Published in Volume
123, Issue 6
(June 3, 2013)J Clin Invest.
Copyright © 2013, American Society for Clinical
Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy
1Department of Neurology, UCSF, San Francisco, California, USA.
2Gladstone Institute of Neurological Disease, San Francisco,
3Howard Hughes Medical Institute, San Francisco,
4Department of Pathology, UCSF, San Francisco,
5Veterans Affairs Medical Center, San Francisco,
Address correspondence to: Louis J. Ptáček, Department
of Neurology, University of California San Francisco, Howard Hughes Medical
Institute, 1550 Fourth Street, UCSF-Mission Bay, Rock Hall 548, San Francisco,
California 94158, USA. Phone: 415.502.5614; Fax: 415.502.5641; E-mail:
firstname.lastname@example.org. Or to: Ying-Hui Fu, Department of Neurology,
University of California San Francisco, 1550 Fourth Street, UCSF-Mission Bay, Rock
Hall 548, San Francisco, California 94158, USA. Phone: 415.502.5614; Fax:
415.502.5641; E-mail: email@example.com.
First published May 15, 2013
Received for publication September 5,
2012, and accepted in revised form March 19,
Adult-onset autosomal-dominant leukodystrophy (ADLD) is a progressive and fatal
neurological disorder characterized by early autonomic dysfunction, cognitive
impairment, pyramidal tract and cerebellar dysfunction, and white matter loss in the
central nervous system. ADLD is caused by duplication of the LMNB1
gene, which results in increased lamin B1 transcripts and protein expression. How
duplication of LMNB1 leads to myelin defects is unknown. To address
this question, we developed a mouse model of ADLD that overexpresses lamin B1. These
mice exhibited cognitive impairment and epilepsy, followed by age-dependent motor
deficits. Selective overexpression of lamin B1 in oligodendrocytes also resulted in
marked motor deficits and myelin defects, suggesting these deficits are cell
autonomous. Proteomic and genome-wide transcriptome studies indicated that lamin B1
overexpression is associated with downregulation of proteolipid protein, a highly
abundant myelin sheath component that was previously linked to another myelin-related
disorder, Pelizaeus-Merzbacher disease. Furthermore, we found that lamin B1
overexpression leads to reduced occupancy of Yin Yang 1 transcription factor at the
promoter region of proteolipid protein. These studies identify a mechanism by which
lamin B1 overexpression mediates oligodendrocyte cell–autonomous
neuropathology in ADLD and implicate lamin B1 as an important regulator of myelin
formation and maintenance during aging.
Myelin defects are characteristic of both common sporadic neurological diseases such as
MS and rare genetic diseases such as adult-onset autosomal-dominant leukodystrophy
(ADLD). Investigation of rare inherited diseases whose pathologic features overlap with
common syndromes often casts light on critical features of common disorders.
Leukodystrophies are a heterogeneous group of rare, usually genetic, disorders
characterized by white matter pathologies. ADLD is a progressive and fatal neurological
disorder with onset typically in the fourth or fifth decade of life. ADLD is
characterized by early autonomic dysfunction and cognitive impairment, followed by
pyramidal tract and cerebellar impairments, and loss of white matter in the brain and
spinal cord on magnetic resonance imaging. ADLD is often misdiagnosed as chronic
progressive MS in its initial phases. ADLD is caused by duplication of the
LMNB1 gene, resulting in increased lamin B1 transcripts and protein
expression (1). The links between lamin B1
overexpression and demyelination are not understood. Improved understanding of ADLD
pathogenesis holds the promise of providing insights into more common sporadic white
Myelin is a lipid-enriched specialized membrane synthesized by oligodendrocytes in the
CNS and Schwann cells in the peripheral nervous system (2). Myelin wraps around axons, leading to a substantial increase in axonal
conductance. Defects in myelin disrupt axonal function and lead to axonal degeneration,
although the precise mechanisms are not known (2).
Several proteins, such as myelin basic protein, myelin-associated glycoprotein, and
proteolipid protein (PLP), are either restricted to, or highly enriched in, the myelin
membrane (3). Mutations of the X-linked
PLP1 gene encoding PLP, the most abundant protein of the CNS myelin
sheath, cause Pelizaeus-Merzbacher disease (PMD), another rare leukodystrophy (4). Mutations in PLP1 ultimately
result in the loss or reduction of PLP in the myelin sheath. PMD patients and rodent
models of PMD show loss of white matter and axonal degeneration, indicating that the
integrity of the myelin-axon unit is highly sensitive to deficits in PLP (5–8).
