Article

Meuer, K. et al. Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis. Cell Death Differ. 14, 651-661

Universitätsmedizin Göttingen, Göttingen, Lower Saxony, Germany
Cell Death and Differentiation (Impact Factor: 8.18). 05/2007; 14(4):651-61. DOI: 10.1038/sj.cdd.4402087
Source: PubMed

ABSTRACT

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.

Full-text

Available from: Gunnar P H Dietz
Cyclin-dependent kinase 5 is an upstream regulator of
mitochondrial fission during neuronal apoptosis
K Meuer
1,4
, IE Suppanz
2
, P Lingor
1,4
, V Planchamp
1,4
,BGo
¨
ricke
1,4
, L Fichtner
3,4
, GH Braus
3,4
, GPH Dietz
1,4
, S Jakobs
2
,MBa
¨
hr*
,1,4
and JH Weishaupt
1,4
Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular
networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We
show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally
contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-
negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates
mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator
contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established
neuronal apoptosis pathways.
Cell Death and Differentiation (2007) 14, 651–661. doi:10.1038/sj.cdd.4402087; published online 12 January 2007
Mitochondria can acquire a broad range of morphologies,
from a punctuate shape to a tubular appearance or even
extended networks. Several proteins, most of them belonging
to the family of large GTPases, have been identified that
energy-dependently regulate mitochondrial morphology and
subcellular distribution by promoting either fission (Fis1,
dynamin-related protein 1 (Drp1), MTP18) or fusion of
mitochondria (Mfn1/2 and Opa1).
1–3
As a potential upstream
regulatory factor, the actin cytoskeleton has been shown to
be a prerequisite for mitochondrial fission, possibly by its
influence on Drp1 recruitment to mitochondria.
4
Apoptotic release of the mitochondrial cytochrome c and
other proapoptotic mitochondrial proteins results in formation
of the so-called apoptosome, which in turn activates down-
stream effector caspases with death executing function.
5
This intrinsic, mitochondrial death pathway is relevant for
most forms of neuronal apoptosis, in contrast to extrinsic, for
example death receptor mediated, activation of programmed
cell death.
6
However, despite the contribution of mitochondrial
dysfunction specifically to the demise of neurons, there is only
scarce knowledge about mitochondrial fission during neuronal
apoptosis, nor about its functional contribution to neuronal
cell death. Furthermore, little is known about upstream
regulatory pathways for mitochondrial morphology in general.
Cyclin-dependent kinase 5 (CDK5) is a member of the CDK
family, but, distinct from other CDKs, it does not participate
in cell cycle regulation.
7
Instead, CDK5 is physiologically
implicated in cytoskeletal functions, as well as regulation of
membrane turnover and morphology, for example cell
migration or endocytosis. Moreover, it is involved in neuron-
specific functions including, for example, synaptic plasticity,
axonal outgrowth or transmitter release.
8–11
In neurological
diseases, deregulated CDK5 turned out to be an apical
instigator of neuronal cell death cascades. Amyloid-toxicity
was shown to induce calpain-mediated cleavage of the
CDK5 activators p35 or p39 to p25 and p29. This leads to
redistribution and overactivation of CDK5 after its association
with p25/p29.
12,13
Similarly, deregulation of CDK5, as well as
neuroprotection by CDK5 inhibition, was demonstrated in
many other models for neuronal cell death.
14–17
CDK5 acts early in the cell death cascade before the onset
of mitochondrial dysfunction, and CDK5 inhibition prevents
the decline of the mitochondrial transmembrane potential.
16
However, CDK5 does not directly translocate to mitochon-
dria,
16
and it remained largely unclear how CDK5 deregulation
can be integrated into longer-established ‘classical’ mitochon-
drial cell death pathways.
We delineate CDK5 as an upstream regulator of mito-
chondrial fission. The change in mitochondrial shape takes
place within minutes, and with a similar time course
independent of the proapoptotic stimulus. We give evidence
that mitochondrial scission is necessary for neuronal apopto-
sis, and show that it contributes to CDK5 neurotoxicity.
Thereby, we provide a link between CDK5 deregulation and
mitochondrial apoptosis pathways.
Results
Early mitochondrial fission is necessary for neuronal
apoptosis. Neuronally differentiated dopaminergic CSM14.1
Received 01.3.06; revised 11.10.06; accepted 08.11.06; Edited by M Piacentini; published online 12.1.07
1
Department of Neurology, University Hospital Go
¨
ttingen, Go
¨
ttingen, Germany;
2
Mitochondrial Structure and Dynamics Group, Department NanoBiophotonics, Max-
Planck-Institute for Biophysical Chemistry, Am Fassberg, Go
¨
ttingen, Germany;
3
Institute for Microbiology and Genetics, University of Go
¨
ttingen, Grisebachstrae,
Go
¨
ttingen, Germany and
4
DFG Research Center of the Molecular Physiologie of the Brain (CMPB)
*Corresponding author: M Ba
¨
hr, Department of Neurology, University Hospital Go
¨
ttingen, Robert-Koch-Str. 40, 37075 Go
¨
ttingen, Germany. Tel: þ 49 551 39 6603;
Fax: þ 49 551 39 14302; E-mail: mbaehr@gwdg.de
Keywords: mitochondrial fission; neuroprotection; cyclin-dependent kinase; neuronal apoptosis; cyclin-dependent kinase inhibitors; neurodegeneration
Abbreviations: CDK5, cyclin-dependent kinase 5; dCSM cells, neuronally differentiated CSM14.1 cells; Drp1, dynamin-related protein 1; dnDrp1, dominant-negative
mutant of dynamin-related protein 1; MPP
þ
, 1-methyl-4-phenylpyridinium ion
Cell Death and Differentiation (2007) 14, 651–661
&
2007 Nature Publishing Group All rights reserved 1350-9047/07
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Page 1
cells (dCSM cells; see also material and methods) and cultured
rat primary midbrain neurons were transfected with the
fluorescent protein dsRed2 fused to a mitochondrial targeting
signal (mito-dsRed2). This resulted in a fluorescent mito-
chondria-specific labelling, showing that the vast majority of
neuronal cells contained tubular or interconnected mito-
chondria under control conditions (Figure 1a; Supplementary
Figure 1a). Time-course of mitochondrial morphology after
apoptosis induction was then studied by time-lapse micro-
scopy. Several cell death inducing agents were tested:
Staurosporine, 1-methyl-4-phenylpyridinium ion (MPP
þ
),
which is the active metabolite of the neurotoxin MPTP used
for in vivo Parkinson’s disease models, and the calcium
ionophore A23187. A rapid decline in mean mitochondrial
length was observed in dCSM cells within minutes after cell
death induction (Figure 1a and b). Moreover, the decay of
mitochondrial length followed very similar kinetics, independent
of the proapoptotic stimulus, and was similarly found also in rat
primary neuronal midbrain cultures (Figure 1a and b). Time-
lapse analysis of individual mitochondria confirmed that the
observed decrease in mean mitochondrial length was due to
mitochondrial fission events, and not caused by shortening or
contraction of individual mitochondria (see also Supplementary
movie available online on Cell Death and Differentiation
web site). Demonstrating the specific involvement of the
mitochondrial fission machinery, mitochondrial fission was
antagonized by co-transfection of a dominant-negative mutant
of the mitochondrial fission protein Drp1 (Drp1
K38A
, tagged with
ECFP; Figure 1b).
