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KIF2A deficiency causes early-onset neurodegeneration
Nuria Ruiz-Reig
a
, Georges Chehade
a
, Janne Hakanen
a
, Mohamed Aittaleb
b
, Keimpe Wierda
c
, Joris De Wit
d
, Laurent Nguyen
e
,
Philippe Gailly
f
, and Fadel Tissir
a,b,1
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD; received June 6, 2022; accepted October 3, 2022
KIF2A is an atypical kinesin that has the capacity to depolymerize microtubules. Patients
carrying mutations in KIF2A suffer from progressive microcephaly and mental disabil-
ities. While the role of this protein is well documented in neuronal migration, the rela-
tionship between its dysfunction and the pathobiology of brain disorders is unclear.
Here, we report that KIF2A is dispensable for embryogenic neurogenesis but critical in
postnatal stages for maturation, connectivity, and maintenance of neurons. We used a
conditional approach to inactivate KIF2A in cortical progenitors, nascent postmitotic
neurons, and mature neurons in mice. We show that the lack of KIF2A alters microtu-
bule dynamics and disrupts several microtubule-dependent processes, including neuronal
polarity, neuritogenesis, synaptogenesis, and axonal transport. KIF2A-deficient neurons
exhibit aberrant electrophysiological characteristics, neuronal connectivity, and function,
leading to their loss. The role of KIF2A is not limited to development, as fully mature
neurons require KIF2A for survival. Our results emphasize an additional function of
KIF2A and help explain how its mutations lead to brain disorders.
neuronal polarity jbrain wiring jneurodevelopment jneurodegeneration
Microtubules (MTs) are important components of the cytoskeleton. They play crucial
roles in cell polarity, adhesion, proliferation, migration, and differentiation (1). MTs
undergo cycles of growth (polymerization) and shrinkage (depolymerization) through
addition or loss of tubulin-dimer subunits. This dynamic is mediated by severing and
depolymerizing enzymes and regulated by MT-binding proteins such as microtubule-
associated proteins (MAPs) and plus-end–tracking proteins (+TIPs), as well as by
tubulin posttranslational modifications (PTMs). Defective MT dynamics are often
associated with neurodevelopmental and neurodegenerative disorders (2, 3).
KIF2A belongs to the kinesin-13 subfamily endowed with an MT depolymerizing
activity (4). In mitotic cells, KIF2A is localized at the kinetochore and centrosome,
where it depolymerizes the MT’s minus-end and regulates mitotic spindle and cilia
assembly/disassembly (5–7). Several studies have investigated the role of KIF2A in
cortical neurogenesis. However, the published results are contradictory. In utero elec-
troporation of KIF2A short hairpin RNA (shRNA) decreases the proliferation of neural
progenitors and promotes differentiation of neurons (8). In contrast, electroporation of
two KIF2A variants (i.e., c.961C >G, p.His321Asp and c.950G >A, p.Ser317Asn;
Gene ID: 3796) causes the opposite effect, increasing proliferation of progenitors and
decreasing the production of neurons (9). Conditional knock-in (KI) mice expressing
one copy of the human KIF2A variant c.961C >G, p.His321Asp have no change in
proliferation but display an increase in early embryonic cell death. Consequently, the
mutant brain is smaller at birth (10). In human brain organoids, KIF2A, which is
expressed in the mother centriole, interacts with CEP170 downstream of WDR62 to
promote disassembly of the primary cilium. Disruption of WDR62-CEP170-KIF2A
signaling depletes the number of progenitor cells, leading to smaller brains (11). KIF2A
is enriched in postmitotic neurons where it forms homodimers that bind to MTs and
move toward the plus end, wherein they remove tubulin subunits in an adenosine
50-triphosphate–dependent manner (4, 12, 13). Functional analysis using Kif2a KO
mice revealed that the protein is necessary for neuronal migration, elongation of axon
collaterals, and axonal pruning (14–16). KIF2A accumulates in the growth cone where
it regulates MT length (14, 17). Early postnatal deletion of KIF2A does not disrupt
proliferation or migration of granular cells in the dentate gyrus, but results in mossy
fiber sprouting and an epileptic hippocampus (18). KIF2A has several splice isoforms
whose expression is developmentally regulated (19, 20), and its activity is regulated by
phosphorylation by different kinases (21). These data emphasize the complexity of
KIF2A function and suggest that it could have different roles depending on isoform,
expression, activity level, and interaction with different partners.
In humans, de novo mutations in KIF2A have been linked to a wide variety of clini-
cal manifestations depending on the affected domain of the protein. Mutations in the
Significance
In this paper, we describe an
additional function for KIF2A in the
postnatal brain and provide
evidence that KIF2A-related
pathologies result from defects in
neuronal connectivity and early-
onset neurodegeneration rather
than from impaired neurogenesis
as commonly assumed. We
conditionally deleted KIF2A from
progenitors, nascent and mature
cortical neurons and showed that
this protein is key for maturation
and maintenance of neurons.
KIF2A deficiency altered
microtubule dynamics and
intracellular transport and
compromised neuronal
connectivity and survival. Our
results shed light on the
mechanisms by which KIF2A
mutations cause brain diseases.
Author affiliations:
a
Laboratory of Developmental
Neurobiology, Institute of Neuroscience, Universit
e
catholique de Louvain, 1200 Brussels, Belgium;
b
College
of Health and Life Sciences, Hamad Bin Khalifa
University, 34110 Doha, Qatar;
c
Electrophysiology Unit,
VIB-KU Leuven Center for Brain & Disease Research,
3000 Leuven, Belgium;
d
VIB-KU Leuven Center for Brain
& Disease Research, Department of Neurosciences, 3000
Leuven, Belgium;
e
Laboratory of Molecular Regulation of
Neurogenesis, GIGA-Stem Cells, Interdisciplinary Cluster
for Applied Genoproteomics, University of Li
ege, 4000
Li
ege, Belgium; and
f
Laboratory of Cell Physiology,
Institute of Neuroscience, Universit
e catholique de
Louvain, 1200 Brussels, Belgium
Author contributions: N.R.-R., G.C., and F.T. designed
research; N.R.-R., J.H., and K.W. performed research;
J.H., M.A., K.W., J.D.W., L.N., P.G., and F.T. contributed
new reagents/analytic tools; N.R.-R., G.C., M.A., J.D.W.,
L.N., P.G., and F.T. analyzed data; and N.R.-R. and F.T.
wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2022 the Author(s). Published by PNAS.
This article is distributed under Creative Commons
Attribution-NonCommercial-NoDerivatives License 4.0
(CC BY-NC-ND).
1
To whom correspondence may be addressed. Email:
ftissir@hbku.edu.qa or fadel.tissir@uclouvain.be.
This article contains supporting information online at
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2209714119/-/DCSupplemental.
Published November 7, 2022.
PNAS 2022 Vol. 119 No. 46 e2209714119 https://doi.org/10.1073/pnas.2209714119 1of11
RESEARCH ARTICLE
|
NEUROSCIENCE
nucleotide-binding domain were associated with malformations
of cortical development, including microcephaly, lissencephaly,
and partial agenesis of the corpus callosum (CC; 22–25). Var-
iants in the motor domain were associated with epilepsy, and
variants in other domains were associated with autism spectrum
disorder (25–27). Finally, overexpression and one rare variant
were associated with Alzheimer’s disease (AD) (28, 29).