Lamins are intermediate filament proteins lining the inner nuclear membrane and
distributed throughout the nucleoplasm. There are 2 major mammalian lamin types, lamin A
and B. A-type lamins are derived from the LMNA gene through alternative
splicing, giving rise to 2 isoforms, A and C. B-type lamins, B1 and B2, are encoded by
different genes (LMNB1 and LMNB2) (9, 10). The
nuclear lamina is implicated in a wide range of human diseases collectively known as
laminopathies (11, 12). Numerous functions are ascribed to nuclear lamina, including
docking sites for chromatin, regulation of gene expression, and nuclear stability (13, 14).
Lamin B1 is known to maintain nuclear integrity and mediate transcriptional regulation
We now report a BAC-mediated transgenic approach to generating a BAC transgenic mouse
model of ADLD (Lmnb1BAC). This approach allows the advantage
of expressing full-length lamin B1 murine homolog (Lmnb1) under the
control of endogenous lamin B1 promoter and regulatory sequences.
Lmnb1BAC mice recapitulated many of the features of
ADLD. In addition, we generated a series of transgenic mice overexpressing
Lmnb1 in specific CNS cell lineages. Our findings indicate that
overexpression in oligodendrocytes is sufficient for the onset of histopathological,
molecular, and behavioral deficits characteristic of ADLD. As in ADLD,
pathophysiological effects become evident in adult animals and progressively worsen with
age. Using Lmnb1BAC mice as the starting point for
transcriptome and proteomic profiling, we discovered that PLP is downregulated in these
animals and that the transcriptional occupancy of Yin Yang 1 (YY1), a transcriptional
activator of Plp1 (18), is
reduced. These results provide a potential link between lamin B1 overexpression and PLP
downregulation. Together, our findings reveal a valid in vivo model for investigation of
how aging and genetic predispositions can cause myelin defects with devastating effects
on health and behavior.
Generation of an ADLD mouse model. To investigate the pathophysiological mechanism of lamin B1 overexpression in ADLD,
we generated BAC transgenic mice carrying additional copies of murine WT lamin B1
(Lmnb1BAC). To maintain the regulatory properties
of the endogenous Lmnb1 gene, we made use of large genomic fragments
containing the entire Lmnb1 locus within the BAC. A genomic insert
containing Lmnb1 (Figure 1A)
was isolated from a mouse BAC genomic library. We generated 2 BAC transgenic lines
containing varying numbers of the entire Lmnb1 locus. We performed
expression analyses of lamin B1 by Western blot and quantitative real-time PCR
(qRT-PCR) from hemibrains of 12-month-old Lmnb1BAC
animals. Western blot analysis of Lmnb1BAC transgenic
animals showed approximately 4- (line no. 1) and 2.5-fold (line no. 2) higher
expression compared with WT littermates (Figure 1, B and C). Consistent with protein expression results, the highest
transcript levels were also found in line no. 1; Lmnb1 mRNA showed
approximately 3.5- (line no. 1) and 1.5-fold (line no. 2) higher expression compared
with WT (Figure 1D). Consequently, all of the
following experiments were performed on line no. 1, the highest expressing line. Both
transgenic lines with lamin B1 duplication were born healthy and indistinguishable
from control littermates. Line no. 2 progressed with a milder and later onset
phenotype compared with line no. 1. Line no. 2 did not exhibit any abnormal pathology
up to 12 months of age.
Generation of Lmnb1BAC mice. (A) Map of the genomic insert in the BAC RP23-460J18 clone (RPCI
library, C57BL/6J) used to generate mice with increased dosage of lamin B1
(Lmnb1BAC mice). The genomic insert is 177.20-kb
long and contains the entire Lmnb1 locus. Black boxes represent
lamin B1–coding sequences, and light gray lines indicate the BAC
vector (pBACe3.6) sequence. Primers used for detection of the transgene are shown
(T7 side: F1R1; SP6 side: F2, R2). (B) Representative Western blot of
lamin B1 and α-tubulin in hemibrain lysates from
Lmnb1BAC lines and WT mice at 12 months of age.
(C) Quantification of Western blots normalized to
α-tubulin. (D) qRT-PCR of lamin B1 transcript levels
normalized to GAPDH from corresponding Lmnb1BAC mice
hemibrains. Error bars represent mean ± SEM from 4 independent
ADLD mouse model exhibits cognitive impairment and age-dependent motor
deficits. Cognitive and motor deficits are both observed in ADLD patients. We performed Morris
water maze (MWM) and passive avoidance (PA) tasks in
Lmnb1BAC and WT mice to assess cognitive impairments
at 12 months and accelerated rotarod and balance beam to measure motor deficits at 12
and 24 months of age. The MWM is an established paradigm for testing spatial memory
in rodents (19–21). Lmnb1BAC mice
exhibited substantial spatial memory deficits in an MWM assay compared with WT
controls. WT mice exhibited a preference for the target quadrant where the platform
was located during training compared with other quadrants (Figure 2A), indicating that they remembered the overall
location of the platform. In contrast, Lmnb1BAC mice were
impaired and did not spend more time in the target quadrant compared with others
(Figure 2A). Analyses of virtual platform
crossings during the probe test revealed that WT animals showed a clear preference
for the target platform location (Figure 2B).