Our videomicroscopy data were confirmed by counting the
proportion of cells that displayed mitochondrial fragmentation
(defined by at least 50% punctuate mitochondria), as in dCSM
cells we observed a several fold increase in neuronal cells
with fragmented mitochondria already 30 min after pro-
apoptotic treatment (Supplementary Figure 1c), that could
be attenuated by Drp1
K38A
. Moreover, similar findings were
made with primary neuronal midbrain cultures (Supplemen-
tary Figure 1d).
Showing a functional contribution of mitochondrial fission
to neuronal apoptosis cascades, we found that the fission
inhibiting Drp1
K38A
-ECFP substantially reduced staurosporine-
induced cell death of both dCSM cells (Figure 1c) and rat
primary midbrain neurons (Figure 1d). However, although
necessary for neuronal cell death, mitochondrial fission
was not sufficient to induce apoptosis, as expression of
Fis1-EGFP resulted only in marginal cell death (about 10%)
without proapoptotic treatment, and did not increase the
sensitivity of dCSM cells to staurosporine-induced apoptosis
(Figure 1c), regardless of more than 90% Fis1-induced
mitochondrial fission within 24 h after transfection (Supple-
mentary Figure 1c).
Nuclear condensation and fragmentation are observed
in the final execution phase of neuronal cell death. Thus,
we asked whether Drp1
K38A
would influence the release
of mitochondrial cytochrome c, a hallmark of the earlier
mitochondrial phase of apoptosis. To be able to judge
cytochrome c distribution within single dCSM cells, we
performed immunocytochemistry using an antibody directed
against cytochrome c. While cytochrome c immunoreactivity
was clearly confined to a mitochondrial distribution in
most control neurons, it changed to a diffuse cytoplasmatic
pattern when staurosporine was added (Figure 1e–g),
which was clearly prevented by Drp1
K38A
-ECFP expression
(Figure 1e–g).
Similar to Drp1
K38A
, the Bcl-2 protein family member Bcl-xL
also interferes with apoptotic cytochrome c release and
has strong anti-apoptotic effects in neurons, acting at the
level of mitochondria.
18
We thus compared Bcl-xL with
Drp1
K38A
, and asked whether Bcl-xL might, directly or
indirectly, have similar effects on mitochondrial morphology
as well. dCSM cells were treated with recombinant Bcl-xL
fused to a Tat protein transduction domain (Tat-Bcl-xL), which
is delivered intracellularly and acts anti-apoptotically as
shown before.
19
Despite the expected anti-apoptotic effect
and prevention of cytochrome c release (Figure 2a and b),
Tat-Bcl-xL did not alter mitochondrial morphology (Figure 2c),
suggesting that Bcl-xL acted downstream or in parallel to the
mitochondrial fission machinery. This finding demonstrates
that preventing mitochondrial dysfunction is not sufficient to
prevent mitochondrial fission.
CDK5 induces caspase-independent mitochondrial
fission. The search for cellular functions upstream of
apoptotic mitochondrial fission in neurons was the primary
aim of our study. Parts of the mitochondrial fission machinery
colocalizes with cytoskeletal structures, and translocation of
Figure 1 . Early mitochondrial fission is necessary for neuronal apoptosis. In order to stain mitochondria, neuronally differentiated CSM cells (dCSM cells) or rat primary
midbrain neurons were transfected with mitochondrially targeted dsRed2. (a) Time-lapse videomicroscopy showing mitochondrial fragmentation in a representative dCSM cell
upon MPP
þ
treatment (upper panels), or a neuron in primary neuronal midbrain cultures upon STS treatment (1 mM; lower panels). (b) Quantification of time-lapse
videomicroscopy reveals mitochondrial fission starting within minutes after treatment with staurosporine, MPP
þ
or the calcium ionophore A23187. Kinetics of mitochondrial
fragmentation was found to be very similar independent of the proapoptotic stimulus. Fragmentation was attenuated by overexpression of Drp1
K38A
-ECFP (Drp1
K38A
). In (b),
mitochondrial length was standardized to values of untreated control cells. (c, d) Staurosporine treatment (STS; 1 mM) for up to 12 h induced nuclear fragmentation (DAPI
staining). Cotransfection with Drp1
K38A
-ECFP prevented nuclear signs of apoptosis. Apoptosis was quantified at the indicated time points after staurosporine treatment as
percentage of transfected cells with pyknotic or fragmented nuclei. Drp1
K38A
-ECFP protected both staurosporine-treated dCSM cells (c) and cultured primary midbrain
neurons (d). (e-g) Drp1
K38A
-ECFP prevents apoptotic cytochrome c release in neurons. Neuronal dCSM cells were stained with a cytochrome c antibody and a Cy3-coupled
secondary antibody. Cytochrome c immunoreactivity was clearly confined to a mitochondrial distribution in most control cells (e; upper panel). After staurosporine treatment
(STS), a change in cytochrome c staining to a diffuse pattern was observed (e, middle panel), which was prevented by Drp1
K38A
-ECFP expression (e, lower panel). Almost all
staurosporine-treated cells expressing Drp1
K38A
-ECFP after transient transfection (arrows in f, right panel) displayed a mitochondrial cytochrome c distribution (visible in the
Cy3 channel shown in the left panel of e; arrows), while lack of Drp1
K38A
-ECFP expression was always accompanied by cytochrome c release in the same culture well (f,
arrowheads). (g) Quantification of staurosporine-induced cytochrome c release in transfected dCSM cells, which is significantly reduced by Drp1
K38A
-ECFP expression.