While the role of KIF2A in neuronal migration is well estab-
lished (14, 15, 20), its roles in embryonic neurogenesis and in
the postnatal brain remain unclear. We conditionally ablated
the gene from cortical progenitors, nascent neurons, and fully
mature neurons. We report that KIF2A is dispensable for
embryonic neurogenesis but crucial for neuronal maturation,
connectivity, and survival.
Results
The Lack of KIF2A Triggers Premature Loss of Neurons. The
role of KIF2A in cortical neurogenesis and its relationship with
microcephaly have been controversial (8–11). To explore this,
we first examined KIF2A expression in the developing and
adult cerebral cortex using in situ hybridization and immuno-
fluorescence in mice. KIF2A is expressed in postmitotic neu-
rons throughout life and to a lesser extent in neural progenitors
(SI Appendix, Fig. S1 A–I). We crossed Kif2a
F/F
mice with
Emx1-Cre to inactivate KIF2A in cortical progenitors starting
from embryonic day (E) 9.5 (15, 30) and generated cortex-
specific conditional knockout mice (hereafter referred to as
Emx1-cKO). In these mice, KIF2A expression persisted in
sparse neurons, presumably cortical interneurons, which do not
express Emx1-Cre (SI Appendix, Fig. S1 Dand I). To assess the
impact of KIF2A deletion on proliferation of cortical progeni-
tors, we evaluated the number of Ki67-positive cells in the
ventricular-subventricular zones, the number of 5-bromo-20-deoxy-
uridine (BrdU)–positive cells, 2 h after intraperitoneal injection
of BrdU in pregnant females at E14.5, and the ratio between
Ki67-positive and BrdU-positive cells. We did not detect any
difference between Emx1-cKO embryos and littermate controls
(SI Appendix,Fig.S2A–C). Furthermore, there was no difference
in number of apical radial glia (Pax6
+
), progenitors undergoing
mitosis (PHH3+), intermediate progenitors (Tbr2
+
), early post-
mitotic neurons (Tbr1
+
), or apoptotic cells (cleaved Caspase 3
[cCasp3-positive]) during embryonic stages (SI Appendix,Fig.S2
D–I). Accordingly, the number of neurons and brain size were
normal at birth, indicating that the loss KIF2A had no effect on
neuronal differentiation (SI Appendix,Fig.S2J–M). However,
the distribution of neurons was altered in Emx1-cKO mice due
to defective radial migration (SI Appendix,Fig.S2J), which is in
line with published data (14). We analyzed cortical layering at
P21 and found that upper layer neurons, labeled with Cux1,
were the most affected (SI Appendix,Fig.S3A–D). To test
whether this effect was cell autonomous or due to abnormalities
in radial glia, we deleted KIF2A only in postmitotic neurons
using Nex-Cre mice (31) (hereafter referred to as Nex-cKO)(SI
Appendix,Fig.S1Eand I). Like Emx1-cKO,Nex-cKO mice dis-
played a widespread distribution of Cux1
+
neurons, extending
from layer I to layer VI (SI Appendix,Fig.S3A–D).
At postnatal day (P) 21, brain size was roughly similar between
control and mutant mice (Fig. 1A). However, closer scrutiny
revealed the presence of few apoptotic cells (cCasp3-positive; Fig.
1B) along with shrinkage of the somatosensory cortex in Emx1-cKO
mice (Emx1-cKO:4.71 ±1.22%, P=0.0026; Nex-cKO:
0.95 ±3.12%, P=0.259; Fig. 1C). At P40, the reduction of cor-
tical thickness was exacerbated (Emx1-cKO mice: 32.07 ±3.9%,
P=0.0003; Nex-cKO:29.37 ±3.35%, P=0.0001) (Fig. 1C).
The mutant brains were markedly smaller (Fig. 1D), and many
cCasp3-positive cells and reactive astrocytes were detected (Fig. 1
Eand F). The upper cortical layers were relatively more affected
than the deep layers (Cux1
+
layer thickness: Emx1-cKO:45.8 ±
7.1%, P=0.003; Nex-cKO:32.6 ±10.4%, P=0.035; Ctip2
+
layers thickness: Emx1-cKO:25.9 ±4.6%, P=0.005;
Nex-cKO:18.4 ±4.4%, P=0.014) (Fig. 1 Gand H). At P120,
the reduction reached 59.16 ±3.30% (P<0.0001) and
62.50 ±4.14% (P=0.0001) in Emx1-cKO and Nex-cKO
mice, respectively (Fig. 1 C,I–K). Given that Nex-Cre recombines
only in neuronal and not in glial cells (31), the neurodegenerative
effect was cell autonomous. Other cortical areas such as the motor
and visual cortices as well as hippocampal formation were also
reduced in Emx1-cKO and Nex-cKO mice (SI Appendix,Fig.S4).
KIF2A Is Essential for Neuronal Function. To function prop-
erly, neurons must form synapses and be integrated into neuro-
nal networks. To test whether the lack of KIF2A could affect
synapse formation, we cultured hippocampal neurons from
control and Kif2a
/
embryos at E18.5 and used immunostain-
ing for pre- and postsynaptic markers VGlut1 and PSD95 to
identify glutamatergic synapses after 15 d in vitro (15 DIV)
(Fig. 2A). Kif2a
/
neurons exhibited lower density of PSD95
terminals (44.76 ±7.37%, P<0.0001) and diminished
colocalization of PSD95 and Vglut1 in dendrites compared
with control neurons (synaptic puncta: 54.27 ±8.78%,
P<0.0001; Fig. 2 Band C). In line with this, Western blot
analysis of cortical extracts showed a reduction in PSD95
(75.45 ±14.8%, P<0.0001) and Vglut1 (45.71 ±18.49%,
P=0.039) in Emx1-cKO mice at P21, suggesting that the loss
of KIF2A disturbed the formation of glutamatergic synapses
(Fig. 2D). We next analyzed the intrinsic electrophysiological
features of neurons by whole-cell recording of pyramidal neurons
in acute brain slices. At P21, neurons were smaller and failed
to maintain the membrane potential (Vm; Fig. 2E). The input
resistance was higher and the maximum Na-current was lower in
KIF2A-depleted neurons compared with controls (Fig. 2E),
potentially due to the decrease in cell size and/or a change in
membrane channel insertion in mutant neurons. Higher input
resistance implies lower membrane conductance so that any cur-
rent entering the neuron could have a larger or faster impact on
membrane potential. Indeed, the rheobase of mutant neurons
was reduced, and less current was required to induce action
potentials (Fig. 2Eand SI Appendix,Fig.S5A–C).