Nonselective preferences for the target platform by
Lmnb1BAC mice indicate that they do not recall the
location of the hidden platform and are impaired in spatial memory retention (Figure
2B). Lmnb1BAC and
WT animals performed equally well during the visible platform test and exhibited
equal velocities, demonstrating that Lmnb1BAC animals
were not visually impaired and that neither their cued reference memory nor their
swimming was impaired (data not shown). In agreement with these findings,
Lmnb1BAC mice exhibited impairments in a
step-through PA task, which reflects long-term spatial associative learning and fear
memory of an aversive experience. WT but not Lmnb1BAC
mice showed substantial increase in the latency to avoid the light and step through
the dark chamber where they received an electric shock the day before
(P < 0.01) (Figure 2C). This indicated that Lmnb1BAC mice have
spatial associative memory deficits compared with WT mice at 12 months of age. During
training, the latencies to step through the dark chamber were not different among
genotypes, indicating that all the mice showed the same level of spontaneous aversion
to light. Together, these studies suggest cognitive and memory impairments in
A mouse model of ADLD exhibited cognitive and motor deficits. Lmnb1BAC and WT mice were behaviorally tested to
assess spatial and fear memory in the MWM and PA tests and motor functions in the
rotarod and balance beam tests. (A and B)
Lmnb1BAC mice exhibited spatial memory deficits
in the MWM at 12 month of age. (A)
Lmnb1BAC mice spent substantially less time in the
target quadrant and (B) made less platform area crossings compared
with WT during the probe trial. (C)
Lmnb1BAC mice failed to recall an aversive
experience made on the previous day on the PA test and exhibited decreased
step-through latencies compared with WT mice at 12 months of age. The bars
indicate the mean latencies to enter the dark compartment on the training day
(white) and 24 hours later on the retention day (black).
(D–F) Progressive deterioration of motor
functions in Lmnb1BAC mice compared with WT mice,
(D) Lmnb1BAC mice spent less time on
the accelerated rotarod across all 8 trials at 24 but not at 12 months of age
compared with WT. (E) Lmnb1BAC mice
showed increased latency to traverse a 5-mm–wide balance beam at 12
months and progressively worsened at 24 month. (F)
Lmnb1BAC mice also exhibited increased hind limb
slips compared with WT controls. Values are expressed as mean ± SEM.
n = 10–14 per genotypic group. *P
< 0.05; **P < 0.01; ***P <
Lmnb1BAC animals exhibited progressive motor impairment on 2 different
motor tasks, the accelerated rotarod and balance beam. The accelerated rotarod did not detect significant motor impairments at 12 months of
age in Lmnb1BAC mice compared with WT. However, by 24
months of age, Lmnb1BAC mice were not able to remain on
the rotarod as long as their WT counterparts (Figure 2D). The balance beam, typically a more sensitive test for gait
impairment, detected gait deficits at 12 months and progressed to 24 months of age.
Lmnb1BAC mice showed increased latency and number
of hind limb slips to traverse a 42-cm beam that was 5 mm in diameter (Figure 2, E and F). Together, these data demonstrate
age-dependent motor deficits in Lmnb1BAC animals.
Lamin B1 overexpression results in seizures in ADLD mice. Recent studies report epilepsy in some patients with myelin diseases, including MS
and inherited leukodystrophies such as PMD (22, 23). Murine models of some
leukodystrophies exhibit epilepsy (24). In
addition, some ADLD patients exhibit epilepsy (unpublished observations, Eric Huang).
We observed frequent behavioral seizures in Lmnb1BAC
animals, which varied from generalized spasms and tremors to generalized clonic
activity with atonia and tail extension. To confirm these observations, we used EEG
recording. Cortical EEG recordings revealed frequent spontaneous epileptic activity,
including epileptiform discharges and seizures, in
Lmnb1BAC mice but not in WT controls. Representative
traces over 2 minutes, 30 seconds, of a 24-hour recording showed that
Lmnb1BAC mice, but not WT mice, displayed frequent
interictal spikes (arrowheads) in both left and right hemispheres (Figure 3A). Lmnb1BAC mice
exhibited an approximately 20-fold increase in the number of interictal spikes
compared with WT mice (Figure 3B). Given a
subconvulsive dose of pentylenetetrazol (PTZ), a GABAA receptor
antagonist, Lmnb1BAC animals were more susceptible to
PTZ-induced seizures compared with WT controls. Seven out of nine
Lmnb1BAC animals exhibited seizures within 5 to 7
minutes of drug administration compared with only 1 out of 9 WT animals (Figure 3C). These findings demonstrate that lamin B1
overexpression induces aberrant brain network activity, such as neuronal
hypersynchrony and hyperexcitability, and predisposes animals to epilepsy.