Expression of the fluorophore-tagged Drp1
K38A
(Drp1
K38A
-ECFP) was verified by respective fluorescence for each cell included in the analysis. Control cells were transfected
with EGFP only. (*): Po0.05 as determined by ANOVA followed by Student–Newman–Keuls test (b) or two-tailed Student’s t-test (c, d, g)
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Drp1 to mitochondria seems to be actin-dependent.
20,4
CDK5 is known to contribute to the regulation of the actin
and tubulin cytoskeleton, as well as regulation of membrane
turnover and morphology, for example cell migration or
endocytosis.
8–10
Altogether, this led us to hypothesize that
CDK5, which is an upstream instigator of neuronal demise in
various neuronal cell death models, might participate in a
signal transduction pathway that links proapoptotic signals to
the dynamic changes of mitochondrial morphology we had
observed.
Consequently, we co-transfected dCSM cells and primary
midbrain neurons with EGFP-tagged CDK5 and p25 or p35.
P25 is the toxic proteolytic product of the physiological CDK5
activator p35, which results in overactivation and subcellular
redistribution of CDK5.
Supporting our hypothesis of an involvement of CDK5 in the
regulation of mitochondrial fission, overexpression of p35/
CDK5, and even more pronounced expression of p25/CDK5,
was sufficient to induce mitochondrial fragmentation both in
dCSM cells and cultured primary midbrain neurons (Figure
3a–c). Moreover, p25/CDK5-induced mitochondrial fission
depended on the above described fission machinery, because
co-expression of the dominant-negatively acting Drp1
K38A
-
ECFP abolished p25/CDK5-induced mitochondrial fission,
both in neuronal dCSM cells and primary neuronal midbrain
cultures (Figure 3a–c). As expected based on its known
neurotoxic effect, p25/CDK5 expression, induced apoptosis
in transfected cells, which could also be suppressed by
co-transfection with Drp1
K38A
-ECFP (Figure 3d and e).
CDK5 initiates neuronal apoptosis cascades, including
activation of caspases, of which most are acting downstream
of mitochondrial dysfunction. As expected, the pan-caspase
inhibitor zVAD-fmk blocked CDK5-initiated apoptosis
(Figure 3d). However, the mitochondrial fission promoting
effect of p25/CDK5 was not significantly reduced by caspase
inhibition, demonstrating that the profission effect of CDK5 is
not mediated by caspases (Figure 3b), and general anti-
apoptotic treatments do not prevent mitochondrial fission.
Staurosporine- and A23187-induced mitochondrial
fission is CDK5-dependent. We next asked whether
CDK5 also contributes to mitochondrial fission when cell
death is not induced by direct upregulation of CDK5 activity
as in the experiments described above, but by other
apoptotic stimuli that induce mitochondrial fission. Before
investigating this question using the staurosporine-treated
dCSM cells, we verified that CDK5 indeed contributed to
cell death in staurosporine-induced apoptosis of dCSM cells.
We confirmed that CDK5 and one of its neuronal activators,
p39, were expressed in dCSM cells as assessed by Western
blot analysis (data not shown). CDK5 activity measured
by a CDK5 kinase assay
16,12
increased upon staurosporine
treatment (Figure 4a). Moreover, expression of a dominant-
negatively acting CDK5 mutant (CDK5N
144
-EGFP), or
treatment with the highly potent CDK5 inhibitor indolinone
A
16
blocked cell death (Figure 4b) as well as caspase3/7
activation (Figure 4c).
Analysis of non-apoptotic control cultures did not reveal a
significant effect of CDK5 inhibition on mean mitochondrial
length in dCSM cells. Mean mitochondrial length was
2.3370.15, 2.0170.10 and 2.0070.18 mm in control-
transfected cells or cells transfected with wild-type CDK5-
EGFP or CDK5N
144
-EGFP, respectively. As a positive
control, Drp1
K38A
-ECFP was transfected, which resulted in a
Figure 2 Bcl-xL is neuroprotective, but does not influence mitochondrial fission.
Staurosporine-treated (1 mM for 2 h) dCSM cell cultures had a significantly
increased proportion of cells with apoptotic nucleus (a), released cytochrome c (b;
determined by cytochrome c immunohistochemistry as in Figure 1) as well as cells
with predominantly fragmented mitochondria (c). Concomitant treatment with the
cell permeable Tat-Bcl-xL fusion protein protected from staurosporine-induced cell
death and cytochrome c release (a, b; Po0.05 compared to staurosporine
exposure alone; two-tailed Student’s t-test), but did not prevent mitochondrial
fragmentation in staurosporine-challenged cultures (c). Control: untreated or only
staurosporine-treated, respectively. Vehicle: Addition of Tat-Bcl-xL elution buffer
was used as vehicle control. n ¼ 3 independent experiments
CDK5 regulates mitochondrial fission
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Cell Death and Differentiation
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significantly increased mean mitochondrial length of
3.4870.21 mm (100 mitochondria from 10 cells per condition
analysed).
In contrast to healthy cells, we observed a strong effect of
CDK5 inhibition on mitochondrial morphology under apoptotic
conditions. Treatment with the highly specific CDK5 small
molecule inhibitor indolinone A (250 nM) reduced stauro-
sporine-induced mitochondrial fission compared to cultures
that were treated with staurosporine alone (Figure 5a).
Moreover, serial live cell images (Figure 5b and c) as well as
blinded counting of the proportion of cells that displayed
predominantly punctuate mitochondria (Figure 5d) confirmed
that indolinone A substantially diminished mitochondrial
fission after both staurosporine and A23187 treatment. The
same effect could be observed in cells that expressed the
dominant-negative CDK5 mutant CDK5N
144
-EGFP (Figure
5e and f). Similarly, staurosporine-induced mitochondrial
fission that was observed in live rat primary midbrain neurons
was attenuated by CDK5N
144
expression, with an even more
pronounced effect compared to dCSM cells (Figure 5g).
