total distance of Phb2
NKO
(n = 12) and Phb2
fl/fl
control mice
(n = 13) travelled in the open field. ***P,0.001. Error bars
indicate SEM. (F) Locomotor activity of 8-week-old Phb2
NKO
and
Phb2
fl/fl
control mice during day-night cycle measured in
metabolic cages. Data represent total beam break counts during
a 12 hour period. n = 4 per group. ***P,0.001. Error bars
indicate SEM. (G) Representative photographs of pathological
hindlimb clasping reflexes during tail suspension in 18-week-old
Phb2
NKO
mice (lower panel) compared to Phb2
fl/fl
controls (upper
panel). (H) Rotarod performance test of Phb2
NKO
(n = 12) and
Phb2
fl/fl
control mice (n = 13) examined at the indicated time
points. *P,0.05; ***P,0.001. Error bars indicate SEM.
(PDF)
Figure S5 Detection of apoptotic DG neurons in Phb2
NKO
mice.
TUNEL staining of DG neurons in 6-week-old Phb2
NKO
mice is
shown (black arrows). Scale bar: 20 mm.
(PDF)
Figure S6 Extensive loss of hippocampal and cortical neurons in
Phb2
NKO
mice. (A) Loss of pyramidal neurons in all hippocampal
layers of 20-week-old Phb2
NKO
mice. Coronal semithin sections of
the indicated cornu ammonis (CA) areas (CA1, CA2 and CA3) from
20-week-old Phb2
NKO
and Phb2
fl/fl
control mice. Scale bars: 20 mm.
(B) Late-onset morphological alterations of cerebral cortex
neurons in 20-week-old Phb2
NKO
mice. Coronal semithin sections
of cerebral cortex from layers I to VI of 20-week-old Phb2
NKO
and
Phb2
fl/fl
control mice. Scale bars: 20 mm.
(PDF)
Figure S7 Immunoblot analysis of forebrain tissue lysates of
Phb2
NKO
mice. Tissue lysates from cortex, striatum und cerebellum
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 11 November 2012 | Volume 8 | Issue 11 | e1003021
of Phb2
NKO
(KO) and Phb2
fl/fl
(WT) control mice of the indicated
age were analyzed by SDS-PAGE and immunoblotting using the
indicated antibodies. Antibodies directed against VDAC and the
70 kDa subunit of complex II were used to monitor equal gel
loading. b/e: long/short OPA1 isoforms.
(PDF)
Figure S8 COX and SDH activities in DG neurons of 6-week-
old Phb2
NKO
mice. Cross-sections of coronal brain regions from 6-
week-old Phb2
NKO
and Phb2
fl/fl
control mice were stained for either
COX or SDH activities or for both. Representative micrographs
are shown. Scale bar: 40 mm.
(PDF)
Figure S9 Monitoring oxidative damage in Phb2
NKO
mice.
Hippocampal lysates of 14-week-old Phb2
NKO
and Phb2
fl/fl
control
mice were analyzed by SDS-PAGE and immunoblotting using the
indicated antibodies. b-actin was used as a loading control. 4-
hydroxynonenal (4-HNE) stainings of coronal sections of the DG
of 14-week-old Phb2
NKO
and Phb2
fl/fl
control mice did not reveal
any signs of lipid oxidation (data not shown).
(PDF)
Figure S10 Tissue-specific mtDNA loss in PHB2-deficient
neurons in vivo. (A) and (B) Relative levels of mtDNA in (A)
striatum and (B) cortex of Phb2
NKO
and Phb2
fl/fl
control mice. Total
DNA was extracted from brain subregions of mice of the indicated
age and genotype and analyzed by quantitative real-time PCR
analysis using primers specific for mtDNA and nuclear DNA. Data
represent average of at least three independent experiments, each
sample assayed in quadruples. mtDNA, mitochondrial DNA.
Error bars represent SEM. **P,0.01.
(PDF)
Text S1 Supporting behavioral studies and supporting methods.
(DOCX)
Acknowledgments
We thank Dr. Hamid Kashkar for the modified lentiviral and pENTR
vectors, Veronica La Mattina for assistance with in situ hybridization
experiments, Dr. Marina Mora for help with electron microscopy, Drs.
Jens Bru¨ning and Paul Brinkko¨ tter for antibodies, and Gudrun Zimmer
and Jens Alber for expert technical assistance.
