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Cdk5rap2 Exposes the Centrosomal Root of Microcephaly
Syndromes
Timothy L. Megraw, James T. Sharkey, and Richard S. Nowakowski
Department of Biomedical Sciences, Florida State University, College of Medicine, 1115 West
Call Street, Tallahassee, FL 32306
Summary
Autosomal Recessive Primary Microcephaly (MCPH) is characterized by small brain size due to
deficient neuron production in the developing cerebral cortex. While MCPH is a rare disease, the
questions surrounding its etiology strike at the core of stem cell biology. The seven genes
implicated in MCPH all encode centrosomal proteins and disruption of the MCPH gene
Cdk5rap2
in mice revealed its role in neural progenitor proliferation and in maintaining normal centriole
replication control. Here, we discuss the impact that centrosome regulation has on neural
progenitors in the developing brain. We integrate the impact of centriole replication defects with
the functions of Cdk5rap2 and other MCPH proteins, propose mechanisms for progenitor loss in
MCPH, and discuss links to two other microcephaly syndromes.
MCPH: A centrosome-based disease
The cerebral cortex is the region of the brain where learning, memory, language and other
cognitive and motor activities are controlled. Although the relative and actual size of the
cerebral cortex varies among mammals, it develops in a similar way [1]. The mouse is
therefore an appropriate model to investigate human cerebral cortex development.
Neurogenesis of the cerebral cortex requires coordinated temporal control of neural
precursor cell divisions during embryonic brain development. In the mammalian brain,
neural progenitors reside adjacent to the lateral ventricles in the ventricular and
subventricular zones. Neuronal patterning of the cerebral cortex occurs by radial migration
of new neurons outward from the ventricular and subventricular zones of the neocortex [1].
The six layers formed during corticogenesis are produced in an “inside-out” manner. The
first neurons populate the innermost layer, while those produced later migrate in the radial
direction past the early neurons to occupy the outermost layers. The ventricular zone is a
pseudostratified columnar epithelial layer surrounding the ventricle and is where early
symmetric divisions expand the apical progenitor pool. Then, between embryonic days
10-12 in the mouse, the polarity of progenitor divisions changes, becoming progressively
more asymmetric. Asymmetric progenitor divisions are neurogenic, producing a daughter
neuron or neuronal precursor and a renewed stem cell [2, 3]. Basal progenitors, produced
from asymmetric division of apical progenitors, undergo a single neurogenic division [4].
There are systematic changes in the proportion of symmetric vs. asymmetric apical
© 2011 Elsevier Ltd. All rights reserved.
Corresponding author
: Megraw, T. L. (timothy.megraw@med.fsu.edu) .
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Author Manuscript
Trends Cell Biol
. Author manuscript; available in PMC 2012 August 01.
Published in final edited form as:
Trends Cell Biol
. 2011 August ; 21(8): 470–480. doi:10.1016/j.tcb.2011.04.007.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
progenitor divisions during neocortical development [5], and control over this process is
critical for the accurate and appropriate production of neurons at the cerebral cortex [6].
Mutations in the MCPH genes reduce the population of neurons in each of the six layers of
the brain during development, yet with little or no overt structural abnormalities other than
reduced thickness of the cerebral cortex [7, 8]. MCPH individuals generally have reduced
cognitive function but no motor deficit. MCPH loci map to at least seven genes, MCPH 1-7
[7-16] (Table 1).
Surprisingly, all the defined MCPH genes encode centrosome or spindle pole proteins
(Table 1), implicating proper centrosome or mitotic microtubule organization as critical to
generating and/or maintaining normal neuron populations in the developing brain. In recent
years, mutations in an expanding number of genes involved in centriole and cilium assembly
and function have been linked to a spectrum of ciliopathies [17-19]. The revelation that
many diseases are based on defects in cilia and centrosomes has highlighted the importance
of understanding the cellular and developmental functions of centrosomes.
The cell biological cause for MCPH, whether it be aberrant cilia, mitotic centrosomes, or
other centrosomal basis has not been demonstrated. Moreover, several MCPH proteins
function in the DNA damage response pathway involving ATR (ATM and Rad3-related).
Thus, there are two common threads emerging in MCPH disease: the centrosome and DNA
damage response. How they connect is not clear. Recently, however, several reports indicate
that
Cdk5rap2
is required for the maintenance of apical neural progenitors in the mouse
brain, for a normal DNA damage response, and to restrict centriole duplication [20-23]. Loss
of the latter activity results in multipolar spindles, and in cells with more than one primary
cilium. This commentary will focus on Cdk5rap2 and how recent findings, in combination
with findings on other MCPH proteins, reveal important clues into the cell biology of
MCPH as a centrosome-based disease.
The centrosome
The centrosome is the primary microtubule-organizing center (MTOC) in animal cells. It is
a large complex consisting of a pair of centrioles surrounded by a pericentriolar matrix
(PCM) and is present in nearly all animal cells (Box1). The centrosome regulates many
cellular processes including cell polarity and cell division, in addition to functioning as a
hub for factors required for cell cycle progression and DNA damage response [24, 25].
The diverse cellular functions of the centrosome portend a range of mechanisms that affect
the pathology of MCPH, such as progenitor mitotic spindle defects or other cell cycle
disruptions, interkinetic nuclear migration in apical progenitors, cilium dysfunction, or
neuronal migration. Since cortical patterning of neurons appears relatively normal in MCPH
brains, neuronal migration is an unlikely etiology. To understand which aspects of
centrosome function are impacted in MCPH it is essential to study the roles of the MCPH
proteins using
in vivo
models. Recent studies on
Cdk5rap2
and other MCPH genes have
provided important clues [20-23, 26-28].