Moreover, we found additional evidence that the effect of
CDK5 on mitochondrial fission is mediated by modulation of
the known fission machinery: co-expression of wild-type
Drp1-ECFP antagonized the reduction of mitochondrial
fission under indolinone A treatment or CDK5N
144
-EGFP
expression (Figure 5b, d–f). Concomitantly, downstream anti-
apoptotic effects of CDK5 inhibition could also be reversed by
Drp1 expression: We found a reduced proportion of cells with
Figure 3 Overexpression of p25/CDK5 is sufficient to induce mitochondrial fragmentation in a caspase-independent manner. Overexpression of EGFP-tagged CDK5/p25
resulted in mitochondrial fission in most transfected cells after 18 h (ac), both in dCSM cells (a, b) and cultured primary midbrain neurons (c). Additional overexpression of
Drp1
K38A
-ECFP blocked this effect (ac). (Co-) expression of the fluorophore-tagged proteins was verified by respective fluorescence for each cell included in the analysis. (d,
e) As expected, p25/CDK5 overexpression induced cell death in dCSM cells (d) and primary neuronal midbrain cultures (e) 18 h after transfection, which was also substantially
attenuated by Drp1
K38A
-ECFP. The pan-caspase inhibitor zVAD-fmk blocked CDK5-mediated apoptosis (d), but not mitochondrial fission (b). (*): Po0.05; two-tailed
Student’s t-test
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apoptotic nuclei or released cytochrome c in cells treated with
indolinone A or expressing CDK5N
144
-EGFP (Figure 6a–c).
Again, this effect was abolished by overexpression of wild-
type Drp1. It is important to note that wild-type Drp1 did not
induce mitochondrial fission or apoptosis when expressed
alone, confirming previous data in non-neuronal mammalian
cells.
1
CDK5 knockdown reduces mitochondrial fission. In the
concentration applied in our experiments, Indolinone A is a
highly specific CDK5 inhibitor.
16
Moreover, our data were
confirmed by the parallel use of a dominant-negative CDK5
mutant. Nevertheless, an unspecific effect of indolinone A or
dnCDK5 on kinases other than CDK5 cannot be completely
excluded, although identical results from the small molecule
CDK5 inhibitor and the dominant-negative CDK5 mutant
make artefacts by unspecific co-inhibition of other kinases
extremely unlikely. However, in order to further strengthen
the central claim of our study, we performed CDK5
knockdown experiments using RNAi. Confirming our
previous results, knockdown of CDK5 protein by RNAi
(Figure 7a) clearly attenuated apoptotic mitochondrial
fission (Figure 7b). Moreover, reminiscent of our data
obtained with CDK5 RNAi in neuronal cells, a yeast
deletion strain lacking pho85, the yeast orthologue of
CDK5, displayed a reduced proportion of fragmented
mitochondria (Supplementary Figure 2).
Discussion
In a systematic time-course analysis, we found that mitochon-
drial fragmentation occurred early during neuronal apoptosis,
and was largely invariant, regardless of the fission-inducing
stimulus. This is reminiscent of the constant time period
required for complete apoptotic cytochrome c release, which
is independent of the mode or strength of the apoptotic
stimulus.
21
Most likely, both mitochondrial fission and release
of mitochondrial cytochrome c are basic mechanisms of
cell death with conserved, invariant time dynamics. Our
findings in apoptotic models are in line with one earlier report
that describes mitochondrial fission in neurons exposed
to excitotoxic conditions.
22
Consistent with few recent studies
that demonstrated the relevance of mitochondrial fission
for apoptosis of non-neuronal mitotic cells
23–26
our study
provides evidence that mitochondrial fission is not a mere
epiphenomenon of neuronal cell death, but that blocking
mitochondrial fission is indeed protective for neurons. How-
ever, our data obtained from Fis1-EGFP overexpression
exemplify that induction of mitochondrial fission, although
necessary, is not sufficient to induce apoptosis. Similarly,
experiments using Bcl-xL showed that apoptosis can at least
Figure 4 CDK5 activity is involved in staurosporine-induced death of dCSM
cells. (a) Staurosporine treatment induced an early transient increase in CDK5
activity in neuronal dCSM cells. Histone H1 was used as CDK5 substrate. LC:
Loading control demonstrating equal amounts of CDK5 protein. The loading control
was performed by Coomassie staining of the same SDS gel that was subsequently
used for autoradiographic detection of CDK5 activity (see Materials and methods).
Staurosporine-induced cell death (b) and caspase-3/7 activity (c) could be blocked
by a dominant-negative CDK5 mutant (CDK5N
144
-EGFP) or the CDK5 inhibitor
indolinone A (250 nM), respectively. In (b), the percentage of cells with apoptotic
nuclei refers to the population of CDK5N
144
-EGFP transfected cells. (*): Po0.05;
two-tailed Student’s t-test
Figure 5 Inhibition of CDK5 attenuates apoptotic mitochondrial fragmentation. (a) Treatment with the CDK5 inhibitor indolinone A prevents mitochondrial fragmentation
upon staurosporine-treatment (STS). dCSM cells were treated with STS for 2 h (1 mM; left panels). Right panels show preserved mitochondrial fragmentation under
concomitant treatment with indolinone A (250 nM). Note that the CDK5 inhibitor did also prevent apoptotic nuclear condensation as shown by DAPI labelling. (b, c) Quantitative
time-lapse microscopy confirmed that indolinone A (250 nM) reduced staurosporine- (b) or A23187-induced (c) mitochondrial fission. The effect of indolinone A could be
antagonized by co-expression with wild-type Drp1 (b). Mitochondrial length was standardized to untreated control cells. (d) The effect of indolinone A was confirmed by blinded
counting of cells displaying predominantly punctuate mitochondria. Indolinone A reduced the percentage of dCSM cells with fragmented mitochondria at different time points
after stauroporine treatment, which was again antagonized by co-expression with wild-type Drp1. (e, f) Effects comparable to indolinone A treatment were observed in cells
expressing the dominant-negative CDK5 mutant CDK5N
144
-EGFP (e: time-lapse microscopy; f: blinded counting of cells with fragmented mitochondria). Also here, the effect
of CDK5N
144
could be antagonized by co-expression with wild-type Drp1 (Drp1; Drp1-ECFP). Note that Drp1 expression did not induce mitochondrial fragmentation by itself.