To understand how KIF2A mutant neurons were integrated
in the cortical network, we examined spontaneous excitatory and
inhibitory postsynaptic currents (sEPSCs and sIPSCs, respec-
tively) (Fig. 2 F–Jand SI Appendix,Fig.S5D–H). KIF2A
mutant neurons had significantly reduced sEPSC frequency
(51.83%, P≤0.0001) and amplitude (14.43%, P=0.008)
(Fig. 2 F–H). The decay time and area were reduced (decay
time: 14.47%, P=0.016; area: 31.40%, P≤0.0001) (Fig. 2
F,I,andJ), indicating that glutamatergic synapses were smaller,
receptor subunit composition was different, and/or the postsynap-
tic channel density was reduced. We also analyzed the sIPSCs in
mutant P21 cortex. Pyramidal neurons receive inhibitory inputs
from cortical interneurons, which are not mutated in Emx1-cKO
mice. The frequency and amplitude of sIPSCs were similar in
control and mutant neurons (frequency: +11.1%, P=0.725;
amplitude: +1.64%, P=0.516) (SI Appendix,Fig.S5D–F).
However, the decay time (28.75%, P≤0.0001) and the total
current (area: 26.98%, P=0.001) were smaller, possibly due to
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differences in the composition of postsynaptic channels (SI
Appendix,Fig.S5D,G,andH).
KIF2A cKO Mice Exhibit Wiring Abnormalities. KIF2A is impli-
cated in axonal pruning during development and affects the
length of axon collaterals (14, 16). Yet, whether this has an impact
on axon targeting and neuronal connectivity is unknown. To
address this, we analyzed axon pathfinding and formation of the
main tracts. Cortical neurons form corticocortical and cortico-
subcortical connections. To evaluate corticocortical projections,
we used in utero electroporation at E15.5 to label prospective neu-
rons in layers II and III and analyzed callosal projections at P10.
In control animals, axons crossed the CC and ran radially through
the contralateral hemisphere, reaching the most upper cortical
layers (Fig. 3A). In sharp contrast, mutant callosal axons, although
they were able to cross the midline, ended their journey in the
white matter or deep cortical layers (Fig. 3A). We evaluated the
distribution of the mean fluorescence intensity and found that
it was greatly reduced in upper layers of the contralateral side
(Fig. 3B). At P21, the CC was thinner in Emx1-cKO mice
compared with control mice (Fig. 3 Cand D). To assess
cortico-subcortical projections, we used the Thy1-YFP trans-
gene to identify pyramidal neurons of layer V and examine
their projections (32). Compared with control mice, the num-
ber of axons in the corticospinal tract (CST) was significantly
reduced in the brain stem, and the pyramidal decussation was
almost absent in Emx1-cKO and Nex-cKO mice (SI Appendix,
Fig. S6 A–C). CST axons were not detected in the spinal cord
in conditional mutant mice (SI Appendix, Fig. S6 A’–C’). The
loss of axons in the mutant cortex was confirmed by a gradual
decrease in Tau-1 protein levels from P1 to P21 (Fig. 3E).
Finally, even though we did not delete KIF2A in the thalamus,
we analyzed thalamocortical projections at P21 to assess how
wild-type thalamic inputs are integrated in the mutant cortex.
Fig. 1. Severe premature neurodegeneration in Kif2a cKO mice. (A) Representative images of whole brains (Top) and Nissl-stained coronal sections (Bottom)ofthe
indicated genotypes at P21. (B) Coronal sections from P21 brains stained for cCasp3. White arrowheads point to cCasp3-positive cells. (C) Cortical thickness at the
level of the somatosensory area (S1) at P21 (control =1,287 ±11 μm, n=7; Emx1-cKO =1,226 ±11 μm, n=6; Nex-cKO =1,275 ±46 μm, n=5); P40 (control =
1,325 ±42 μm, n=4; Emx1-cKO =874 ±16 μm, n=3; Nex-cKO =936 ±14 μm, n=4); and P120 (control =1,217 ±16 μm, n=3; Emx1-cKO =497 ±37 μm, n=3;
Nex-cKO =457 ±48 μm, n=3). (D) Representative images of whole brains (Top) and Nissl-stained coronal sections (Bottom) at P40. (E) Coronal sections from P40
brains stained for cCasp3. (E’and E’’)Magnifications of the boxed areas in Efrom Emx1-cKO and Nex-cKO, respectively, showing cCasp3-positive cells. (Fand G) Coro-
nal sections from P40 brains stained for GFAP (F)andCux1andCtip2(G). The yellow lines in Goutline the thickness of upper cortical layers (II to IV), whereas the
white lines delineate the thickness of deep cortical layers (V and VI). (H) Quantification of thickness of Cux
+
/upper layers (control =460.3 ±23.4 μm; Emx1-cKO =
249.4 ±23 μm; Nex-cKO =310 ±41.8 μm) and Ctip2
+
/deep layers (control =524 ±20.2 μm; Emx1-cKO =388.1 ±13.5 μm; Nex-cKO =427 ±11.1 μm); n=3 animals
for each genotype. (I) Representative images of whole brains (Top) and Nissl-stained coronal sections (Bottom)oftheindicatedgenotypesatP120.(Jand K)Higher
magnification of brain coronal sections stained with Nissl (J)oranti–NeuN antibodies (K)fromcontrol(Left), Emx1-cKO (Middle), and Nex-cKO (Right) mice. Scale bars:
(A,D,andI)2mm(Top), 1 mm (Bottom); (Band E–G)100μm; (E’)50μm; (J)500μm; and (K) 200 μm. Data are represented as mean ±SEM. Values were obtained by
unpaired. Student’st-test; *P<0.05, **P<0.01, and ***P<0.001. Dapi, 40,6-diamidino-2-phenylindole; GFAP, Glial fibrillary acidic protein.
PNAS 2022 Vol. 119 No. 46 e2209714119 https://doi.org/10.1073/pnas.2209714119 3of11
In contrast to control animals, more thalamic axon terminals
(labeled with Vglut2) were found in deep cortical layers (Fig.
4A, dashed line), and none reached layer I (Fig. 4A, arrow-
heads). A reduced number of terminals were observed in layer
IV, where they shaped lessened barrel fields (Fig. 4A), but stel-
late cells were not correctly organized along the walls of barrel
fields (Fig. 4B). We tested whether layer IV neurons were
nevertheless activated by thalamic afferents using expression of
retinoic acid–related orphan receptor Beta (Rorβ), a thalamic
activity-dependent transcription factor (33, 34). Rorβimmuno-
reactivity was considerably decreased in mutant layer IV com-
pared with control littermates (Fig. 4C), and this was further
confirmed by in situ hybridization that emphasized a robust
reduction of Rorβmessenger RNA (mRNA) (32.40 ±4.95%,
P=0.0002) (Fig. 4 Dand E).