Cdk5rap2 function at the centrosome
Cdk5rap2 is a centrosomin family protein. Centrosomin orthologs have been extensively
characterized in the fruit fly
Drosophila melanogaster
and the fission yeast
S. pombe,
where
these proteins are essential for MTOC activity (Box 2). RNAi knockdown and mutation of
Cdk5rap2 reveals diverse centrosomal functions and its requirement for neural progenitor
division or survival (Table 2). From these investigations, a variety of activities are attributed
to Cdk5rap2 including centrosome cohesion, centriole engagement, centriole replication
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control, MTOC activity, gamma tubulin recruitment, attachment of centrosomes to spindle
poles, binding to the dynactin complex, DNA damage response, Chk1 kinase recruitment,
microtubule plus end dynamics, checkpoint control, and spindle orientation in neural
progenitors. Linking these cellular phenotypes to function in the developing brain using
vertebrate model systems is critical to understand the mechanisms of cerebral cortex
development and the basis of MCPH.
Mouse models for MCPH
Three mouse mutants for
Cdk5rap2
were reported recently. One mutation, originally
associated with Hertwig’s anemia (
an
) mutant mouse, is a gamma radiation-induced
mutation that inverts exon 4 [22, 29]. The inverted exon is spliced out of the transcript,
resulting in a 37 amino acid deletion that removes most of CM1 (Box 2, Figure I).
Homozygous
Cdk5rap2an
mice have microcephaly. The small cerebral cortex correlates with
multipolar spindles, misalignment of spindles in neural precursors, and loss of neural
progenitors [22].
Cdk5rap2an
mice show loss of primordial germ cells and infertility, anemia
with hematopoietic precursor proliferation defects, aneuploidy, increased generation of
histiocytic sarcoma, and radiation sensitivity. While rescue with a
Cdk5rap2
transgene
would confirm the linkage of these phenotypes, linkage is likely because the
an
mutation has
been backcrossed over dozens of generations and the
an
allele failed to complement another
Cdk5rap2
mutant (
Cdk5rap2RRO242
)[22, 29]. Surprisingly, microcephaly was strain-
dependent, as were the other
Cdk5rap2an
phenotypes [22, 29]. The existence of such
background effects in humans is an intriguing possibility.
Human MCPH is not associated with anemia or early onset cancers, possibly reflecting
differences between the
Cdk5rap2an
mouse model and human disease. Notably, there are no
reports on the fertility of MCPH individuals for comparison to the
Cdk5rap2an
mouse. A
possible explanation for the phenotype differences between human and
Cdk5rap2an
mouse
could be the types of mutations. The
Cdk5rap2an
mouse has an in-frame deletion affecting
CM1, yet the mutant protein is expressed and localized at centrosomes. In contrast, the
human alleles are conceptual protein truncations (Box2, Figure I) [10]. In
Drosophila
, a
deletion mutation within the CM1 domain of
cnn
, similar to
Cdk5rap2an
, causes centrosome
separation failure in embryos, a dominant and neomorphic phenotype [30]. The
Cdk5rap2an
mutation was not dominant, yet a neomorphic function cannot be excluded. In contrast to the
mouse
Cdk5rap2an
mutation, a similar CM1 deletion mutation in chicken DT40 cells did not
cause centrosome amplification or multipolar spindles, but rather centrosome disjunction
from the spindle and defective DNA damage response ([23] and Table 2).
In another study, two slice trap mutations, resulting in 64 and 435 amino acid truncations,
designated
Cdk5rap2RRU031
and
Cdk5rap2RRF465
respectively, were characterized in mice
[20]. These alleles are similar to the two mapped human
CDK5RAP2
mutations (Box 2,
Figure I) [10]. Neither mutant, however, displayed microcephaly or other obvious defects in
brain development, although background effect, as seen for the
Cdk5rap2an
mouse, was not
tested. Of the two mutations, one (
Cdk5rap2RRU031
) was leaky, producing about 7%
expression of full-length protein relative to wild type in cultured embryonic fibroblasts
(MEFs). Despite low expression, the protein was detected at centrosomes. On the other
hand,
Cdk5rap2RRF465
appears fully penetrant, expressing only the truncated protein. Both
mutants were defective in centrosome cohesion coincident with reduced centrosomal signal
for rootletin (Box 1), yet
Cdk5rap2RRF465
also resulted in centriole amplification. Loss of
centriole engagement (Box 1) was the evident cause of amplification in
Cdk5rap2RRF465
mutant MEFs, but
de novo
centriole biogenesis [31] was not excluded as an alternative
mechanism. The consequences of centriole amplification in
Cdk5rap2RRF465
mutant MEFs
are multipolar spindles and supernumerary primary cilia. How these phenotypes may
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contribute to MCPH is discussed below. Comparison of
Cdk5rap2
mutant phenotypes to
other mouse MCPH mutants will be important to establish whether common or disparate
mechanisms account for MCPH.
In addition to
Cdk5rap2
, mouse mutants for
Aspm
and
Mcph1
were constructed. Two mouse
mutants, one short (
Aspm1-7
) and one long (
Aspm1-25
) truncation of
Aspm
, produced
modest microcephaly (
Aspm1-7
had a neocortex that was 86% the thickness of control) [26].
Embryonic
Aspm
mutant brains showed no significant differences in the symmetry of
progenitor divisions. In contrast,
Aspm
knockdown by RNAi
in utero
increased the ratio of
asymmetric to symmetric divisions of neural progenitors, an effect that would conceptually
curtail expansion of the apical progenitor pool in an
in vivo
context [32]. The
Aspm
mutants
also showed reduced numbers of germ cells and reduced male and female fertility.