Author Contributions
Conceived and designed the experiments: CM PM AK HSB EIR TL.
Performed the experiments: CM PM AK MM HSB SDJ. Analyzed the
data: CM PM AK MM HSB SDJ. Wrote the paper: CM EIR TL.
References
1. Westermann B (2010) Mitochondrial fusion and fission in cell life and death. Nat
Rev Mol Cell Biol 11: 872–884.
2. Chen H, Chan DC (2010) Physiological functions of mitochondrial fusion.
Ann N Y Acad Sci 1201: 21–25.
3. de Brito OM, Scorrano L (2010) An intimate liaison: spatial organization of the
endoplasmic reticulum-mitochondria relationship. The EMBO journal 29:
2715–2723.
4. Rugarli EI, Langer T (2012) Mitochondrial quality control: A matter of life and
death for neurons. EMBO J in press.
5. Chen H, McCaffery JM, Chan DC (2007) Mitochondrial fusion protects against
neurodegeneration in the cerebellum. Cell 130: 548–562.
6. Ishihara N, No mura M, Jofuku A, Kato H, Suzuki SO, et al. (2009)
Mitochondrial fission factor Drp1 is essential for embryonic development and
synapse formation in mice. Nature cell biology 11: 958–966.
7. Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, et al. (2000) OPA1,
encoding a dynamin-related GTPase, is mutated in autosomal dominant optic
atrophy linked to chromosome 3q28. Nat Genet 26: 211–215.
8. Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, et al. (2000) Nuclear
gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in
dominant optic atrophy. Nat Genet 26: 207–210.
9. Zu¨ chner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, et al.
(2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-
Marie-Tooth neuropathy type 2A. Nat Genet 36: 449–451.
10. Song W, Chen J, Petrilli A, Liot G, Klinglmayr E, et al. (2011) Mutant
huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1
and increases its enzymatic activity. Nature medicine 17: 377–382.
11. Su B, Wang X, Zheng L, Perry G, Smith MA, et al. (2010) Abnormal
mitochondrial dynamics and neurodegenerative diseases. Biochimica et
biophysica acta 1802: 135–142.
12. Knott AB, Perkins G, Schwarzenbacher R, Bossy-Wetzel E (2008) Mitochon-
drial fragmentation in neurodegeneration. Nat Rev Neurosci 9: 505–518.
13. Merkwirth C, Dargazanli S, Tatsuta T, Geimer S, Lower B, et al. (2008)
Prohibitins control cell proliferation and apoptosis by regulati ng OPA1-
dependent cristae morphogenesis in mitochondria. Genes Dev 22: 476–488.
14. Kasashima K, Sumitani M, Satoh M, Endo H (2008) Human prohibitin 1
maintains the organization and stability of the mitochondrial nucleoids. Exp Cell
Res 314: 988–996.
15. Sato S, Murata A, Orihara T, Shirakawa T, Suenaga K, et al. (2011) Marine
Natural Product Aurilide Activates the OPA1-Mediated Apoptosis by Binding to
Prohibitin. Chem Biol 18: 131–139.
16. Osman C, Merkwirth C, Langer T (2009) Prohibitins and the functional
compartmentalization of mitochondrial membranes. J Cell Sci 122: 3823–3830.
17. Artal-Sanz M, Tavernarakis N (2010) Opposing function of mitochondrial
prohibitin in aging. Aging 2: 1004–1011.
18. Tatsuta T, Model K, Langer T (2005) Formation of membrane-bound ring
complexes by prohibitins in mitochondria. Mol Biol Cell 16: 248–259.
19. Osman C, Haag M, Potting C, Rodenfels J, Dip PV, et al. (2009) The genetic
interactome of prohibitins: coordinated control of cardiolipin and phosphati-
dylethanolamine by conserved regulators in mitochondria. J Cell Biol 184: 583–
596.
20. Tavernarakis N, Driscoll M, Kyrpides NC (1999) The SPFH domain: implicated
in regulating targeted protein turnover in stomatins and other membrane-
associated proteins. Trends Biochem Sci 24: 425–427.
21. Browman DT, Hoegg MB, Robbins SM (2007) The SPFH domain-containing
proteins: more than lipid raft markers. Trends Cell Biol 17: 394–402.
22. Artal-Sanz M, Tsang WY, Willems EM, Grivell LA, Lemire BD, et al. (2003)
The mitochondrial prohibitin complex is essential for embryonic viability and
germline function in Caenorhabditis elegans. J Biol Chem 278: 32091–32099.