KIF2A Regulates Neuritogenesis. While analyzing cortical lam-
ination and connectivity, we noticed a substantial reduction of
layer I in Emx1-cKO and Nex-cKO mice (SI Appendix, Fig. S3
Cand D, orange lines). This layer comprises apical dendrites
of pyramidal neurons that receive long-range inputs and few
inhibitory interneurons. We stained cortical sections with the
dendritic marker MAP2 and observed an abnormal distribu-
tion. While MAP2 delineated radially oriented apical dendrites
in control mice, it accumulated in cell bodies in Emx1-cKO
and Nex-cKO mice (Fig. 5 Aand B). To investigate this
further, we electroporated a green fluorescent protein (GFP)–
expressing plasmid at E15.5 and analyzed neuronal morphol-
ogy at postnatal stages. At P10, KIF2A-deficient neurons
exhibited smaller dendritic trees compared with control
neurons (SI Appendix,Fig.S7A). At P21, mutant dendrites
frequently displayed swellings and dendritic spines could
barely be recognized (Fig. 5C). These abnormalities were not
secondary to defects in neuronal migration since they were
not restricted to mislocalized neurons. To test if the effect
of KIF2A was cell autonomous, we electroporated Cre-
and floxed GFP-expressing plasmids in cortices of Kif2a
F/F
embryos to delete KIF2A only from sparse cortical neurons,
identified by Cre-dependent GFP expression. Mutant neurons
in control background displayed defects in neuronal shape,
suggesting that the function of KIF2A is cell autonomous (SI
Appendix, Fig. S7B). Layer V pyramidal neurons labeled with
Fig. 2. KIF2A is essential for glutamatergic synapse maintenance and neuronal function. (A) Hippocampal neurons from Kif2a
+/+
and Kif2a
/
embryos at
15 DIV, immunostained with anti-Vglut1, -PSD95, and -MAP2 to visualize glutamatergic synapses. (B) Representative images of a dendrite from Kif2a
+/+
(Top)
and Kif2a
/
(Bottom) pyramidal neurons showing presynaptic boutons (Vglut1, green) and postsynaptic terminals (PSD95, red). (C) Quantification of PSD95
and synaptic puncta per 100 μm (PSD95: Kif2a
+/+
=66.62 ±3.56, Kif2a
/
=36.80 ±3.39; Puncta: Kif2a
+/+
=40.41 ±2.79, Kif2a
/
=18.48 ±2.2; n=30 neu-
rons for each genotype). (D) Western blot analysis and quantification of the relative amount of PSD95 and Vglut1 from P21 control (Ctl) and Emx1-cKO cortex
extracts. (E) Means ±SEM and Pvalues of intrinsic physiological characteristics of cortical somatosensory L2/3 pyramidal neurons recorded in P21 brain
slices. (F)Top: Example traces of sEPSCs in control (blue) and Emx1-cKO (gray) recordings. Bottom: 10 Superimposed individual (shaded) sEPSCs and the
averaged (bold) sEPSC in control (blue) and Emx1-cKO (gray) neurons. (G) sEPSC frequency (control =12.27 ±0.9 Hz; Emx1-cKO =5.91 ±1.3 Hz). (H) sEPSC
amplitude (control =17.46 ±0.9 pA; Emx1-cKO =14.94 ±0.9 pA). (I) sEPSC decay time (control =2.31 ±0.1 ms; Emx1-cKO =1.98 ±0.1 ms). (J) sEPSC area
(control =45.89 ±3.3 fC; Emx1-cKO =31.48 ±2.5 fC). Sample size for G–J, control n=21 and Emx1-cKO n =28 neurons from four different animals. Scale
bars: (A)25μm; (B)5μm. Data are represented as mean ±SEM (Cand D) or as tukey plot (G–J). Values were obtained by unpaired Student’st-test; *P<0.05,
**P<0.01, and ***P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Max., maximum; Vm, membrane potential.
4of11 https://doi.org/10.1073/pnas.2209714119 pnas.org
Thy1-YFP also exhibited aberrant morphology (SI Appendix,
Fig. S6 Dand E).
To explore further the function of KIF2A in dendrite forma-
tion, we used primary cultures of hippocampal neurons. At
15 DIV, MAP2 was distributed in dendrites in control but not
in mutant neurons (Fig. 5D). Some portions were swollen and
contained dense signal, whereas others were devoid of MAP2
signal (Fig. 5E). Primary dendrites were significantly thinner
in KIF2A-depleted neurons (26.89 ±2.48%, P<0.0001)
(Fig. 5F). We conducted a Sholl analysis and found that
Kif2a
/
neurons formed more proximal (near the soma) and
less distal intersections compared with control neurons (Fig.
5G), indicating a defect in dendritic branching. Mutant neu-
rons formed more but shorter primary dendrites compared to
wild-type neurons (number: +37.78 ±7.8%, P<0.0001;
length: 43.7 ±5.14%, P<0.0001) (Fig. 5 H–J). Mutant
neurons had significantly fewer and shorter secondary dendrites
than controls (number of secondary dendrites per primary den-
drite: 43.25 ±9.1%, P<0.0001; secondary dendrite length:
33.92 ±5.46%, P<0.0001; Fig. 5 H,K, and L).
The presence of longer axon collaterals in Kif2a KO mice (14)
prompted us to assess axonogenesis in KIF2A-deficient neurons.
We used TRIM46 antibodies that label the proximal axon and
found that 48.44 ±8.52% of mutant neurons versus 10.27 ±
0.71% of control neurons had two or more axons (Fig. 6 A
and B;P=0.0043). A key determinant of axon versus dendrite
formation is the stabilization of MTs (35). During the first steps
of neuronal polarization, the neurite which accumulates more sta-
ble MTs, forms the axon (35). Since KIF2A regulates MT
dynamics (13, 15, 16), we analyzed the PTMs of tubulin using
immunostaining for acetylated tubulin (long-lived/stable MT)
and tyrosinated tubulin (freshly polymerized/dynamic MT). At
10 DIV, Kif2a
/
hippocampal neurons accumulated more acety-
lated tubulin and less tyrosinated tubulin than control neurons
(Fig. 6C). Acetylated tubulin was not evenly distributed along
dendrites in the mutant neurons. Instead, it clustered locally,
forming a pearl necklace-like structure (Fig. 6C, arrowheads). The
axon’s growth cone was considerably larger and contained more
stable MTs (Fig. 6C).Westernblotanalysisofcorticalextracts
from control and Emx1-cKO mice at P1 confirmed an increase
in stable MTs in the mutant (acetylated tubulin: +69.4 ±35%,
P=0.088; polyglutamylated tubulin: +18.1 ±7%, P=0.035)
(Fig. 6D). We assessed the level of CLASP1 and EB3, two +TIP
proteins important for MT dynamics (36). Both were significantly
reduced in the mutant cortex (CLASP1: 42.4 ±15%, P=0.02;
and EB3: 64 ±26%, P=0.034) (Fig. 6E). These results
Fig. 3. Defective connectivity in Kif2a cKO mice.
(A)Left: P10 coronal sections from control and
Emx1-cKO mice that were electroporated with a
GFP-expressing plasmid at E15.5. (Right) Higher
magnifications of the boxed areas emphasizing
the electroporated side (Ipsi) and contralateral
somatosensory cortex (Contra). (B) Mean fluores-
cence intensity per bin in control and Emx1-cKO
mice in the electroporated (Ipsilateral, Left) and
(contralateral, Right) sides. (C) Luxol fast blue
(myelin) staining of brain coronal sections at P21
underscoring the absence of callosal commissure
in Emx1-cKO mice. (D) Immunofluorescence for cal-
bindin (CB) on coronal sections from control and
Emx1-cKO mice at the level of the CC at P21. (E)
Western blot analysis and quantification of Tau-1
levels at P1 (+29 ±52%, P=0.613), P10 (40 ±
13%, P=0.022), and P21 (38.2 ±9%, P=0.001)
in cortical extracts from control and Emx1-cKO
mice. Scale bars: (A,Left and C) 500 μm; (A,Right
and D) 100 μm. Data are represented as mean ±
SEM. Values were obtained by unpaired Student’s
t-test; n.s., not significant, *P<0.05, **P<0.01,
and ***P<0.001. IUE, In utero electroporation; Ctl,
control; Dapi, 40,6-diamidino-2-phenylindole;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
PNAS 2022 Vol. 119 No. 46 e2209714119 https://doi.org/10.1073/pnas.2209714119 5of11
emphasize the importance of KIF2A for MT dynamics, neuronal
polarization, and neurite morphogenesis.