Interestingly, human
Aspm
rescued microcephaly in the mouse mutant (to a normal mouse-
sized cerebral cortex) [26].
Two
Mcph1
mutants, one a targeted knockout of exon 2 (
Mcph1−
) [28], the other a splice
trap insertion (
Mcph1gt
) [27] were generated. The
Mcph1gt
mutant displayed no
microcephaly or DNA damage sensitivity. The
Mcph1gt
mutation had leaky expression
through the splice trap insertion (28% mRNA expression relative to wild type in the brain),
yet still displayed the premature chromosome condensation phenotype associated with
MCPH1
mutation in humans [7, 8, 11]. The
Mcph1−
mutant, on the other hand, had reduced
birth rate, slow growth, radiation sensitivity, defective DNA damage response, and male
infertility due to meiotic arrest and apoptosis of spermatocytes. Brain size was not examined
in the
Mcph1−
mouse. Centrosome aberrations were not reported in the
Aspm
or
Mcph1
mouse mutants. Thus, despite the generation of several MCPH mouse models, a clear
etiology for MCPH has not emerged.
What common functions emerge from MCPH models?
Given that MCPH proteins reside at centrosome or spindle poles, a plausible basis for
MCPH lies with centrosome function and/or spindle assembly. On the other hand, defective
response to DNA damage is another common feature. Whether these two features are related
is unclear. Examination of centrosomal and DNA damage response phenotypes among all
animal models with microcephaly will be important to determine if these are universal
MCPH phenotypes.
Centrosome amplification occurs in
Cdk5rap2an
and
Cdk5rap2RRF465
mice, in
MCPH1
mutant human and chicken cells [33, 34], and may also occur in
CEP152
mutant human
cells [35]. This shared phenotype suggests that centrosome amplification may be a common
link in MCPH. Centrosome amplification can disrupt the fidelity and timing of mitosis,
spindle orientation during stem cell division (Box 3), and be tumorigenic [19, 36-38]. In
Cdk5rap2an
and
Cdk5rap2RRF465
mutants, amplified centrosomes assemble multipolar
spindles in MEFs [20] and neural progenitors [22], with the latter result correlated to
progenitor loss in the
Cdk5rap2an
embryonic brain. In addition, excess centrioles lead to
supernumerary primary cilia in affected cells [20].
While posing a threat to chromosomal stability, multipolar spindles activate the mitotic
checkpoint, allowing time for extra centrosomes to cluster and for the spindle to resolve into
a bipolar structure [38]. Nevertheless, aneuploidy can result due to inappropriate
microtubule-chromosome attachments, leading in most cases to cell death [37]. Whether
chromosomal instability, or other cell cycle effects, is responsible for MCPH in
Cdk5rap2
mutants is unclear. However, in the
Cdk5rap2an
mouse, neural progenitor cells were
progressively lost, coincident with premature cell cycle exit and elevated apoptosis [22].
Similarly,
in utero
RNAi of
Cdk5rap2
in embryonic brains caused premature cell cycle exit
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and loss of apical progenitors, with a resulting increase of basal progenitors and
differentiated neurons during the 3-day time-course of the experiments [21]. Notably,
Cdk5rap2RRF465
MEFs and
Cdk5rap2
mutant chicken cells had premature senescence [20,
23]. Therefore cell cycle disruption is a possible factor in MCPH, a role implicated for
Mcph1 [11, 27, 33, 39]. Consistent with this scenario, when cell cycle exit is manipulated
directly using p27Kip1 overexpression, neocortical cell numbers are reduced similarly to
MCPH [40].
Spindle orientation
Another possible effect of centrosome amplification is an impact on mitotic spindle
orientation in neural progenitors. Spindle polarity may influence the control and timing of
cell fate determination [6, 41, 42] (Box 3). Spindle orientation becomes random in neural
progenitors when centrosomes are inactivated, or upon disruption of microtubule asters that
link the spindle to the cell cortex, or disruption of the Lis1/dynactin complex that links
asters to the cell cortex to regulate spindle rotation [42]. Supernumerary centrosomes,
induced by Plk4 overexpression, lead to spindle misorientation in
Drosophila
[36]. In
normal
Drosophila
neuroblasts the apical centrosome is a “dominant” MTOC, with a larger
PCM (Figure 1) [43, 44]. This centrosome asymmetry was lost among amplified
centrosomes in Plk4-overexpressing neuroblasts. Moreover, neuroblasts overproliferated,
presumably due to increased symmetric, rather than asymmetric, divisions [36]. An opposite
effect, a shift from symmetric to asymmetric division, might be expected with mammalian
progenitors under similar conditions (Figure 1,Box 3).
During neurogenesis of the mouse cerebral cortex, neural progenitors initially divide
symmetrically to expand the progenitor pool, and then switch to an asymmetric phase for
neurogenesis. Since the apical cortical aspect is small in apical epithelial progenitors (Figure
1), even a slight skew in the cleavage plane angle can promote asymmetric inheritance of
cortical determinants [6]. Mutation of
Cdk5rap2
or
Nde1
(a Lis1/dynactin complex
component) or RNAi of
Aspm
alters spindle orientation in apical progenitors. These
phenotypes correlated with progenitor depletion (and reduced cerebral cortex in the case of
Nde1
and
Cdk5rap2
) [22, 32, 45]. One interpretation of these results is that precocious
asymmetric division produces neurons early at the expense of progenitor pool expansion. In
this model, an active centrosome-dependent process drives the symmetric proliferative
divisions, the loss of which results in asymmetric division. In conflict with this idea,
however, depletion of LGN, a non-centrosomal polarity determinant, caused randomized
spindle orientation and apical progenitors were displaced from the mouse neuroepithelium.