(Co-) expression of CDK5N
144
-EGFP and Drp1-ECFP protein was verified by EGFP and ECFP fluorescence for each cell included in the analysis. (g) Similar to the results
obtained in the neuronal dCSM cells, staurosporine-induced mitochondrial fission of cultured primary midbrain neurons was inhibited by CDK5N
144
-EGFP, as shown by
quantification of time-lapse microscopy data. (*): Po0.05 as determined by ANOVA followed by Student–Newman–Keuls test (b, c, g) or two tailed student’s t test (d, f)
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temporarily be blocked without preventing mitochondrial
fission.
After we had shown the requirement of mitochondrial fission
for neuronal apoptosis, we proceeded to study the involve-
ment of CDK5 in apoptotic mitochondrial fission, the main
focus of our study. Disruption of the actin cytoskeleton
precludes mitochondrial fission in non-neuronal cell lines
4
and neurons (our unpublished observation). The findings of an
implication of cytoskelettal proteins in mitochondrial fission
prompted us to examine whether CDK5 was a modulator of
mitochondrial fission in neurons. CDK5 is an apical instigator
in many neuronal cell death cascades
15,14,27,17
and phos-
phorylates various proteins implicated in cytoskeletal organi-
zation.
28,29
In addition, CDK5 has previously been shown
to be implicated in various cellular events involving turnover
of biological membranes, for example cell migration, axonal
outgrowth or endocytosis.
10,27,11
These cellular functions also
require cytoskelettal structures, and we considered that, albeit
a simplifying view, the process of mitochondrial fission shares
mechanistic similarities with the cleavage of cell membranes
during ‘pinching off’ of endocytotic vesicles.
Although it is known that CDK5 acts upstream of mito-
chondrial dysfunction in neuronal cell death paradigms,
16
Figure 6 Neuroprotective effects of CDK5 inhibition are reversed by Drp1.
Indolinone A treatment (a) or CDK5N
144
-EGFP expression (b) prevented nuclear
signs of apoptosis at different time points after stauroporine treatment. This effect
could be antagonized by co-expression of wild-type Drp1. Importantly, Drp1 did not
induce apoptosis by itself. (c) CDK5N
144
-EGFP expression suppressed cytochrome
c release in dCSM cells after 2 h staurosporine exposure (STS; 1 mM). This effect
was abolished by co-expression of wild-type Drp1. Percentage values represent the
proportion of cells with diffuse cytoplasmatic cytochrome c staining compared to
cells with labeling restricted to mitochondria (see also Figure 2). (*): Po0.05; two
tailed students t-test
Figure 7 (a) Western blot demonstrating specific downregulation of the
endogenous CDK5 protein. dCSM cells were transfected with 33 nM anti-CDK5
siRNA or for control condition with 33 nM anti-EGFP siRNA. At 3 days after
transfection cells were lysed and Western blots were performed. All lanes were
loaded with the same amount of total protein as indicated by b-tubulin detection. (b)
Quantification of time-lapse microscopy shows mitochondrial fragmentation induced
by staurosporine treatment (1 mM) is prevented by CDK5 siRNA. Mitochondrial
length was standardized to untreated control cells
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Cell Death and Differentiation
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it remained unclear how CDK5 could be conceptually
integrated in established mitochondrial cell death cascades.
Our data not only delineate the first signal transduction kinase
regulating cell death-associated mitochondrial fission in
mammalian cells, but also link CDK5 to ‘classical’ apoptosis
pathways with mitochondrial disintegration and subsequent
cytochrome c release.
We have not identified here the direct substrate of CDK5
that is relevant for mitochondrial fission. The pivotal direct
CDK5 targets to induce mitochondrial fission could well be
among already known CDK5 substrates, possibly including
components of the cytoskeleton. In our study, we show that
the effect of CDK5 is at least partially mediated via the known
fission protein Drp1, implicating the mitochondrial fission
machinery in the observed changes in mitochondrial morpho-
logy. Intriguingly, CDK5 has previously been demonstrated
to play a critical role in synaptic vesicle endocytosis by
phosphorylation of dynamin 1.
11
Similarly, the dynamin-
related protein 1 (Drp1) may be directly modulated by
CDK5. However, CDK5 could also indirectly regulate
Drp1 function or localization by phosphorylation of one or
several other CDK5 substrates, for example via cytoskeletal
alterations.
Furthermore, reduced mitochondrial fission under CDK5
inhibitory treatment cannot be explained as a secondary effect
due to general preservation of mitochondrial function.
Supporting this view, the anti-apoptotic protein Bcl-xL, despite
its well-established protective effects at the mitochondrial
level, does not prevent mitochondrial fission (James et al.
this study). In contrast to Bcl-xL, Bcl-2 was shown to block
mitochondrial fission.
30
However, in this case a direct
interaction of Bcl-2 with the fission machinery, and not the
general preservation of mitochondrial functions, was shown to
be responsible for the reduction in mitochondrial fission.
30
Finally, we found that caspase-inhibition with a pan-caspase
inhibitor prevented cell death, but not mitochondrial fission
induced by p25/CDK5. This further excludes unspecific
effects of anti-apoptotic treatments on mitochondrial fission,
and shows that CDK5-induced fission is caspase-indepen-
dent.
In principle, increased fusion instead of decreased
fission could explain the effect of CDK5 inhibition on
mitochondrial shape. However, the effect of CDK5 both on
mitochondrial shape and on apoptosis was mediated via the
mitochondrial fission protein Drp1. As Drp1 is only involved
in mitochondrial fission, but not in fusion, we can conclude that
the protective effects of CDK5 inhibition are mediated via the
mitochondrial fission machinery.
Based on our current knowledge, it is possible that apart
from CDK5 additional upstream signal transduction pathways
may also contribute to the regulation of apoptotic mitochon-
drial fission in neurons. Vice versa, depending on the
cell death paradigm, additional mechanisms other than
mitochondrial fission are likely to contribute to CDK5 toxicity.
For instance, nuclear translocation of CDK5 and a nuclear
pathway leading to inactivation of the protective transcription
factor MEF2 by nuclear CDK5 has recently been described.
31
Accordingly, nuclear CDK5 toxicity could have at least
contributed to CDK5-mediated cell death in our paradigm,
although inhibition of specifically nuclear CDK5 activity was
protective in excitotoxic cell death, but not, for example, in the
purely apoptotic model of camptothecin-induced neuronal cell
death.