Lamin B1 overexpression results in seizures. Spontaneous EEG activity over the parietal cortex of
Lmnb1BAC and WT mice was recorded for 24 hours.
(A) EEG recordings from the left and right hemisphere,
respectively, are shown for a representative transgenic mouse overexpressing lamin
B1 (top traces) or a WT control (bottom traces). Arrowheads mark interictal
spikes. (B) Quantification of interictal spikes over 24-hour
recordings indicates the occurrence of spontaneous epileptiform activity in
Lmnb1BAC mice specifically. n =
10 animals per genotypic group. (C) Histogram quantifying percentage
of animals seizing after the administration of 30 mg/kg of PTZ. n
= 9 animals per genotypic group. Values are expressed as means ± SEM.
*P < 0.05.
Lamin B1 overexpression results in aberrant myelin formation, axonal
degeneration, and demyelination. To address whether lamin B1 overexpression is associated with myelin pathology, we
examined myelin integrity by electron microscopy in 12- and 24-month-old
Lmnb1BAC and WT mice. Because pathologic studies
show greatest loss of white matter in pyramidal tracts of ADLD patients (25), we performed ultrastructural analysis on
pyramidal tracts in the brain region of the pons. Although these ultrastructural
analyses did not identify overall demyelination in 12-month-old animals assessed by g
ratio (the ratio between the diameter of the inner axon and the total outer diameter
[Figure 4, A and B]), they did identify such
demyelination, however, by 24 months compared with WT controls (Figure 4, C and D). Ultrastructural analyses reveal a
variety of aberrant structures, which were generally dominated by outfoldings,
extensions, and invaginations originating from the myelin sheath. Aberrant myelin
loops consisted of multiple loops within the same myelinated axon, which may result
from a retrograde inversion of a single infolding, since axonal material can be seen
in the gap between the inner and outer infoldings (Figure 4, F and G, 12-month-old animals; Figure 4H, 24-month-old Lmnb1BAC animals).
These morphological abnormalities were not seen in WT mice (Figure 4, E and Q, 12 and 24-month-old animals,
respectively). Apart from these abnormalities of the myelin sheath, axonal
disintegration and even degradation of the entire axon-myelin unit was observed at a
significantly higher level compared with that in WT controls (Figure 4, I and J, 12-month-old animals; Figure 4, M and N, 24-month-old animals). Additionally,
hypermyelinated fibers (Figure 4, K and L,
12-month-old animals) were also observed. Frequent dying and degenerating
oligodendrocytes were also noted in the 24-month-old
Lmnb1BAC animals (Figure 4P), which corresponded with demyelinated axons (Figure 4R) compared with WT controls (Figure 4, O and Q). Toluidine blue staining of adjacent
sections showed similar findings of demyelination and aberrant structures in
24-month-old Lmnb1BAC animals (Supplemental Figure 1;
supplemental material available online with this article; doi:
10.1172/JCI66737DS1). Quantification of approximately 150 to 200 axons
for each group at 12 months of age showed significant axonal degeneration and
aberrant myelin formation in Lmnb1BAC animals compared
with WT controls (Figure 4, S and T), similar to
observations found in some other myelin disorders (26–28). Because
inflammation is seen in active MS lesions, we additionally assessed Iba1 and GFAP
immunostaining for microglia activation and reactive astrocytes, respectively, in
various regions of the brain, including cortex, hippocampus, and cerebellum in
12-month-old Lmnb1BAC and control animals. There were no
differences in the cortex (Supplemental Figure 2), hippocampus, or cerebellum (data
not shown). These data are consistent with ADLD patient data showing no obvious
Lamin B1 overexpression results in aberrant myelin formation, axonal
degeneration, and demyelination. Ultrastructural analysis was performed on pyramidal tracts of 12- and 24-month-old
Lmnb1BAC and corresponding WT mice.
(A and B) g ratio analyses at 12 and (C
and D) 24 months, at which time demyelination is prominent in
Lmnb1BAC mice. P < 0.05.
(E) WT controls at 12 months. (F and G)
Ultrastructural analyses revealed numerous aberrant structures, which included
outfoldings, extensions, and invaginations originating from the myelin sheath at
12 months and (H) at 24 months. (I and J)
Axonal disintegration and degradation of the entire axon-myelin unit was observed
at a significantly higher level at 12 months and (M and
N) at 24 months compared with WT controls. (K and
L) Hypermyelinated fibers and dying axons were also observed in
Lmnb1BAC mice at 12 months of age.