Nevertheless, this did not affect progenitor pools, which relocated basally, or cerebral cortex
development [46, 47]. Moreover, disruption of aPKCλ in mouse brain disrupts apical
adherens junctions, interkinetic nuclear migration, and the neuroepithelial tissue architecture
[48]. Despite these disruptions, cortical neurogenesis is not impeded. Therefore, while
centrosome disruption may impact spindle orientation in mouse neural progenitor cells, it is
clear that spindle misorientation is not sufficient to disrupt neurogenesis and cause MCPH.
Centrosome asymmetry
While the centrosome pair can regulate spindle orientation in polarized cells, they are also
asymmetric, one being older than the other. Centrosome asymmetry was first described in
Drosophila
male germline stem cells where the older “mother” maintains association with
the apical cortex [49]. In
Drosophila
neuroblasts, on the other hand, the younger “daughter”
centrosome is maintained apically and the older “mother” is inherited by the neuronal
precursor cells [50]. This relationship may not be important, however, because asymmetric
division occurs successfully when the centrosomes are experimentally manipulated into
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switched positions [51]. In the embryonic mouse brain, the older mother centriole is retained
in the renewed apical progenitor, while the younger mother is distributed into differentiated
cells [52]. Depletion of ninein, a mother centriole-specific marker, by
in utero
RNAi
disrupted this asymmetry and neural progenitors were depleted [52]. Together, these data
suggest that centrosome asymmetry may be an important regulator of neurogenesis,
somehow impacting progenitor fate or survival. Imbalance of the mother and daughter
centriole numbers or ratios could, under this scenario, disrupt progenitor division polarity
and survival in mouse
Cdk5rap2
mutant neural progenitors (Figure 1).
The centrosome in neural progenitor maintenance: a role for cilia
Centriole disruption or amplification could impact neural progenitor survival by means other
than mitotic spindle or cell cycle disruption. Primary cilia are key signaling centers for
hedgehog (hh) and other signaling pathways [17, 18], and have established roles in neural
progenitor maintenance at the neocortex [53]. Most human cells contain a single primary
cilium, whose assembly is templated by the mother centriole. Consistent with a cilium basis
for MCPH, the
Stil
mutant mouse displayed defects in Sonic hh signaling, resulting in neural
tube and left-right patterning defects consistent with cilium dysfunction [7, 8].
MEFs from
Cdk5rap2RRF465
mice exhibited multiple primary cilia in cells with amplified
centrioles. Whether this is also true for the neural progenitor cells, particularly in the
microcephalic
Cdk5rap2an
mutant, was not investigated. Supernumerary cilia could create a
quantitative change in intercellular signaling, thereby impacting neural progenitors. In
support of this model, a recent study showed that, in cells with multiple primary cilia
induced by Plk4 overexpression, that hh signal reception was reduced by approximately
50% in cells with two primary cilia compared to cells with one (Tim Stearns, personal
communication). Thus, supernumerary cilia may dampen rather than amplify hh signaling,
and this could impact survival of progenitor cells. Regulation of cilium assembly and
function is therefore a possibility for all the MCPH proteins, given the connection they share
to the centrosome. Among the MCPH proteins, Cdk5rap2, Cep152, CPAP or their orthologs
have been shown to impact cilium assembly or function in some form. It is noteworthy that
pericentrin (see below) and ninein are also required for cilium assembly [54, 55].
If indeed the basis for MCPH is centrosomal, it remains to be shown whether the cause is
disruption of cilia, spindle regulation at mitosis, or some other yet unknown basis. Given the
diverse functions attributed thus far to the seven known MCPH proteins, multiple causes are
a possibility [7, 8]. Recently, however, several of the MCPH orthologs (Table 1) were found
to reside in complexes in
Drosophila
(Cnn, Asl, and Sas-4) and human cells (CEP152 and
CPAP) [56-58]. With MCPH proteins assembled together, it is plausible they function
through a common pathway to impact brain development. In addition, the centrosomal
protein pericentrin (Pcnt) [54] binds directly to Cdk5rap2 [21, 59], and associates with
several MCPH proteins or their
Drosophila
orthologs (Box 4) [39, 58].
MCPH has links to SCKL and MOPD II syndromes
PCNT
is mutated in microcephalic osteodysplastic primordial dwarfism Majewski type II
(MOPD II, MIM 210720) [60, 61]. Primordial dwarfism (proportionately small overall
stature) and microcephaly are among the clinical features of MOPD II. These features
overlap with Seckel syndrome (SCKL1, MIM 210600), and some patients with lesions in
PCNT
were diagnosed as SCKL. A consensus is emerging, however, that mutations in
PCNT are assigned only to MOPD II [62, 63]. Recently,
CPAP
and
CEP152
mutations,
originally mapped in MCPH, were also mapped to SCKL syndrome [35, 64]. Other MCPH
mutations also cause short stature [7, 12], yet this is a prominent feature in SCKL and
MOPD II. Interestingly, there appears no correlation between the perceived severity of the
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lesions mapped to
CPAP
and its association with MCPH versus SCKL, and may reflect
genetic background effects [64]. The similarities of MCPH, SCKL and MOPD II syndromes
have been noted frequently [7, 8, 54, 64], and the recent genetic and molecular findings
underscore relatedness among these microcephaly syndromes (Figure 2). Among these
syndromes, there is a common thread to DNA damage response.