31
Moreover, while the EGFP tag itself results in partial
nuclear localization of respective fusion proteins, we never
observed an increase in nuclear EGFP-tagged CDK5 or p25
in our paradigm, and always noted a substantial degree of
cytoplasmatic localization for both proteins (data not shown).
The fact that also the only cytoplasmatically localized
p35/CDK5 resulted in mitochondrial fission is in agreement
with the cytoplasmatic CDK5 activity directly influencing mito-
chondrial morphology. However, this does not conflict with
concepts of nuclear mechanisms of CDK5 toxicity, for
example inactivation of the transcription factor MEF2, as both
effects may contribute to neuronal cell death at the same time.
The observation that also the cytosolic physiological
p35/CDK5 resulted in mitochondrial fragmentation suggests
that p35/CDK5 activity may also contribute to mitochondrial
fission occurring under several physiological circumstances
which are not accompanied by p25 generation and cell death,
for example during cell cycle.
Similar to our observations on CDK5 inhibition in neurons,
lack of its orthologue Pho85
40
resulted in less fragmented
mitochondria in Saccharomyces cerevisiae. Our findings are
principally intriguing in light of the conserved functions of
Pho85/CDK5 that have been identified so far: similar to CDK5,
Pho85 contributes to organization of the actin cytoskeleton in
yeast,
32,33
and plays a role in the cellular stress response.
34,35
The fact that CDK5 inhibition and Pho85 deletion resulted
in parallel effects on mitochondrial morphology could be an
indication for at least partially conserved kinase pathways
modulating mitochondrial morphology. However, despite
principally similar effects on mitochondrial morphology, one
also has to emphasize that there are profound differences
between yeast and higher eukaryotes, for example concern-
ing their cytoskeleton.
Recapitulating, we show that mitochondrial fragmentation
is necessary for neuronal apoptosis and takes place within
minutes after induction of apoptosis with similar kinetics,
independent of the type of proapoptotic treatment. We
identified CDK5 as a signal transduction kinase modulating
mitochondrial fission, integrating CDK5 into established
neuronal apoptosis pathways. Vice versa, we show that
mitochondrial fission is required and a mediator for neurotoxic
CDK5 action, although basal p35/CDK5 activity may also
contribute to mitochondrial fission under physiological circum-
stances. Thus, our study contributes to the understanding
of CDK5 action in neuronal cell death cascades, but also
highlights the mitochondrial fission machinery as a potential
target for therapeutic approaches against neurodegenerative
diseases.
Materials and Methods
Chemical reagents. Cell culture reagents were purchased from PAA
Laboratories. Staurosporine, nocodazole and MPP
þ
were obtained from Sigma-
Aldrich (Co
¨
lbe, Germany), A23187 from Alexis (Gru
¨
nberg, Germany). Z-VAD-FMK
were obtained from Bachem (Weil am Rhein). Indolinone A was provided by
Boehringer Ingelheim Pharma KG, Ingelheim, Germany.
Expression vectors and Tat-Bcl-xL fusion protein. Expression
plasmids for mito-dsRed2 and EGFP-N
2
were purchased from Clontech, hFis1-
EGFP and Drp1
K38A
-ECFP were kind gifts from J-C Martinou
,
constructs for
CDK5 regulates mitochondrial fission
K Meuer et al
659
Cell Death and Differentiation
Page 9
CDK5N
144
-EGFP and p25-EGFP were generously provided by L-H Tsai. Tat-Bcl-xL
was expressed and the fusion protein purified as described previously.
19
Cell culture and transfection. CSM14.1 cells are derived from the ventral
mesencephalic region of an E14 rat, and immortalized with the temperature-
sensitive Large T antigen.
36
When cultured at 331C (permissive temperature),
CSM14.1 cells express the large T antigen, proliferate, have a flat neuroepithelial-
like morphology and express the neural stem cell marker nestin.
36
When transferred
to 391C, the temperature-sensitive large T antigen is inactivated and proliferation
ceases. Moreover, CSM14.1 cells acquire a neuron-like morphology, extend
neurites, and differentiate into neuronal cells expressing the dopaminergic markers
Nurr1, TH and ALDH2. Grafted differentiated CSM14.1 cells have been successfully
used to alleviate symptoms in hemiparkinsonian animals.
37
For our study, CSM14.1
cells were cultured and differentiated for 6–8 weeks in complete DMEM
supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and
streptomycin at 391Cin5%CO
2
(called dCSM cells in this paper).
For time-lapse microscopy and cell death assays, dCSM cells were grown in
0.8 cm
2
chamber slides (eight-well Lab-Tek chambered cover glass system; Nalge
Nunc, Naperville, IL). Cells (2 10
6
per well) were transfected with plasmid DNA
using the Amaxa electroporation system according to the manufacturer’s
instructions (kit primary neurons, program 0-03).
Mitochondria were visualized by transfection with mito-dsRed2. Co-labelling with
the small molecule mitochondrial stain mitotracker green confirmed the specific
mitochondrial distribution of the dsRed2 (data not shown). As CDK5N
144
, Drp1
K38A
,
p25 or Fis1 were fluorophore-tagged (ECFP or EGFP), we cotransfected EGFP
along with mito-dsRed2 in control conditions. 24 h after transfection respective
compounds were applied at the following concentrations: 1 mm staurosporine,
50 nM A23187, 1 mM MPP
þ
, 250 nM indolinone A and 500 nM Tat-Bcl-xL.
siRNA transfection. Anti-CDK5 siRNA was chemically synthesized by Qiagen
(Hilden, Germany) and Cy3 conjugated at the 5
0
end. The following sequence was
used: anti-CDK5-sense 5
0
GAG GAU CUU UCG ACU GCU A-3
0
, anti-CDK5-
antisense 5
0
UAG CAG UCG AAA GAU CCU C-3
0
. Anti-EGFP siRNA was also
chemically synthesized by Qiagen (Hilden, Germany) and had the following
sequences: anti-EGFP-sense 5
0
GCA AGC UGA CCC UGA AGU UCA U-3
0
, anti-
EGFP-antisense 5
0
GAA CUU CAG GGU CAG CUU GCC G-3
0
. Anti-EGFP siRNA
was used as a control. Lyophilized siRNA was reconstituted following the
manufacturer’s instructions. For targeting of endogenous genes by cationic lipid-
mediated transfection, siRNA was complexed with Lipofectamine 2000 (Invitrogen,
Karlsruhe, Germany) according to manufacturer’s instructions. Per well of a 24-well
culture plate, 1 ml Lipofectamine 2000 was diluted in 50 ml Opti-Mem
s
I Reduced
serum medium and combine with 20 pmol siRNA (resulting in a final concentration
of 33 nM) diluted in 50 ml Opti-Mem
s
I Reduced serum medium after 5 min of
incubation at room temperature. The formulation was continued for 20 min at room
temperature and the mixture was applied to the culture wells. Three days after
transfection protein lysates for Western blot analysis or time lapse videomicroscopy
were performed.