(O) Normal oligodendrocyte and (Q) normal compact
myelin at 24 months of age in control animals. (P) Representative of
frequent dying and degenerating oligodendrocytes in 24-month-old
Lmnb1BAC mice. Dying oligodendrocytes correlate
with increased (R) irregular and demyelinated fibers in 24-month-old
Lmnb1BAC animals. (S and
T) Quantification of approximately 150 to 200 axons for
12-month-old Lmnb1BAC mice exhibited significant
axonal degeneration and aberrant myelin formations compared with WT controls.
n = 3 animals per genotype. Values are expressed as means
± SEM. **P < 0.01, ***P
< 0.001. Scale bars: 2 μm.
Lamin B1 overexpression induces cell-autonomous neuropathology in
oligodendrocytes. An interesting feature of ADLD is specific demyelination in the presence of
ubiquitously overexpressed lamin B1 protein. To investigate the pathologic
consequences of lamin B1 overexpression on different cell lineages in the brain, we
overexpressed a FLAG-tagged lamin B1 selectively in oligodendrocytes, neurons, and
astrocytes driven by cell-specific promoters (Plp1,
Camk2a, and GFAP, respectively). Each of these
lines showed expression levels of lamin B1 that were similarly higher than their WT
counterparts, which were quantified by Western blot analyses and qRT-PCR at 12 months
of age (Figure 5, A–C). Western blot
analysis of total lamin B1 showed on average a 2.2-, 2.4-, and 2.3-fold increase in
mice where Lmnb1 was driven by Plp1,
Camk2a, and GFAP, respectively, compared with WT
controls (Figure 5, A and B).
Overexpression levels were consistent with what was observed in ADLD patients, which
exhibit on average a 2-fold increase (1, 11). For reasons we do not completely understand,
endogenous lamin B1 appeared to increase as a result of lamin B1 overexpression
compared with WT controls (Supplemental Figure 3A). mRNA by qRT-PCR
analyses also exhibited marked and comparable increases in all 3 cell-specific lines
(Figure 5C). Only animals overexpressing lamin
B1 in oligodendrocytes (PLP-LMNB1Tg) exhibited
age-dependent motor deficits beginning at 10 months (observed) and rapidly
progressing to 12 months of age, at which time these animals became moribund. At 5
months, PLP-LMNB1Tg animals showed no differences on the
accelerated rotarod or on an 11-mm–wide balance beam compared with WT
controls (Figure 5, D–F). However,
by 12 months, PLP-LMNB1Tg animals exhibited considerable
decreased latency to fall on the accelerated rotarod (Figure 5D) and showed marked increased latency to traverse the balance
beam compared with WT controls (P < 0.001) (Figure 5E). Additionally,
PLP-LMNB1Tg animals exhibited more hind limb slips
(Figure 5F). These motor deficits observed in
the PLP-LMNB1Tg mice were accompanied by demyelination,
aberrant myelin formation, and axonal degeneration (Figure 6, B–D). Among aberrant myelin loops in
PLP-LMNB1Tg animals, myelin-axon structure was also
irregular and nonuniform compared with WT controls (Figure 6, E and F). Interspersed within areas of demyelinated axons
(e.g., Figure 6E, arrow) were axons with normal
myelin thickness (e.g., Figure 6E, arrowhead).
The heterogeneity of myelin thicknesses is represented by the g ratio (Figure 6B). WT controls did not exhibit demyelination or
display aberrant myelin loops (Figure 6, A and
F). Toluidine blue staining of adjacent sections showed similar findings of
demyelination and aberrant structures in PLP-LMNB1Tg mice
(Supplemental Figure 1). The neuropathology and age-dependent motor deficits observed
in PLP-LMNB1Tg animals may more accurately reflect
mechanisms related to degeneration in ADLD. Consistent with the global overexpressing
PLP-LMNB1Tg animals also exhibited spontaneous
seizures (data not shown). Animals overexpressing lamin B1 specifically in neurons
(CAM/tet-Lmnb1) and astrocytes (GFAP/tet-Lmnb1)
showed no motor deficits on the balance beam and accelerated rotarod at 12 months and
no overt behavioral abnormalities beyond 1 year (Supplemental Figure 3, B and C,
respectively). Together, these results indicate that oligodendrocytes are inherently
more vulnerable to the overexpression of lamin B1 than other cell types.
Lamin B1 overexpression in oligodendrocytes results in progressive motor
impairment and myelin deficits. (A) Representative Western blot for lamin B1, FLAG-tagged lamin B1,
and α-tubulin in hemibrain lysates from transgenic mouse lines
overexpressing lamin B1 selectively in oligodendrocytes, neurons, and astrocytes
driven by cell-type–specific promoters (Plp1,
Camk2a, and GFAP, respectively) vs. control
mice at 12 months of age (lanes are discontinuous). Dual color detection with IR
fluorescence for antibody against lamin B1 (green) and antibody against
FLAG-tagged lamin B1 (red; top panel). Individual detection against FLAG (second
panel), lamin B1 (third panel), and α-tubulin (bottom panel).