Cell cycle arrest in response to DNA damage is a critical checkpoint at S-phase or G2/M to
ensure repair of insults prior to cell cycle resumption. These responses require the ATM
(ataxia telangiectasia, mutated) and ATR kinase signaling cascades acting through the
effector checkpoint kinases Chk1 and Chk2 [65]. Chk1 and Chk2 localize to centrosomes,
where Chk1 inhibits cyclin-dependent kinase 1 (Cdk1) activation and halts the cell cycle at
the G2/M transition in response to DNA damage [65].
Centrosome amplification is a hallmark response to DNA damage or to aberrant DNA
damage signaling [65]. Amplification of centrosomes in response to ionizing radiation is
Chk1-dependent [25, 66]. SCKL cells, whether mutant in
ATR
or other genes, exhibit
centrosome amplification [67]. It will be informative to survey cells from
CPAP
and
CEP152
SCKL patients, and from MCPH patients, for amplified centrosomes and impaired
DNA damage response to determine if either or both of these effects are shared in these
syndromes.
Mcph1, Cdk5rap2, Cep152 and Aspm, like PCNT and ATR [39, 60], have roles in, or
responses to, the DNA damage response [7, 8, 23, 33, 35, 39, 68, 69]. MCPH1 coordinates
with PCNT to recruit CHK1 to centrosomes where it inhibits CDC25B [39]. Mcph1 acts
upstream of ATR but downstream of γ-H2AX in foci formation on ionizing radiation-
induced DNA damage in chicken cells [68]. Importantly,
MCPH1
mutant or knockdown
cells show centrosome amplification, depending on the cell type, with hyper-amplification
upon DNA damage [33, 34]. Moreover, DNA damage-induced centrosome duplication
appears to involve centriole disengagement [70], similar to the proposed mechanism for
centriole amplification in
Cdk5rap2RRF465
mouse cells [20].
The centrosome amplification and DNA damage response impairment observed in Seckel,
MOPD II, and MCPH cells, and the multiple interactions found among the proteins
involved, link these syndromes at a cellular and molecular level. These links
notwithstanding, it is unclear what causal connection exists between the DNA damage
response and microcephaly diseases. Does impaired DNA damage response have a direct
impact on neural progenitors, or is the effect an indirect one due to centriole amplification or
other centrosome disruption? On the other hand, the possibility that amplification or other
centrosome effects is an inconsequential side effect of impaired DNA damage signaling in
MCPH cannot be excluded.
While the MCPH mutations produce small brains, the MOPD II and SCKL mutations also
produce small overall stature, suggesting that while MCPH affects primarily neural
progenitors, the MOPD II and SCKL mutations affect the stem cells for multiple organs. In
support of this, a conditional knockout for
ATR
causes progeria in adult mice and a
progressive loss of stem cells in multiple tissues [71]. Since
ATR
mutant SCKL cells have
amplified centrosomes [33], reminiscent of
Cdk5rap2
mutant mouse cells [20, 22], perhaps a
related mechanism depletes stem cells in both mutants and between syndromes.
Future Directions
Deciphering the functions of CDK5RAP2 and the other MCPH proteins will reveal
fundamental aspects of neural stem cell biology. The findings that mutations in
Cdk5rap2
mutant mice produce amplified centrosomes and neural progenitor loss supports previous
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models whereby MCPH arises from a centrosome-based defect in neural stem cell division
or maintenance. Drawing on recent findings in a variety of systems regarding MCPH protein
functions and the roles of centrosomes in neural stem cells, several models are invoked to
explain how MCPH may disrupt neural progenitor cells. The distinguishing features of these
prevailing models involve centrosome roles in i) mitotic spindle orientation, ii) centrosome
asymmetry iii) cilium assembly or function, and iv) DNA damage response. In the neocortex
one or more of these likely contribute to neuron reduction in MCPH by affecting cell
proliferation, specifically cell cycle exit, or possibly also cell survival. While excess
centrosomes are a feature of
Cdk5rap2, MCPH1
, and
CEP152
mutant cells, it remains to be
seen if this is common among MCPH mutations, or if some other common feature unites the
etiology. Exciting discoveries are on the horizon as the mechanisms that link the MCPH
proteins to the biology of neural stem cells are exposed. Experimental linkage of the
molecular deficits to the specific changes in the behavior of the neural stem cells that
produce the neocortex presents an exciting challenge for MCPH and also for MOPD II and
SCKL. The full impact of centrosome disruption in
Cdk5rap2
mutant cells
in vivo
remains
to be understood, but it is clear that the regulatory roles of centrosomes are crucial for the
mechanisms of stem cell control in organogenesis.
Acknowledgments
We thank Ling-Rong Kao and Tomer Avidor-Reiss for comments and discussion, and funding support from NIH
grant R01GM068756, and an ARRA supplement to support GM068756, from the National Institute of General
Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institute of General Medical Sciences or the National Institutes of Health.
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Box 1. The Centrosome
A typical cell contains one or two centrosomes, depending on the cell cycle stage. Each
centrosome contains a pair of centrioles. The centriole is a microtubule-based cylindrical
structure with nine-fold radial symmetry [24] (Figure I). A nascent “daughter” centriole
assembles at the proximal end of the “mother” centriole and remains tightly engaged to
the mother centriole until anaphase, when the mother-daughter bond is disengaged in a
process that requires Plk1 and separase [80], yet the pair remain linked via a fibrous
linker consisting of rootletin, C-Nap1 and Cep68, and regulated by Nek1 kinase, protein
phosphatase 1, Cdk5rap2 and other proteins ([74] and references therein) (Figure I).