KIF2A Is Essential for Transport of Lysosomes. MTs serve as a
railway for organelle transport. Thus, any change in MT com-
position or dynamics can affect intracellular transport. To test
whether transport is affected in the absence of KIF2A, we
labeled lysosomes in primary cultures of hippocampal neurons
with Lysotracker DND-99 and evaluated their movement
along a segment of the axon (Fig. 7A, white arrowheads) from
time-lapse videos. Particle displacements were converted to
kymographs, and different transport parameters were analyzed
(Fig. 7B). The mean speed in single anterograde runs was sig-
nificantly lower in mutant neurons compared with controls
(42.6 ±9.3%, P<0.0001) (Fig. 7C). The speed of retro-
grade movements was also reduced, but this was not statistically
significant (13.21 ±7.7%, P=0.089) (Fig. 7C). The
pausing time (% of the time the lysosomes were static) was
higher in mutant neurons than in controls (+50.14 ±11.14%,
P<0.0001) (Fig. 7D). In line with reduced velocity and
increased pausing time, KIF2A mutant neurons showed a sig-
nificant reduction in single run length (anterograde movement:
28.79 ±10.8%, P=0.008; retrograde movement: 15.79 ±
7.1%, P=0.025) (Fig. 7E). Many lysosomes were trapped in
axonal beadings (yellow arrows in Fig. 7B), but the overall den-
sity of lysosomIs in the axon was similar between control and
mutant neurons (Fig. 7F). These results show that the velocity
and traveled distance were reduced in mutant neurons and that
more lysosomes were static, suggesting that intracellular trans-
port is affected by the loss of KIF2A.
Deletion of KIF2A in Mature Neurons Is Sufficient to Cause
Their Loss. The expression of KIF2A is maintained in the adult
brain (SI Appendix, Fig. S1 Hand I; Allen brain atlas: https://
mouse.brain-map.org/). To assess the protein’s function in
mature neurons and circumvent the developmental effect, we
ablated it from glutamatergic neurons of the cerebral cortex
and hippocampus using CamKIIa-Cre
ERT2
, while expressing
Tomato (Ai14) in Cre-expressing neurons (37). We injected
CamKII-cKO (Kif2a
F/F
; tdTomato; CamKII-Cre
ERT2
) and con-
trol (Kif2a
+/+
; tdTomato; CamKIIa-Cre
ERT2
) mice with tamox-
ifen for 5 consecutive d starting from P40 and analyzed their
brains 8 wk after the last injection (Fig. 8 Aand B). CamKII-
cKO brains (post-tamoxifen injections) were markedly smaller,
and the neocortex was thinner compared with control brains
(16.7 ±2%, P<0.0001; Fig. 8 C–E). The number of
NeuN-positive cells in the somatosensory cortex was sig-
nificantly reduced in mutant compared with control mice
(18.9 ±2.7%, P=0.0005; Fig. 8 Fand G). Moreover, we
observed reactive astrocytes in mutant cortex, suggesting an
increase of astrogliosis, likely secondary to cell death (Fig. 8H).
As expected, neuronal migration was not affected (Fig. 8 I
and J). However, the mutant hippocampus had a lower neuro-
nal density especially in the CA1 (20 ±6.7%, P=0.02;
Fig. 8 Jand K). These results indicate that KIF2A, independent
of its developmental function, is necessary for neuronal survival.
Discussion
KIF2A Regulates MT Dynamics and Intracellular Transport.
KIF2A catalyzes tubulin disassembly, and its loss not only
reduces depolymerization of the MT but also disrupts its func-
tion, since KIF2A-depleted neuroblasts display slower displace-
ment of EB3 comets (15). Down-regulation of KLP10A, the
Drosophila ortholog of KIF2A, reduced MT dynamics and
increased MT pausing time in Drosophila S2 cells (38). Abnor-
malities in MT dynamics correlate with enhanced PTM of
tubulin, such as acetylation and polyglutamylation. +TIP pro-
teins interact with the labile domain of the MT (39). There-
fore, increased acetylation and polyglutamylation along with
decreased levels of +TIP proteins CLASP1 and EB3 indicate
that the lack of KIF2A triggers changes in the composition of
the MT, enhancing stable MTs at the expense of dynamic
MTs. CLASP1 modulates MT dynamics and promotes neurite
extension (40, 41). EB proteins bind the MT plus-end, mediate
MT–protein interactions, and increase growth velocity (42–44).
In KIF2A mutant neurons, MAP2 accumulates in the cell body
instead of dendrites. MAP2 is important for MT assembly, and
its loss results in shorter dendrites (45, 46), a phenotype found
in Kif2a
/
neurons. Higher levels of stable MTs and lower lev-
els of MAP2, CLASP1, and EB3 could account for the aberrant
polarization, supernumerary axons (35), and dendritic abnormal-
ities. The delicate balance between tubulin PTM and MAPs is
important for intracellular transport (47–50). For instance, an
increase in tubulin polyglutamylation leads to a reduction in axo-
nal transport and neurodegeneration (51–53). Mutant neurons
for spastin, an MT-severing protein, have increased levels in
tubulin polyglutamylation that, in turn, reduce KIF5 binding to
Fig. 4. Sensory input processing in Kif2a cKO mice. (A) Immunofluorescence
for Vglut2 on coronal sections from control and Emx1-cKO mice at P21. White
dashed lines delineate lower cortical layers innervated by thalamocortical
inputs, whereas yellow arrowheads point to the lack of thalamocortical inner-
vation of layer I in Emx1-cKO mice. (B)Immunofluorescence for Vglut2 in
transversal sections of P21 control and Emx1-cKO mice emphasizing barrel
fields. White arrowheads depict the walls of barrel fields formed by stellate
cells. (C)Rorβimmunostaining of P21 coronal sections from control and
Emx1-cKO mice. (D) Coronal sections of P21 brains from control and Emx1-
cKO mice hybridized with a Rorβriboprobe showing mRNA expression in the
cortex (Cx) and suprachiasmatic nucleus (SCh). (E) Mean intensity of the signal
in Cx relative to SCh (Control: 1.05 ±0.04; Emx1-cKO:0.71±0.02; n=3for
each genotype) and distribution of Rorβ-mRNA signal across cortical layers of
control (blue) and Emx1-cKO (gray) mice at P21. Orange dashed lines delimit
layer IV of the cortex. Scale bars: (A–C)100μm; (D) 1 mm. Data is represented
as mean ±SEM. Values were obtained by unpaired Student’st-test; *P<0.05,
**P<0.01, and ***P<0.001. Dapi, 40,6-diamidino-2-phenylindole.
6of11 https://doi.org/10.1073/pnas.2209714119 pnas.org
MTs (54). Given that the lysosomes are transported by KIF5,
the aberrant lysosome transport observed in KIF2A-depleted
neurons could be due to increased polyglutamylation. Therefore,
KIF2A is critical to maintain MT dynamics, intracellular trans-
port, and neuronal survival.