23. Park SE, Xu J, Frolova A, Liao L, O’Malley BW, et al. (2005) Genetic deletion
of the repressor of estrogen receptor activity (REA) enhances the response to
estrogen in target tissues in vivo. Mol Cell Biol 25: 1989–1999.
24. Artal-Sanz M, Tavernarakis N (2009) Prohibitin couples diapause signalling to
mitochondrial metabolism during ageing in C. elegans. Nature 461: 793–797.
25. Schleicher M, Shepherd BR, Suarez Y, Fernandez-Hernando C, Yu J, et al.
(2008) Prohibitin-1 maintains the angiogenic capacity of endothelial cells by
regulating mitochondrial function and senescence. J Cell Biol 180: 101–112.
26. Song Z, Chen H, Fiket M, Alexander C, Chan DC (2007) OPA1 processing
controls mitochondrial fusion and is regulated by mRNA splicing, membrane
potential, and Yme1L. J Cell Biol 178: 749–755.
27. Ishihara N, Fujita Y, Oka T, Mihara K (2006) Regulation of mitochondrial
morphology through proteolytic cleavage of OPA1. EMBO J 25: 2966–
2977.
28. Griparic L, Kanazawa T , van der Bliek AM (2007) Regulation of the
mitochondrial dynamin-like protein Opa1 by proteolytic cleavage. J Cell Biol
178: 757–764.
29. Duvezin-Caubet S, Jagasia R, Wagene r J, Hofmann S, Trifunovic A, et al.
(2006) Proteolytic processing of OPA1 links mitochondrial dysfunction to
alterations in mitochondrial morphology. J Biol Chem 281: 37972–37979.
30. Ehses S, Raschke I, Mancuso G, Bernacchia A, Geimer S, et al. (2009)
Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease
isoenzymes and OMA1. J Cell Biol 187: 1023–1036.
31. Steglich G, Neupert W, Langer T (1999) Prohibitins regulate membrane protein
degradation by the m-AAA protease in mitochondria. Mol Cell Biol 19: 3435–
3442.
32. Piechota J, Kolodziejczak M, Juszczak I, Sakamoto W, Janska H (2010)
Identification and characterization of high molecular weight complexes formed
by matrix AAA proteases and prohibitins in mitochondria of Arabidopsis
thaliana. The Journal of biological chemistry 285: 12512–12521.
33. Casari G, De-Fusco M, Ciarmatori S, Zeviani M, Mora M, et al. (1998) Spastic
paraplegia and OXPHOS impairment caused by mutations in paraplegin, a
nuclear-encoded mitochondrial metalloprotease. Cell 93: 973–983.
34. DiBella D, Lazzaro F, Brusco A, Battaglia G, A P, et al. (2008) AFG3L2
mutations cause autosomal dominant ataxia SCA28 and reveal an essential role
of the m-AAA AFG3L2 homocomplex in the cerebellum. Annual meeting of the
American Society of Human Genetics. Philadelphia, Pennsylvania.
35. Pierson TM, Adams D, Bonn F, Martinelli P, Cherukuri PF, et al. (2011) Whole-
exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-
neuropathy syndrome linked to mitochondrial m-AAA proteases. PLoS Genet 7:
e1002325. doi:10.1371/journal.pgen.1002325
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 12 November 2012 | Volume 8 | Issue 11 | e1003021
36. Minichiello L, Korte M, Wolfer D, Kuhn R, Unsicker K, et al. (1999) Essential
role for TrkB receptors in hippocampus-mediated learning. Neuron 24: 401–
414.
37. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter
strain. Nat Genet 21: 70–71.
38. Akepati VR, Muller EC, Otto A, Strauss HM, Portwich M, et al. (2008)
Characterization of OPA1 isoforms isolated from mouse tissues. Journal of
neurochemistry 106: 372–383.
39. Giaccone G, Marcon G, Mangieri M, Morbin M, Rossi G, et al. (2008) Atypical
tauopathy with massive involvement of the white matter. Neuropathology and
applied neurobiology 34: 468–472.
40. Hanger DP, Anderton BH, Noble W (2009) Tau phosphorylation: the
therapeutic challenge for neurodegenerative disease. Trends in molecular
medicine 15: 112–119.
41. Mazanetz MP, Fischer PM (2007) Untangling tau hyperphosphorylation in drug
design for neurodegenerative diseases. Nature reviews Drug discovery 6: 464–
479.