In contrast to mutations in their mammalian counterparts, mutations in some MCPH
ortholog genes in
Drosophila
(Table 1) result in complete loss of centrioles. Centrioles
are absent in
Sas-4
[73] or
asterless
(
asl
) [81] loss-of-function mutant flies. Moreover,
Asl/CEP152 serves an essential early role in centriole biogenesis [56, 57, 81, 82]. In
contrast to
Drosophila
, where development to adults can be achieved without centrioles,
mouse development arrests early if centrioles are severely disrupted. This is because
centrioles are required for assembly of cilia, which are essential for hedgehog and other
signaling pathways [17, 18]. Yet, in humans, mutations in the
asl
and
Sas-4
orthologs
CEP152
and
CPAP
result in MCPH or SCKL. Moreover, loss of
asp
in
Drosophila
causes mitotic arrest and lethality, while severe loss-of-function mutations in
ASPM
are
viable [11]. In addition, in
Drosophila
, mutants for the single
CDK5RAP2
ortholog
cnn
fail to assemble PCM and mitotic MTOCs are severely deficient [83], a very different
phenotype from
Cdk5rap2
mutant cells which have apparently normal, albeit amplified,
mitotic centrosomes [20]. Since MCPH proteins are conserved and likely share functional
attributes, there are probably paralogs in human that supply functional redundancy, but
for which neural progenitors are uniquely sensitive [11] (Table 1).
Paralogs for
CDK5RAP2
and
WDR62
, have been reported (
MYOMEGALIN
and
MAPKBP1,
respectively). Table 1 shows the results of paralog searches conducted using
iterative BLAST (PSI BLAST), revealing the presence of potential paralogs for
CEP152,
ASPM
and
CPAP
(Table 1). Therefore, tissue-specific or paralog-specific functions may
be ascribed to Cdk5rap2 and other MCPH genes in neural progenitors [11]. A thorough
survey of the expression patterns of these genes will be necessary to determine if paralog
redundancy limits the effects of MCPH mutations.
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Box 2. Functions of centrosomin family proteins
Centrosomin family members are conserved in eukaryotes, yet appear absent from plants.
The founding member, centrosomin (CNN), was discovered in
Drosophila melanogaster
.
Centrosomin proteins have extensive coiled-coil domains, and two highly conserved
domains, Centrosomin Motifs 1 and 2 (CM1 and CM2) (Figure II). The functions for
centrosomins are most extensively characterized for the
Drosophila
and the yeast
S.
pombe
orthologs, CNN and Mto1, respectively. Null mutations in
cnn
disrupt assembly
of the pericentriolar matrix (PCM) surrounding the centrioles in the early embryonic
centrosomes, and at mitosis in somatic cells [83]. A similar loss of MTOC activity occurs
in
S. pombe mto1
mutants ([84] and references therein). Centrioles detach from spindle
poles in
cnn
mutants [30, 85], similar to what occurs in
Cdk5rap2
mutant chicken cells
[23]. The CM1 domain is required for centrosome separation in
Drosophila
embryos, and
for efficient recruitment of γ-tubulin, TACC and Msps [30]. In chicken DT40 cells,
Cdk5rap2 CM1 was required to recruit AKAP450 and the p150Glued subunit of dynactin
to centrosomes [23]. In
S. pombe
, CM1 is necessary but not sufficient to recruit γ-TuC to
MTOCs [86]. Human CDK5RAP2 CM1, on the other hand, was reported to bind directly
to γ-TuRC and activate it [87]. Mto1 acts cooperatively with Mto2, which has no
obvious orthologs in flies or vertebrates, to recruit γ-tubulin [86]. Since vertebrates have
a clear paralog to Cdk5rap2 in myomegalin, it will be interesting to see if depletion of
both causes loss of centrosomal MTOC activity as occurs with mutation of the single
ortholog in
Drosophila
and
S. pombe
.
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Box 3. Regulation of neural stem cell division polarity
A fundamental tenet of stem cell biology is the control of polarized division during
organogenesis to produce two cells destined with different fates. One cell differentiates,
while the other becomes a renewed stem cell that continues the division process to
populate differentiated tissues. Spindle orientation during neural progenitor cell divisions
requires both cortical determinants that capture astral microtubules, and functional
centrosomes that generate the astral microtubules (see Figure 1). Spindle orientation
becomes random in neural progenitors when centrosomes are inactivated. In
Drosophila
,
determinants that polarize neuroblasts and the centrosomes that respond to polarization
cues are essential for normal stem cell proliferation and for the coordinated asymmetric
division that leads to production of neural precursors and stem cell self-renewal [6, 41,
42]. Disruption of polarization or spindle orientation cues can result in increased
symmetric divisions and metastatic tumorigenesis [88]. A similar paradigm functions in
mouse neural progenitors, except that polarization cues and spindle orientation appear to
actively control symmetric rather than asymmetric divisions. The loss of which may
result in increased neurogenic asymmetric divisions [6, 41, 42]. Disruption of LGN, a
conserved neural stem cell polarizing component, however, caused randomized spindle
orientation in mouse and chick [46, 47], yet had no impact on brain size in mice [46].
Thus, the importance of spindle orientation in progenitor cells is called into question [6,
41, 42, 46, 47, 89].
Centrosome asymmetry, on the other hand, may be a conserved feature of stem cell
biology. In
Drosophila
, male germline stem cells maintain the older “mother” centrosome
near the apical cell membrane proximal to the niche, a microenvironment that maintains
stem cells [90]. Similarly, in neuroblasts one centrosome is maintained near the apical
membrane and has MTOC activity, while the other is inactive until late in prophase when
it migrates to the basal side of the cell [43, 44]. In contrast to germ cells, the apically-
anchored centrosome is the younger centrosome in
Drosophila
neuroblasts [50]. Recent
studies [52] show that, opposite to
Drosophila
neuroblasts, mouse neural progenitors
maintain the older mother centriole when they divide asymmetrically in the mouse brain,
and may rely on centrosome asymmetry to maintain progenitor pools. Therefore, the
principle of centrosome asymmetry may be a common regulatory mechanism for
polarized stem cell division.