(B) Quantification of Western blots of total lamin B1 normalized
to α-tubulin. (C) qRT-PCR of lamin B1 transcript levels
normalized to GAPDH from corresponding cell-specific hemibrains at 12 months of
age. Error bars represent mean ± SEM from 3–4 independent
experiments. (D–F) Animals overexpressing
lamin B1 selectively in oligodendrocytes (PLP-LMNB1Tg)
exhibited progressive motor deficits on the accelerated rotarod and balance beam.
(D) PLP-LMNB1Tg mice had shortened
latency to fall on the accelerated rotarod and (E) exhibited
increased latency to traverse an 11-mm–wide balance beam with
(F) hind limb slips. Values are expressed as means ±
SEM, n = 10–12 per group. ***P
PLP-LMNB1Tg mice exhibit demyelination, aberrant
myelin formation, and axonal degeneration. (A and B) PLP-LMNB1Tg
animals exhibited demyelination compared with WT controls, quantified by g ratio
of 150–200 axons, P < 0.05. (C
and D) PLP-LMNB1Tg animals showed
aberrant myelin formation and axonal degeneration. (E and
F) Demyelinating axons (e.g., arrow) and axons with normal myelin
thickness (e.g., arrowhead) were frequently observed in
PLP-LMNB1Tg mice (E) but not in WT
controls (F). Myelin-axon structure also appeared irregular and
nonuniform in PLP-LMNB1Tg mice compared with WT
controls. n = 4 animals per genotype. Scale bars: 2
Lamin B1 overexpression results in significant decrease of the highly abundant
myelin protein PLP. We used quantitative mass spectrometry iTRAQ labeling to compare hindbrain protein
levels from 12-month-old Lmnb1BAC and WT mice. Out of 500
proteins identified at 95% confidence interval of high-quality scoring peptide
candidates, only 4 proteins showed substantial changes (P <
0.05). The heterogeneous nuclear ribonucleoprotein (HNRNPN) and histone H4 (H4) were
increased by 20% and 30%, respectively, while neurofilament medium polypeptide
protein (NEFM) was reduced by 20%. Interestingly, PLP was reduced by 30% (Figure
7A). Decreased Plp1
expression was confirmed by qRT-PCR (Figure 7B).
Mice overexpressing lamin B1 specifically in oligodendrocytes
(PLP-LMNB1Tg) exhibited further reduction in PLP
transcript levels (Figure 7C). However, mice
overexpressing lamin B1 specifically in neurons (CAM/tet-Lmnb1) did
not show any changes in Plp1 levels compared with WT controls
(Supplemental Figure 3D).
Lamin B1 overexpression results in the decrease of the highly abundant myelin
protein PLP. (A) Quantitative mass spectrometry iTRAQ labeling of hindbrains from
12-month-old Lmnb1BAC mice and WT mice identified PLP
at 95% confidence interval of high-quality scoring peptide candidates to be 30%
downregulated in Lmnb1BAC animals compared with WT
controls. (B) This finding was validated by qRT-PCR. (C)
Mice overexpressing lamin B1 specifically in oligodendrocytes
(PLP-LMNB1Tg) exhibited a further reduction in
Plp1 transcript levels. Values are expressed as means
± SEM. n = 3 per group. **P <
0.01; ***P < 0.001. (D) Transcriptome
analysis performed on purified oligodendrocytes validated proteomic changes
including decreased Pol II binding to the Plp1 promoter in
Lmnb1BAC mice compared with WT controls. Lamin
B1 also regulates additional genes. x axis defined as M: log
differential-expression ratio (log fold change); y axis defined
as A: average log intensity (reads per kb of gene length) from oligodendrocytes
between Lmnb1BAC mice and WT controls.
In addition, we performed transcriptome analysis by identifying Pol
II–binding sites on a genome-wide scale from oligodendrocytes purified
from Lmnb1BAC and WT control mice. Pol II binding was
changed on promoters of a wide variety of genes. However, Pol II binding validated
proteomic changes, including identifying decreased Pol II binding to the
Plp1 promoter in Lmnb1BAC mice
compared with WT controls (Figure 7D). Together,
these 2 independent approaches suggest that lamin B1 overexpression results in
altered gene expression and specifically suggest that a highly abundant myelin
protein gene, Plp1, is reduced at the transcriptional level. This
lowered Plp1 transcription level serves as an entry point for
studies aimed at understanding pathophysiology in ADLD. We hence sought to further
examine how lamin B1 overexpression leads to altered PLP1
Loss of binding occupancy of YY1 to Plp1 promoter. Alteration of gene transcription can result from many mechanisms, including changes
in transcription factor binding and occupancy in the promoter region of the affected
gene. In an effort to identify potential factors that contribute to the altered gene
regulation of Plp1, we performed quantitative ChIP using known
transcription factor candidates of the Plp1 promoter from hindbrains
of 12-month-old Lmnb1BAC and WT mice. We identified YY1,
a known transcriptional activator of Plp1 (18), as binding and occupying the Plp1 promoter
significantly less in Lmnb1BAC hindbrains than in WT
controls (Figure 8A). No differences were found
in ChIP reactions performed by IgG antibody or real-time PCR for
Plp1 3′ UTR, demonstrating the specific changes of
YY1 binding to the Plp1 promoter (Figure 8A). We next asked whether decreased binding of YY1 to the
Plp1 promoter resulted from reduced YY1 levels. We measured total
protein levels of YY1 in hindbrains of 12-month-old mice and found no difference
among genotypes (Figure 8, B and C). Taken
together, these results indicate that overexpression of lamin B1 affects
myelin-producing cells through the misregulation of Plp1 gene
expression, in part via the transcription factor YY1.