42. Schon EA, Przedborski S (2011) Mitochondria: the next (neurode)generation.
Neuron 70: 1033–1053.
43. Reddy PH (2011) Abnormal tau, mitochondrial dysfunction, impaired axonal
transport of mitochondria, and synaptic deprivation in Alzheimer’s disease.
Brain research 1415: 136–148.
44. Kasashima K, Ohta E, Kagawa Y, Endo H (2006) Mitochondrial functions and
estrogen receptor-dependent nuclear translocation of pleiotropic human
prohibitin 2. J Biol Chem 281: 36401–36410.
45. Zhou P, Qian L, D’Aurelio M, Cho S, Wang G, et al. (2012) Prohibitin reduces
mitochondrial free radical production and protects brain cells from different
injury modalities. The Journal of neuroscience: the official journal of the Society
for Neuroscience 32: 583–592.
46. George SK, Jiao Y, Bishop CE, Lu B (2011) Mitochondrial peptidase IMMP2L
mutation causes early onset of age-associated disorders and impairs adult stem
cell self-renewal. Aging cell 10: 584–594.
47. Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, et al.
(2004) Premature ageing in mice expressing defective mitochondrial DNA
polymerase. Nature 429: 417–423.
48. Artal-Sanz M, Tavernarakis N (2009) Prohibitin and mitochondrial biology.
Trends in endocrinology and metabolism: TEM 20: 394–401.
49. Bogenhagen DF, Rousseau D, Burke S (2008) The layere d structure of human
mitochondrial DNA nucleoids. J Biol Chem 283: 3665–3675.
50. Birner R, Nebauer R, Schneiter R, Daum G (2003) Synthetic lethal interaction
of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the
prohibitin complex of Saccharomyces cerevisiae. Mol Biol Cell 14: 370–383.
51. Ballatore C, Lee VM, Trojanowski JQ (2007) Tau-mediated neurodegeneration
in Alzheimer’s disease and related disorders. Nature reviews Neuroscience 8:
663–672.
52. Ittner LM, Fath T, Ke YD, Bi M, van Eersel J, et al. (2008) Parkinsonism and
impaired axonal transport in a mouse model of frontotemporal dementia.
Proceedings of the National Academy of Sciences of the United States of
America 105: 15997–16002.
53. Shahpasand K, Uemura I, Saito T, Asnao T, Hata K, et al. (2012) Regulation of
Mitochondrial Transport and Inter-Microtubule Spacing by Tau Phosphoryla-
tion at the Sites Hyperphosphorylated in Alzheimer’s Disease. J Neurosci 32:
2430–2441.
54. Kopke E, Tung YC, Shaikh S, Alonso AC, Iqbal K, et al. (1993) Microtubule-
associated protein tau. Abnormal phosphorylation of a non-paired helical
filament pool in Alzheimer disease. The Journal of biological chemistry 268:
24374–24384.
55. Ferreira A, Lu Q, Orecchio L, Kosik KS (1997) Selective phosphorylation of
adult tau isoforms in mature hippocampal neurons exposed to fibrillar A beta.
Molecular and cellular neurosciences 9: 220–234.
56. Zhu X, Raina AK, Rottkamp CA, Aliev G, Perry G, et al. (2001) Activation and
redistribution of c-jun N-terminal kinase/stress activated protein kinase in
degenerating neurons in Alzheimer’s disease. Journal of neurochemistry 76:
435–441.
57. Mandelkow EM, Mandelkow E (2012) Biochemistry and cell biology of tau
protein in neurofibrillary degeneration. Cold Spring Harbor perspectives in
medicine 2: a006247.
58. Martinelli P, La Mattina V, Bernacchia A, Magnoni R, Cerri F, et al. (2009)
Genetic interaction between the m-AAA protease isoenzymes reveals novel roles
in cerebellar degeneration. Hum Mol Genet 18: 2001–2013.
59. Tiveron MC, Hirsch MR, Brun et JF (1996) The expression pattern of the
transcription factor Phox2 delineates synaptic pathways of the autonomic
nervous system. The Journal of neuroscience: the official journal of the Society
for Neuroscience 16: 7649–7660.
Prohibitins Protect against Neurodegeneration
PLOS Genetics | www.plosgenetics.org 13 November 2012 | Volume 8 | Issue 11 | e1003021