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Box 4. Cdk5rap2 and Pericentrin are partners
Pericentrin (Pcnt) is a conserved centrosomal protein mutated in MOPD II [54]. Evidence
from studies in fungi,
Drosophila
and human cells point to roles for Pcnt and its orthologs
in regulation of microtubule assembly into the mitotic spindle. It is also required for
cilium assembly, which appears to be its critical function in
Drosophila
[54]. Interactions
between Cdk5rap2 and PCNT were shown for the human, mouse and
Drosophila
homologs ([21, 58, 59] and our unpublished data). The Cdk5rap2-Pcnt association was
shown to be direct for the mouse and human counterparts. How Pcnt and Cdk5rap2 are
functionally related is unclear, but RNAi knockdown studies in mouse neural progenitors
and in cell lines showed that Pcnt is required for Cdk5rap2 localization to centrosomes,
while Cdk5rap2 is not required for Pcnt localization [21, 59, 74], except in HeLa cells
where they are mutually dependent [74, 77].
Pcnt
RNAi affected neural progenitor
proliferation similarly to
Cdk5rap2
knockdown [21].
PCNT
mutant cells did not show
amplified centrosomes, but they did show a reduction or absence of the microtubule
nucleator γ-tubulin at centrosomes, as did mutation of the gene for MCPH1, which also
associates with PCNT [39, 60]. PCNT and MCPH1 cooperate to recruit CHK1 kinase to
centrosomes to regulate mitotic entry. While Pcnt is not reported to affect centrosome
replication, disruption of its partner
MCPH1
causes centriole amplification in some cell
types [34]. Tying these connections together will be important to understand the
functional significance of the CDK5RAP2-PCNT interaction. Moreover, the intimate
association between CDK5RAP2 and PCNT suggests a shared etiology between MCPH
and MOPD II.
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Figure I. Forms of centrosomal bondage
Centriole mother-daughter pairs are tightly bound in the “engaged” state until their release is
triggered, which normally occurs at mitosis. The spot vacated by the procentriole is shown
in pink, and is a trigger to license replication. The older mother (M) is marked with distal
(yellow) and subdistal (red) appendages. The daughter (D) is shown as a small procentriole.
A linker (purple) comprised of the related proteins C-Nap1 and Rootletin connects each
centriole pair. The linker connects the mother centrioles between centrosomes. Loss of
cohesion and engagement normally occur in the cell cycle at G2 and anaphase, respectively.
In homozygous
Cdk5rap2RRF465
mutant cells, cohesion and engagement are both disrupted.
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Figure II. Cdk5rap2 is a centrosomin family member
Schematic diagram shows the relative domain structures of centrosomin proteins from
S.
pombe
(Mto1),
Drosophila
(Centrosomin), mouse (Cdk5rap2), and both human paralogs
(Cdk5rap2 and Myomegalin). Mice and other vertebrates also have Myomegalin (not
shown). The relative positions of conserved domains are shown, and positions of mutations
are indicated for mouse and human Cdk5rap2. The C-terminal box in Mto1 (shaded in olive
green) is not conserved with CM2 in the metazoans shown, but it is required for localization
to MTOCs, similar to the function of CM2.
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Figure 1. Models for control of progenitor division symmetry by centrosomes
Centrosomes assemble astral microtubules that link the spindle to polarizing determinants
localized at the cell cortex. Astral microtubules thereby link spindle orientation to cell
polarity. (a) In
Drosophila
neuroblasts the centrosomes are asymmetric, where the younger
“daughter” centrosome assembles a larger PCM and a larger aster while the older “mother”
centrosome undergoes PCM loss in prophase, followed by PCM restoration at
prometaphase, and then the older centrosome segregates into the ganglion mother cell [43,
44, 50, 72]. (b) When centrosomes are amplified, this asymmetry is lost and neuroblasts
divide symmetrically [36]. (c) When astral microtubules or the dynactin complex are
disrupted, the spindle is misoriented early in mitosis but gets corrected towards telophase
[41, 42]. Neuroblasts frequently divide symmetrically, however, when centrosomes are
completely absent [19, 41, 42, 73].
Neural progenitor cells in mouse divide symmetrically during the proliferative phase (d),
and then switch to an asymmetric mode of division upon neurogenesis (e,f). If the spindle is
misaligned during the proliferative divisions the cell may divide asymmetrically (e,f) [3, 6,
32, 41, 45]. The centrosomes are asymmetrically inherited, with the older mother centriole
(yellow centrosome) retained in the apical progenitor cell (the opposite to
Drosophila
, where
the younger centriole is retained in the progenitor cell) [52].
How amplified centrosomes or rearrangement of mother centrioles will impact progenitor
division in mice is not known. (g) Symmetric division is disrupted if centrosomes or the
dynactin complex are disrupted. The drawings in (h) and (i) are speculative impacts that
centrosome amplification may have. In (h), imbalance of positioning of mother centrioles
could impact spindle rotation, while in (i), centrosome asymmetry may be lost, similar to
what was observed in
Drosophila
(b).
In (a-i), the basal cortical determinants are labeled red, and the apical green. The
centrosomes are red, the microtubules green, and the chromosomes blue. In (d-i), the
projection on top of the cells is the basal process, shown here not to scale (it is relatively
Megraw et al. Page 19
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much longer). The dotted line delineates the cleavage furrow, the adherens junctions are
indicated in light blue.