Lamin B1 overexpression results in reduced binding occupancy of YY1 to
Plp1 promoter. (A) Quantitative ChIP coupled to PCR revealed a reduction in YY1
binding to the Plp1 promoter in hindbrains from 12-month-old
Lmnb1BAC mice compared with WT controls
normalized to 2% input. Primers were designed to amplify 150–200 bp
containing either a consensus YY1-binding site or 3′ UTR.
3′ UTR and IgG were used as controls. (B) Representative
blot of YY1 total protein levels normalized to α-tubulin in hindbrains
of 12-month-old Lmnb1BAC and WT mice. (C)
Quantification of YY1 protein level relative to α-tubulin was found
not to be markedly different between Lmnb1BAC and WT
mice. n = 4 per group of at least 2 independent experiments.
P > 0.05.
Duplication of the gene encoding lamin B1 causes ADLD. Here, we demonstrate that
overexpression of lamin B1 in mice recapitulates many key features of ADLD.
Lmnb1BAC mice exhibit spontaneous seizures, cognitive
impairments, and age-dependent motor deficits similar to what is observed in ADLD
patients. Ultrastructural analysis revealed aberrant myelin foldings eventually leading
to myelin and axonal degeneration in Lmnb1BAC mice. Although
lamin B1 is broadly expressed in Lmnb1BAC mice, we found
that specific overexpression of lamin B1 in oligodendrocytes
(PLP-LMNB1Tg mice) was sufficient to induce similar
abnormalities, suggesting a cell-autonomous mechanism linking lamin B1 to myelin
defects. Quantitative proteomic analysis of Lmnb1BAC mice
revealed substantial reduction in PLP protein levels relative to WT controls
(P < 0.05). Consistent with these results, we found that both
RNA Pol II binding on the Plp1 promoter and transcript levels of
Plp1 were downregulated in Lmnb1BAC
animals. Finally, using quantitative ChIP assays, we discovered that YY1, a
transcriptional activator of Plp1, was found at markedly reduced levels
at Plp1 promoters. Taken together, these data suggest that the effects
of lamin B1 overexpression are manifested, at least in part, through altered
transcription of a gene known to play a key role in maintenance of the myelin sheath.
Although seizures have long been recognized to be part of the disease spectrum of MS,
epileptiform activity is not a major feature in inherited leukodystrophies. One
explanation may be that these are rare diseases with a general lack of EEG data in
affected individuals. A more careful search for electrographic abnormalities is leading
to an increase awareness of seizures in some leukodystrophies (22, 29). Among inherited
leukodystrophies in children, 49% were noted to have epilepsy (30). The precise pathogenesis of epileptic seizures in inherited
leukodystrophies is not well understood. Cognitive decline has been shown to correlate
with cortical pathological severity and seizures in MS (31). An increased risk of seizures may also be associated with ADLD; a
possible mechanism could be damage to the brain parenchyma leading to seizure
susceptibility. Alternatively, the effects of demyelination on the cortex could cause
cortical hyperexcitability resulting from altered axonal signaling.
PMD is an X-linked demyelination disorder typically caused by duplications of
PLP1. How duplication of PLP1 leads to PMD remains
unclear. However, recent studies showed that much of the PLP being produced from the
higher gene dosage fails to be incorporated into the myelin sheath in a mouse model of
PMD duplication (32). Consistent with this
finding, we found that overexpression of lamin B1 also results in substantial decrease
of PLP. Disease characteristics of ADLD and PMD, while overlapping, are not identical,
suggesting that pathophysiology of these diseases is not solely due to decreased PLP.
Our observation that Plp1 transcript levels are reduced as early as 11
weeks (data not shown), while behavioral impairments and myelin defects are not seen
until 1 year of age in Lmnb1BAC animals, further suggests
that PLP downregulation alone is insufficient to explain the ADLD-like phenotypes.
Rather, PLP downregulation is likely to initiate a pathological myelin state that is
exacerbated as animals age and trigger multiple mechanisms.