Western blot analysis. For preparation of protein lysates, dCSM14.1 were
plated on six-well plates. After 80–90% confluence was reached, cells were lysed in
lysis buffer
16
on ice for 15 min, and cell debris was pelleted at 13 000 g for 30 min.
Western blotting was performed as described earlier.
16
CDK5 (C-8) and p35 (C-19)
antibodies (both rabbit) were purchased from Santa Cruz. P39 antibody (polyclonal,
rabbit) was kindly provided by L-H Tsai.
Primary midbrain neuron cultures and transfection. The mesen-
cephalic floor plate was dissected from E14 Wistar rat embryos and further
processed for establishing dissociated cell cultures as previously described.
38
For
DNA transfection, cell pellets consisting of 2 10
6
cells each were resuspended in
electroporation medium (Amaxa biosystems; Frankfurt) and 2 mg of plasmid DNA
was added to the solution. The primary neuron solution was then transfected using
the Nucleofector device (Amaxa biosystems; Frankfurt) according to manufacturer’s
instructions. Transfected cells were seeded on poly-L-ornithine/laminin (Sigma)-
coated eight-well chamber slides at a density of 175 000 cells/cm
2
. Cultures were
maintained at 371C in a humidified atmosphere and 5% CO
2
in DMEM/F12 plus the
N1 supplements and antibiotics for 2–4 days. Neurons were identified by their
typical morphology and axonal processes.
Analysis of mitochondrial fragmentation in neurons. Eight-well
chamber slides that allow the parallel imaging of four separately transfected cell
populations of the same preparation and under identical experimental condition
were used throughout the study. At 24 h after transfection and at least 30 min
before application of cell death inductors (staurosporine, MPP
þ
, A23187) time-
lapse images were collected at intervals of 1–15 min. Cells were incubated in a
microscope climate chamber for live cell imaging (371C, 5% CO
2
) on a Zeiss
Axioplan inverted microscope (Carl Zeiss). A 63 1.4 NA oil immersion objective
(Carl Zeiss) was used, and images captured using a CCD camera (Carl Zeiss).
Mitochondrial length was measured using Axiovision software. At least 15
randomly chosen mitochondria per cell from different cytoplasmatic regions were
measured in mm, and then standardized to and given as percentage compared to
untreated control cells. Data were obtained from time-lapse videomicroscopic
pictures of at least 5–10 cells from different preparations per condition. In further
experiments, mitochondria of fixed cells were scored as normal (tubular, elongated)
or fragmented (450% of the mitochondria in a given cell appeared punctuate), and
results expressed as percentage of cells with predominantly fragmented
mitochondria.
Cell death assay. At 24 h after transfection with respective expression
plasmids cells were treated with staurosporine (1 mM) and fixed with 4% para-
formaldehyde/PBS. For experiments using Tat-Bcl-xL, cells were preincubated for
2 h with Tat-Bcl-xL before induction of cell death. For experiments using zVAD-fmk
(100 mM), cells were preincubated over night with zVAD-fmk before induction of cell
death. Nuclei were stained with DAPI and imaged with a Zeiss fluorescent
microscope. Cells were scored as normal or apoptotic (i.e. pyknotic or fragmented)
nuclei. At least 200 transfected cells from at least 3 independent wells per condition
were counted and values calculated as percentage of cells with apoptotic nuclei
compared total cell counts.
CDK5 activity assay. dCSM14.1 were plated on 15 cm dishes. After 80–90%
confluence was reached, cells were incubated with staurosporine [1 mM] for 1, 5, 10,
30 min. After incubation with staurosporine cells were lysed in lysis buffer on ice
for 15 min, and cell debris was pelleted at 13 000 g for 30 min. 50% bead slurry
(30 ml) were added to 500 mg of protein lysate for 1 h at 41C to remove proteins
unspecifically binding to the beads. The beads were then collected by centrifugation,
the pellet was discarded and the supernatant was incubated with 10 ml of CDK5
(Santa Cruz, C-8) antibody for 1 h at 41C followed by incubation with another 30 ml
beads. The beads were collected by centrifugation and washed three times with
lysis buffer and three times with kinase buffer (10 mM MgCl
2
, 50 mM Hepes, 1 mM
DTT, 1 mM ATP). Washed beads were incubated with 10 mg histone H1 (Sigma,
Steinberg, Germany). The reaction was initiated at 301C after addition of
32
P-
labeled g-ATP (Amersham, Uppsala, Sweden), allowed to proceed for 30 min at RT,
and stopped by addition of sample buffer and heating to 951C for 5 min. After SDS-
PAGE, gels were fixed with 40% (v/v) ethanol, 10% (v/v) acetic acid for 60 min. After
two washing steps with water for 10 min, gels were stained with 80% colloidal
coomassie (0,1% (w/v) Coomassie Brillant Blue G250, 2% (w/v) ortho-phoshoric
acid, 10% (w/v) ammoniumsulfat) and 20% (v/v) methanol for 1 h at RT. Gels were
destained in 1% (v/v) acetic acid, dried and subjected to Phosphoimager-analysis.
Immunocytochemistry. Cells grown on glass coverslips were fixed with 4%
paraformaldehyde/PBS and subsequently permeabilized with 0.1% Triton X-100 for
20 min. To block unspecific immunoreactivity, cells were then incubated with PBS
containing 5% NGS and 1% BSA for 30 min. Anti-cytochrome c mouse monoclonal
antibody (PharMingen) was used at 1 : 1000 dilution in PBS. The secondary Cy3-
labelled antibody was obtained from Dianova and used at 1 : 1000 dilution.