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Figure 2. Convergence of three centrosome-based microcephaly diseases
A Venn diagram showing that MCPH, MOPD II and SCKL Syndromes have mutations in
common genes, and exhibit physical interactions (connecting lines) and indirect interactions
(via CHK1, the effector kinase for ATR; dashed lines) that connect these diseases into a
group of with possible common centrosomal etiology. Not shown are additional interactions
shown in
Drosophila
, that link the orthologs of CDK5RAP2, CPAP, CEP152 and PCNT in a
common complex.
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Megraw et al. Page 22
TABLE 1
The MCPH genes
MCPH
gene OMIM
251200 Map
location Other names Paralog† Drosophila
ortholog Centrosome
or spindle
pole
DNA
damage
response
MCPH1 251200
607117 8p23 Microcephalin,
BRIT1 None MCPH1 Yes Yes
MCPH2 604317
613583 19q13.12 WDR62 MAPKBP1 CG7337 Yes ND
MCPH3 604804
608201 9q33.3 Cdk5rap2,
Cep215 Myomegalin/
PDE4DIP Centrosomin
(Cnn) Yes Yes
MCPH4 604321
613529 15q21.1 Cep152 C10orf118/
CTCL tumor
antigen L14-2
Asterless
(Asl) Yes Yes
MCPH5 608716
605481 1q31 Aspm,
Calmbp1 hSfi1/ Sfi1 Abnormal
spindle (Asp) Yes Yes
MCPH6 608393
609279 13q12.2 CPAP, CENPJ,
Sas-4 TCP10 Sas-4 Yes ND
MCPH7 612703
181590 1p32 STIL, SIL,
Sas-5 None? Ana2 Yes ND
†
Except for Myomegalin/PDE4DIP and MAPKBP1, the paralogs listed here are proposed based upon similarity searches by the authors. ND: not determined.
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Megraw et al. Page 23
TABLE 2
Cdk5rap2 depletion phenotypes
Method
of
depletion System Knockdown or mutant phenotypes and other activities Reference
Mutation Human Microcephaly. Centrosomal defects not determined. [10]
RNAi U2OS,
A549 and
hTERT-
RPE1
Cell culture
Loss of cohesion. Pcnt was required for Cdk5rap2 recruitment. [74]
RNAi HeLa,
U2OS and
MRC-5
Loss of MTOC activity, and failure to recruit γ-TuRC to centrosomes. CM1
domain binds to γ-TuRC. [75]
RNAi HeLa Mitotic spindle checkpoint defective. Cdk5rap2 associates with Cdc20. Binds to
checkpoint gene promoters in nuclei and regulates their expression. [76]
RNAi HeLa Modest reduction in γ-tubulin recruitment to mitotic centrosomes. Co-dependence
of Cdk5rap2 and pericentrin for centrosome localization. Plk1 required for
recruitment of Cdk5rap2 to centrosomes.
[77]
RNAi U2OS Cdk5rap2 binds to microtubule plus ends and is dependent on EB1, a direct
binding partner. Activity is not conserved in rodents. [78]
RNAi HeLa Cdk5rap2 recruits dynein to centrosomes and depends on dynactin for its
recruitment to centrosomes. [79]
Mutation /
RNAi Targeted
disruption in
chicken
DT40 cells/
HeLa
CM1 and CM2 domains each required for attachment of centrosomes to spindle
poles. Cdk5rap2 recruits AKAP450 and p150glued/dynactin to centrosomes. Subtle
reduction of γ-tubulin at Cdk5rap2 CM1 mutant centrosomes. Defective DNA
damage response. Reduced Chk1 kinase recruitment to centrosomes. Loss of
cohesion (HeLa, RNAi).
[23]
RNAi
in
utero
Mouse
embryonic
brain
Premature neuronal differentiation. Reduced proliferation and increased cell cycle
exit by neural progenitors. Loss of apical but increase in basal progenitors.
Cdk5rap2 and pericentrin interact. Pericentrin recruits Cdk5rap2 to centrosomes,
but not vice-versa.
[21]
Originally identified as Hertwig’s anemia (
an
), with hematopoietic progenitor
proliferation deficiency, elevated aneuploidy, radiation sensitivity, lack of
primordial germ cells, and early onset histiocytic sarcomas. Phenotypes are strain
dependent.
[29].
Mutation Mouse Mutation (
an
) mapped to an inversion of exon 4 that deletes most of CM1. Severe
microcephaly, but is strain dependent. Cortical neurons are most deficient in later-
forming superficial layers. Reduction in neural progenitors, especially basal
progenitors at later stages of neurogenesis. Apparent mitotic delay. Multipolar
spindles in neuronal precursors. Defective spindle orientation in cortical
progenitors. Mitotic centrosomes recruit Aurora A kinase. Premature cell cycle
exit and increased cell death of neuronal progenitors.
[22]
Mutation Mouse Two gene trap mutations (
Cdk5rap2RRU031
and
Cdk5rap2RRF465
) that produce
truncated proteins. Centriole amplification in embryonic fibroblasts. Loss of
centriole cohesion and engagement. Multipolar spindles and supernumerary cilia.
Mitotic delay. Mitotic centrosomes recruit γ-tubulin and other PCM components
and are apparently normal MTOCs. No microcephaly observed.
[20]
HeLa, U2OS, A549, hTERT-RPE1 and MRC-5 are human cell lines.
CM1 and CM2 are centrosomin motifs 1 and 2, respectively.
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