Loss of KIF2A Triggers Neurodegeneration. Our results show
that KIF2A is important during development for polarization,
maturation, and survival of neurons. Conditional ablation of
KIF2A from progenitor or early postmitotic neurons triggers
neurodegeneration, leading to severe shrinkage of the cerebral
cortex (less than half the volume of a normal cortex by the age
of 4 mo) and cortical dysfunction. Deletion of KIF2A in the
mature brain also triggers neurodegeneration, although to a lesser
extent, indicating that KIF2A is not only necessary during
developmental but also in the mature brain. Neurodegenerative
diseases have in common defective MTs, loss of functional
synapses, axon degeneration, and disintegration of neuronal net-
works. All these features are found in KIF2A conditional knock-
outs. Deletion of KIF2A affects the intracellular transport of
lysosomes, and defective lysosome transport was associated with
autophagic stress and neurodegeneration (55, 56). KIF2A was
recently linked to AD (28, 29), and its activity depends on phos-
phorylation by different kinases such as CDK5, PKA, and
TTBK2 (21, 57). Importantly, all these kinases have been impli-
cated in Tau phosphorylation and neurodegenerative diseases.
KIF2A Links Neurodevelopment to Neurodegeneration. While
the function of KIF2A in neuronal migration is well established
(14, 58), its role in neurogenesis remains controversial. In utero
electroporation of KIF2A shRNA decreases the proliferation of
progenitors and promotes neuronal differentiation (8). In contrast
Fig. 5. KIF2A shapes neuronal morphology. (A) Immunofluorescence for MAP2 on P21 coronal sections from control, Emx1-cKO, and Nex-cKO mice. (B) Fire
look-up table (LUT) representation of MAP2 staining in the cortex underscoring the distribution, intensity, and shape of the signal. (C) Coronal sections of
P21 brains from control and Emx1-cKO mice electroporated at E15.5 with a GFP-expressing plasmid. Pyramidal neurons are distributed in the upper layers
in control and scattered in all layers in Emx1-cKO. In addition, the dendritic arborization is altered with the presence of swellings and absence of spines. (D)
Hippocampal primary culture from E18.5 Kif2a
+/+
and Kif2a
/
embryos immunolabeled with anti-MAP2 antibody at 15 DIV and represented in fire LUT to
highlight the difference in fluorescence intensity and distribution. (E) Representative images of dendrites from Kif2a
+/+
and Kif2a
/
at 15 DIV. (F) Quantifica-
tion of dendritic thickness (Kif2a
+/+
=2.38 ±0.05 μm, n=43 dendrites; Kif2a
/
=1.74 ±0.04 μm, n=50 dendrites). (G) Sholl analysis showing the number
of intersections every 20 μm from the cell body until 380 μm(n=32 and 47 neurons for Kif2a
+/+
and Kif2a
/
, respectively). (H) Illustration of primary (green)
and secondary (magenta) dendrites in control and mutant neurons. (I) Average number of primary dendrites per neuron (Kif2a
+/+
=6.1 ±0.3, n=21
neurons; Kif2a
/
=8.46 ±0.3, n=26 neurons). (J) Mean length of primary dendrites (Kif2a
+/+
=272.3 ±12.8 μm, n=20 neurons; Kif2a
/
=153.4 ±6μm,
n=19 neurons). (K) Number of secondary dendrites per primary dendrite per neuron (Kif2a
/
=3.6 ±0.3, n=21 neurons; Kif2a
+/+
=2.1 ±0.1, n=26
neurons). (L) Mean length of secondary dendrites (Kif2a
/
=110.4 ±5, n=21 neurons; Kif2a
+/+
=72.9 ±3.5, n=19 neurons). All the values are represented
as mean ±SEM except for graphs I and K, represented as a Tukey plot. Scale bars: (A) 100 μm; (B)50μm; (C,Left) 100 μm; (C,Right Top)50μm; (C,Right
Bottom)5μm; (D)50μm; and (E)5μm. Data are represented as mean ±SEM (F, G, J and L) or as tukey plot (Iand K). Values were obtained by unpaired
Student’st-test; *P<0.05, **P<0.01, and ***P<0.001. Dapi, 40,6-diamidino-2-phenylindole; IUE, In utero electroporation.
PNAS 2022 Vol. 119 No. 46 e2209714119 https://doi.org/10.1073/pnas.2209714119 7of11
electroporation of KIF2A variants c.961C >G/p.His321Asp and
c.950G >A/p.Ser317Asn causes the opposite effect, enhancing
the proliferation of progenitor cells and decreasing the produc-
tion of neurons (9). KI mice expressing one copy of the first
variant (c.961C >G, p.His321Asp; Gene ID: 3796) have no
change in proliferation but an enhanced embryonic cell death.
Consequently, the mutant brain is smaller at birth (10). In the
current work, we deleted KIF2A specifically in cortical progeni-
tors and did not observe any change in either proliferation or sur-
vival of these cells. Mutant mice were born with normal cortical
size. Hence, human variants could have a dominant negative
activity and/or could behave as a gain-of-function mutation dur-
ing embryonic neurogenesis, since the mutant protein interacts
with different partners compared with the wild type (20). Adult
(4-mo-old) mice carrying the human variant c.961C >G,
p.His321Asp exhibited a slight reduction of cortex and hippo-
campal area that was ascribed to embryonic cell death. Kif2a
cKO mice (Emx1-cKO and Nex-cKO), by contrast, displayed a
severe neurodegeneration at the same age (4 mo). The somatosen-
sory cortex was reduced by 60%, whereas the motor and visual
cortex and hippocampal area were reduced by 75%. Postnatal
deletion of KIF2A confirmed that KIF2A, independent of its
role during development, is essential for neuronal survival. It is
worth mentioning that microcephaly due to KIF2A mutations in
patients worsens postnatally (25), and in some cases, patients are
diagnosed with secondary microcephaly (DECIPHER patient
280143, c.1762G >T, p.Val588Phe), suggesting that these
mutations affect postnatal processes such as neuronal maturation
or survival and not only embryonic development.
Materials and Methods
Animals. All animal procedures were carried out in accordance with European
guidelines (2010/63/UE) and were approved by the animal ethics committee of
Fig. 6. Polarization and MT content of KIF2A-deficient neurons. (A) Hippo-
campal neurons from Kif2a
+/+
and Kif2a
/
embryos at 10 DIV, immunola-
beled with anti-TRIM46 and anti-MAP2 antibodies to highlight the proximal
axon (white arrowheads) and dendrites, respectively. (B) Percentage of neu-
rons with two or more axons (Kif2a
+/+
=10.27 ±0.7%, n=79 neurons;
Kif2a
/
=48.44 ±8.52%, n=94 neurons from four different experiments).