Analysis of mitochondrial fragmentation in yeast. Growth and
manipulation of yeast was carried out according to standard procedures. The wild-
type strain BY4741 and the isogenic pho85 deletion strain were obtained from
Euroscarf (Frankfurt, Germany). The disruption was confirmed by polymerase chain
reaction.
To label the mitochondrial matrix with GFP, the cells were transformed with the
plasmid pVT100U-mtGFP.
39
Cells were grown in SC medium with 2% glucose at
301C to logarithmic growth phase. For imaging and phenotypic analysis cells were
chemically fixed in 10% formaldehyde for 10 min. Mitochondrial phenotypes were
counted in blinded experiments by two observers. Each experiment, comprising
more than 100 analyzed cells per strain, was repeated at least six times.
CDK5 regulates mitochondrial fission
K Meuer et al
660
Cell Death and Differentiation
Page 10
Statistical analysis. All values are expressed as mean7S.E.M. The two-
sided t-test, or ANOVA followed by Student–Newman–Keuls test was used to
determine significance as appropriate (NCSS Software, NCSS, Kaysville, Utah).
Acknowledgements. We thank Jean-Claude Martinou and Manuel Rojo
for the gift of Fis1 and Drp1 plasmids, and Pawel Kermer for CSM14.1 cells. p25,
p35 and CDK5 constructs as well as p39 antibody were generously provided by
Li-Huei Tsai. We also thank Christine Poser for excellent technical assistance.
We thank Stefan W Hell for his continuous support (SJ). Funded by Deutsche
Forschengsgemeinschaft through the DFG-Research Center for Molecular
Physiologie of the Brain.
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  • Source
    • "demonstrated that the absence of mitochondrial fragmentation in mutant parkin fibroblasts derived from PD patients results in accumulation of damaged mitochondria not targeted by mitophagy; such a condition could increase oxidative stress levels and lead to cellular dysfunction and death (Zanellati et al. 2015). Genetic inhibition of Drp1 or overexpression of mitofusin 1 (Mfn1), a regulator of mitochondrial fusion, prevent cell death induced by neurotoxins (Barsoum et al. 2006; Meuer et al. 2007; Gomez-Lazaro et al. 2008). Genetic or pharmacological inactivation of Drp1 can also decrease mitochondrial dysfunction and fragmentation associated with mutations in parkin and PINK1 (Lutz et al. 2009; Cui et al. 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by cardinal motor signs such as rigidity, bradykinesia or rest tremor that arise from a significant death of dopaminergic neurons. Non-dopaminergic degeneration also occurs in other brain areas, where it seems to induce the deficits in olfactory, emotional and memory functions that precede the classical motor symptoms in PD. Despite the majority of PD cases being sporadic, several genes have previously been associated with the hereditary forms of the disease. Some of these genes, including α-synuclein, DJ-1 and parkin, are modified by SUMO (small ubiquitin-like modifier), a post-translational modification that regulates a variety of cellular processes. Among the several pathogenic mechanisms proposed for PD is mitochondrial dysfunction. Recent studies suggest that SUMOylation can interfere with mitochondrial dynamics, which is essential for neuronal function, and may play a pivotal role in PD pathogenesis. Here, we present an overview of recent studies on mitochondrial disturbance in PD and the potential SUMO-modified proteins and pathways involved in this process. This article is protected by copyright. All rights reserved.
    Full-text · Article · Mar 2016 · Journal of Neurochemistry
    • "Previous studies from our group have demonstrated that the increase in cell death induced by D1R activation in mutant huntingtin striatal cells is related with an increase in the activity of the Ser/Thr kinase Cdk5 [2]. Interestingly, Cdk5 has been involved in mitochondrial dysfunction by increasing oxidative stress [28] or acting as an upstream regulator of mitochondrial fragmen- tation [21] . In this scenario, we tried to integrate both pathways hypothesizing that in mutant huntingtin striatal cells the increase in Cdk5 activity induced by D1R activation was responsible for the increase in mitochondrial fragmentation. "
    [Show abstract] [Hide abstract] ABSTRACT: The molecular mechanisms underlying striatal vulnerability in Huntington's disease (HD) are still unknown. However, growing evidence suggest that mitochondrial dysfunction could play a major role. In searching for a potential link between striatal neurodegeneration and mitochondrial defects we focused on cyclin-dependent kinase 5 (Cdk5). Here, we demonstrate that increased mitochondrial fission in mutant huntingtin striatal cells can be a consequence of Cdk5-mediated alterations in Drp1 subcellular distribution and activity since pharmacological or genetic inhibition of Cdk5 normalizes Drp1 function ameliorating mitochondrial fragmentation. Interestingly, mitochondrial defects in mutant huntingtin striatal cells can be worsened by D1 receptor activation a process also mediated by Cdk5 as down-regulation of Cdk5 activity abrogates the increase in mitochondrial fission, the translocation of Drp1 to the mitochondria and the raise of Drp1 activity induced by dopaminergic stimulation. In sum, we have demonstrated a new role for Cdk5 in HD pathology by mediating dopaminergic neurotoxicity through modulation of Drp1-induced mitochondrial fragmentation, which underscores the relevance for pharmacologic interference of Cdk5 signaling to prevent or ameliorate striatal neurodegeneration in HD. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Jul 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
  • Source
    • "Activation of Cdk5 plays an early role in the cell death cascade before the initiation of mitochondrial dysfunction, and Cdk5 inhibition prevents the mitochondrial damage and cell death caused by Prx2 inactivationmediated oxidative stress (Sun et al. 2008). Interestingly, Cdk5 also regulates mitochondrial fission during neuronal apoptosis as an upstream signaling kinase (Meuer et al. 2007). However, the precise mechanism by which Cdk5 regulates mitochondrial morphology is still unclear. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial dysfunction is implicated in age-related degenerative disorders such as Alzheimer's disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Aβ oligomers (AβOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AβOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphology-related proteins in N2a cells stably expressing the swedish mutation of amyloid precursor protein (APP), which is known to increase AβO production. We demonstrated that mitochondrial fragmentation by AβOs accompanies reduced Mfn1 and Mfn2 levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2-related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, overexpression significantly inhibits the AβO-mediated cell death pathway. Therefore, these results indicate that AβO-mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5-induced Prx2 phosphorylation.
    Full-text · Article · Oct 2014 · Journal of Neurochemistry
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