(C) Hippocampal neurons from Kif2a
+/+
and Kif2a
/
embryos at 10 DIV,
immunolabeled with anti-tyrosinated tubulin (Tyr-Tub), anti-acetylated
tubulin (Act-Tub), and anti–F-Actin antibodies. White arrowheads indicate
accumulation of acetylated-tubulin in dendrites of Kif2a
/
neurons. (D)West-
ern blot analysis and quantification of the relative amount of Act-Tub and pol-
yglutamylated tubulin (Poly-E) in P1 cortical extracts from control (Ctl) and
Emx1-cKO.(E) Western blot analysis and quantification of the relative amount
of CLASP1 and EB3 from P21 control and Emx1-cKO cortical extracts. Scale
bars: (A)25μm; (C,Left)25μm; and (C,Right)5μm. Data are represented as
mean ±SEM. Values were obtained by unpaired Student’st-test; n.s., not
significant, *P<0.05, **P<0.01, and ***P<0.001.
Fig. 7. Disrupted lysosomal transport in Kif2a
/
neurons. (A) Hippocampal
neuron at 5 DIV transfected with LysoTracker (red). White and yellow arrow-
heads point to the axon and dendrites, respectively. (B) Kymograph gener-
ated from 1.5-min live imaging of lysosomes moving anterogradely (green
lines), moving retrogradely (red lines), or static (blue lines). Yellow arrows
depict trapped lysosomes. (C) Distribution and mean of the speed of ante-
grade and retrograde movements (anterograde: Kif2a
+/+
=0.621 ±0.05 μm/s,
n=221 runs; Kif2a
/
=0.357 ±0.03 μm/s, n=160 runs; retrograde: Kif2a
+/+
=
0.564 ±0.03 μm/s, n=293 runs; Kif2a
/
=0.490 ±0.03 μm/s, n=296 runs).
(D) Pausing time of lysosomes (Kif2a
+/+
=17.54 ±2.5%, n=131 runs; Kif2a
/
=
35.18 ±3.1%, n=113 runs). (E) Mean of length of anterograde and retrograde
movements of single runs (anterograde: Kif2a
+/+
=2.78 ±0.25 μm, n=
264 runs; Kif2a
/
=1.98 ±0.14 μm, n=215 runs; retrograde: Kif2a
+/+
=3.8 ±
0.2 μm, n=499 runs; Kif2a
/
=3.2 ±0.17 μm, n=517 runs). (F) Lysosome
density in the axon (Kif2a
+/+
=17.21 ±0.8, n=20 axons; Kif2a
/
=17.04 ±0.9,
n=15 axons). Scale bar: (A)10μm. Data are represented as mean ±SEM. Val-
ues were obtained by unpaired Student’st-test; n.s., not significant, *P<0.05,
**P<0.01, and ***P<0.001.
8of11 https://doi.org/10.1073/pnas.2209714119 pnas.org
the Universit
e catholique de Louvain under agreement 2019/UCL/MD/006. Mice
were housed in a standard 12-h dark/12-h light cycle. The temperature was
between 20 and 24°C and humidity between 40% and 60%. We used the fol-
lowing mouse lines: Emx1-Cre (30), Nex-Cre (31), CamKIIa-Cre
ERT2
(37) Kif2a
F/F
(15), Thy1-YFP (32), Ai14 (59), and Kif2a
/
.To generate the Kif2a
+/
mouse
line, we crossed Kif2a
F/+
males with PGK-Cre females (60) and intercrossed
Kif2a
+/
females and males to obtain Kif2a
/
and Kif2a
+/+
embryos. To pro-
duce Kif2a
FlF
;Emx1-Cre (Emx1-cKO)andKif2a
FlF
;Nex-Cre (Nex-cKO)mice,we
crossed Emx1-Cre;Kif2a
F/+
and Nex-Cre;Kif2a
F/+
males with Kif2a
F/F
females.
Kif2a
F/F
littermates (without Cre) were considered as control mice. To produce
Kif2a
FlF
;CamKII-Cre
ERT2
(CamKII-cKO) mice, we crossed CamKII-Cre
ERT2
;Kif2a
F/+
males with Kif2a
F/+
females. CamKII-Cre
ERT2
Kif2a
+/+
littermates were consid-
ered control mice. The day of the vaginal plug was considered E0.5. Tamoxifen
(Sigma-Aldrich, T5648) was diluted in 10% ethanol in sunflower seed oil (Sigma-
Aldrich, S5007) (30 mg/mL) and intraperitoneally injected at 180 mg/kg/d for
5consecutived.
Brain Tissue Preparation and Sectioning. Embryos were fixed in 4% parafor-
maldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4, at room temperature
(RT) for 2 h. Mice were perfused transcardially with PFA 4% in 0.1 phosphate-
buffered saline (PBS), pH 7.4. Brains were harvested and postfixed in the same fix-
ative for 2 h at RT for immunohistochemistry and overnight (ON) at 4 °CforIn situ
hybridization (ISH), Nissl, and myelin staining. Embryonic and postanal brains were
washed in PBS after fixation, embedded in 4% agarose diluted in PBS, and
sectioned with a Leica VT1000S vibratome (80 μmand40μmforembryonicand
postnatal brains, respectively). For barrel field visualization, P21 cerebral cortices were
separated from basal ganglia and other brain tissue, flattened between two slides,
postfixed ON at 4 °C, washed with PBS, and sectioned at 80 μm using a vibratome.
Immunofluorescence. Immunohistochemical staining was performed as pre-
viously described (61). Briefly, vibratome floating sections were blocked in 4%
bovine serum albumin (Amresco, 9048–46-8), 3% goat serum (Sigma-Aldrich,
G9023), and 0.2% Triton X-100 (Sigma-Aldrich, T8787) in PBS at RT for 1 h and
incubated with the primary antibodies diluted in blocking solution at 4 °CON
and with the secondary antibodies for 2 h.
BrdU Injection and Immunolabeling. Pregnant females were injected intra-
peritoneally at E14.5 with BrdU (50 mg/kg body weight; Sigma-Aldrich, B5002).
Embryos were collected after 2 h, fixed in PFA 4% in PB 0.1 M. Vibratome
sections were pretreated with HCl 2 N for 30 min, followed by a 10-min incuba-
tion with borate buffer and immunodetection using Rat anti-BrdU (Serotec,
MCA2060GA; 1:200).
In utero Electroporation. Plasmids used in this study were as follows: pCag-
DsRed (Addgene, #11151; 2 μg/μL), pCag-GFP (Addgene, #11150; 1 μg/μL),
Fig. 8. Postnatal deletion of KIF2A causes neuronal loss. (A) Schematic diagram for experimental procedure used to induce KIF2A deletion. Tamoxifen
(TAM) or vehicle (VEH) was injected for 5 consecutive d starting from P40, and brains were analyzed 8 wk after the last injection (P96). (B)Westernblot
analysis of KIF2A from cortical (Cx) and hippocampal (HC) protein extracts in control and mutant mice. (C) Representative images of whole brains of the
indicated genotypes. (D) Nissl-stained coronal sections in control and mutant mice. Right panels are higher magnification of the yellow areas in left
panels of the primary somatosensory cortex (S1) for each genotype. (E) Quantification of the S1 thickness (CamKII-Ctl TAM P40 =1,148 ±19.9 μm, n=5;
CamKII-cKO TAM P40 =968.3 ±10.3 μm, n=4). (F) NeuN staining in the S1 of control and mutant mice. (G) Number of NeuN-positive cells per 390-μm-
wide stripes (CamKII-Ctl TAM P40 =1,179 ±20.30 cells, n=4; CamKII-cKO TAM P40 =956 ±25.23 cells, n=4). (H) GFAP staining in coronal sections
showing reactive astrocytes in mutant cortex. (I) Cux1 immunostaining in coronal sections. (J)Left: NeuN immunostaining in the hippocampus. Right:
Magnification of the areas boxed in Left panels showing NeuN–positive cells in the pyramidal layer of the CA1 area. (K) Quantification of NeuN-positive
cell density in the CA1 area (CamKII-Ctl TAM P40 =82.31 ±2.2 cells per 0.1 mm
2
,n=5; CamKII-cKO TAM P40 =65.83 ±5.6 per 0.1 mm
2
,n=4). Scale
bars: (C)1.7mm,(D,Left) 1 mm; (D,Right); 100 μm, (F,H,andI)100μm; (J,Left)200μm; and (J,Right)100μm. Data are represented as mean ±SEM.
Values were obtained by unpaired Student’st-test; *P<0.05, **P<0.01, and ***P<0.001. Dapi, 40,6-diamidino-2-phenylindole; GFAP, Glial fibrillary
acidic protein.
PNAS 2022 Vol. 119 No. 46 e2209714119 https://doi.org/10.1073/pnas.2209714119 9of11
pCALNL-GFP (Addgene, #13770; 1 μg/μL), and pCag-Cre (Addgene, #13775;
4ng/μL) (62, 63). Plasmids were purified with a Maxiprep Endofree Kit
(Macherey-Nagel) and diluted in 1x PBS. pCAG-DsRed was added to the mix of
electroporation as a control of efficiency. The DNA solution (with 0.05% Fast-
green) was injected into the lateral ventricle of E15.5 embryos using pulled glass
pipettes. The embryos were electroporated using tweezer-type electrodes
(CUY650-5). Five square electric pulses were passed (40V, 50-ms interval cycle
length, 950-ms interval pause). Electroporated animals, expressing DsRed, were
selected using a fluorescent flashlight lamp (Nightsea, DFP-1).
Quantification of Callosal Axons. Slices from controls and Emx1-cKO electro-
porated brains (n=3 mice for each genotype) were analyzed with a laser scan-
ning confocal microscope (Olympus, Fluoview FV1000). To quantify fluorescence,
we analyzed an area of 350 μm width per 1,150 μm height in Fiji (ImageJ).
This frame was subdivided into 100 equal bins of 350 μm width per 11.5 μm
height, and fluorescence intensity in each bin was quantified. The mean fluores-
cence intensity for each bin was calculated using three animals and represented
as mean ±SEM.
Axonal Transport of Lysosomes. Intracellular transport was assessed using
an adapted protocol from ref. 64. Hippocampal neurons from Kif2a
+/+
and
Kif2a
/
littermate embryos were cultured in a glass-bottomed cell culture dish
35/10 mm (627860, CELLview, Greiner bio-one) coated with laminin (5 μg/mL;
Sigma-Aldrich, L2020) and poly-lysine (100 μg/mL; Sigma-Aldrich, P2636). At
5 DIV, the medium was replaced by warm Neurobasal (NB) containing 50 nM
LysoTracker (LysoTracker Red DND-99; Thermo Fisher Scientific, L7528). Neurons
were incubated for 20 min at 37°C with LysoTracker, washed with 1 mL warmed
NB medium, and supplemented with 1 mL NB medium. They were imaged
using an inverted Zeiss Axio Observer microscope equipped with an environ-
mental chamber (37 °C, 5% CO
2
; Pecon) and Zeiss Axiocam 503 mono camera
and 63x (numerical aperture =1.2) water immersion objective (Zeiss). Time-
lapse series were captured every 1 s for 1.5 min from LysoTracker–positive neu-
rons. Neurons with a pyramidal shape were considered. Images were analyzed
using Fiji software (ImageJ). First, the axon of the positive neuron was straight-
ened from the cell body to the distal axon using the “segmented line”tool of
Fiji. We used the KymoToolBox plugin (65) to analyze the movement of lyso-
somes. Stationary vesicles (moving <0.1 μm/s) and run length of less than
0.3 μm (the size of a lysosome) were not considered for analysis. We assessed
the mean speed and length of individual runs and percentage of time pause of
each vesicle. Eleven neurons with pyramidal shape from each genotype were
analyzed from three independent experiments.
Imaging, Quantification, and Statistical Analyses. Images were captured
with a digital camera coupled with an inverted Zeiss Axio Observer microscope
or in a Laser Scanning confocal microscope (Olympus, Fluoview FV1000). Figures
were prepared using Adobe Photoshop and Adobe Illustrator CC 2019, and
two-dimensional mosaic reconstructions were produced when needed using the
Photomerge tool of Photoshop software package. Cell counting was conducted
manually using Fiji software (ImageJ). For NeuN counting in the cortex, images
were pretreated in Fiji with gray scale attribute filtering (MorpholibJ plugin) and
watershed process before automatic counting. Cells were counted in a 180-μm-
wide stripe (E14.5), 340-μm-wide stripes (P1), and 390-μm-wide stripes
(P21 and mature brains). A minimum of three animals and three slices of each
animal were used for all analyses and quantifications. The exact sample size is
specified in the figure legends. Prism 9 (GraphPad) software was used for statisti-
cal analysis. We described the percentage of difference between control
and mutant conditions in the Results. The mean value and the SEM for each
quantification are indicated in the figure legends. All the quantifications are
represented as mean ±SEM except for graphs in Figs. 2 G–Jand 5 IandKand
SI Appendix,Fig.S5E–H(Tukey plot). Values were obtained by unpaired two-
tailed ttest, two-tailed Mann-Whitney rank sum tests for sEPSC and sIPSC fre-
quency, amplitude, and area, and unpaired two-tailed ttest for sEPSC and sIPSC
decay time. ns, not significant; * P<0.05; ** P<0.01; and *** P<0.001.
The Pvalues are reported in the Results. Observations without quantification were
validated and successfully reproduced in a minimum of three different animals.
Data, Materials, and Software Availability. Mouse lines and other resour-
ces used in the study are available through material transfer agreements. All
data generated during this study have been included in the manuscript and
SI Appendix.
ACKNOWLEDGMENTS. We thank Isabelle Lambermont and Clarisse Fouss for
technical assistance. This work was supported by the following grants: Belgian
Fund for Scientific Research (FNRS) PDR T00075.15, FNRS PDR T0236.20, FNRS-
FWO EOS 30913351, Fondation M
edicale Reine Elisabeth, and Fondation Jac-
ques Et Dani
ele Espinasse (JED)-Belgique. G.C. and N.R.-R are Research Fellow
and Postdoctoral Researcher, respectively, at the Belgian FNRS. F.T. is an Honorary
Research Director at FNRS.
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