Considering that lamin B1 overexpression is likely to affect transcriptional activity of
a number of genes, ADLD is probably the consequence of misregulation of a subset of
genes, including PLP, which together bring about the features of ADLD.
Consistent with this hypothesis, our transcriptome analysis points to several other gene
candidates: for instance, we found that superoxide dismutase 1 (Sod1),
an important regulator of superoxide radicals, was transcriptionally downregulated while
the oligodendrocyte lineage transcription factor 2 (Olig2), a
transcription factor known to be important for oligodendrocyte development, was
upregulated upon lamin B1 overexpression (Figure 7D).
The precise mechanism through which lamin B1 overexpression leads to a specific
downregulation of PLP is not yet known. However, nuclear lamins are known to regulate
gene expression, in part through interactions with chromatin and repression of
transcription. Previous studies have shown that nuclear lamins interact with DNA,
histones, transcription factors, and chromatin proteins. Direct interaction of DNA with
nuclear lamins causes downregulation of genes locally (33–35). Moreover,
depletion of lamin B1 in Drosophila led to an upregulation of
lamina-associated genes (36), specifically
showing a direct role for lamin B1 in gene repression. We found a reduction in YY1
occupancy at Plp1 promoters in lamin B1–overexpressing
animals with no change to the overall total protein level of YY1. It is therefore
plausible that overexpression of lamin B1 pulls the Plp1 promoter
region into a heterochromatin-like state and reduces accessibility of these regions to
the YY1 transcription factor. Additionally, YY1 has previously been proposed to
facilitate the attachment of the PLP1 promoter region to
transcriptionally active domains of the nuclear matrix (18). Our observation that histone H4 levels were increased upon lamin B1
overexpression also supports the idea that lamin B1 overexpression leads to repressed
transcription in specific genomic regions.
Together, these findings implicate lamin B1 as a modulator of myelin and genes involved
in premature myelin breakdown, particularly in the aging process. Lamin B1 and its
myelin-specific regulatory targets are therefore potential therapeutic avenues for
remyelination in various demyelinating disorders.
In summary, we provide a valid murine model of ADLD that recapitulates human ADLD.
Collectively, our results implicate an important role for lamin B1 in modulation of
genes involved in normal myelin regulation, which may provide further elucidation of the
molecular mechanisms that underlie a spectrum of myelin-related disorders including
leukodystrophies, laminopathies, and multiple sclerosis. Ultimately, there is hope that
such insights may provide novel potential therapeutic strategies for disorders of
Animals and generation of transgenic mice
All animals were housed in cages grouped by sex and provided with food and water ad
libitum. Animals were housed under specific pathogenic–free conditions
with a 12-hour light/12-hour dark cycle maintained at 23°C. Behavioral
testing occurred between 8 am and 5 pm during the light cycle. Experiments were blind
to mouse genotype and treatment during testing.
Lmnb1BAC. For transgenesis, a large genomic insert (177.20 kb) containing the entire mouse
Lmnb1 locus and cloned into the BAC vector pBACe3.6 was
isolated from a mouse BAC genomic library (BAC RP23-460J18 clone; RPCI library,
C57BL/6J). Purified BAC DNA was used for microinjection into the pronuclei of
fertilized oocytes of C57BL/6J mice and was maintained on a C57BL/6J genetic
PLP-LMNB1Tg. We generated transgenic mice encoding a FLAG-tagged human lamin B1 cDNA, which was
cloned into a PLP promoter-exon expression cassette as previously
described (37). The purified construct was
then used for microinjection into the pronuclei of fertilized oocytes of FVB mice
and was maintained on an FVB genetic background.
CAM/tet-Lmnb1 and GFAP/tet-Lmnb1. Double-transgenic mice (CAM/tet-Lmnb1 and
GFAP/tet-Lmnb1) were generated by crossing
Camk2atTA and GFAP-tTA mice (38, 39), respectively
(B6/CBA genetic background), to a FLAG-tagged tetracycline-responsive element
(TRE)-Lmnb1 mice (FVB). Both lines were maintained separately
and crossed to generate F1 mice hemizygous for each transgene.
MWM. Lmnb1BAC and WT mice were tested using the MWM, as
described (40). In brief, over a number of
trials, animals were trained to find a hidden platform in the MWM using spatial
cues. Memory was tested 24 hours afterwards by a probe trial in which the hidden
platform was removed and the amount of time and number of platform area crossings
PA. The PA test was used as previously described (41). A light/dark chamber with a sliding trap door separating the 2
chambers was used. Briefly, animals were allowed to acclimate for 15 seconds in
the light compartment. Once mice stepped through into the dark compartment they
received a shock of 0.3 mA for a maximum of 2 seconds. The latency to enter the
dark compartment was recorded. The memory-retention test was performed 24 hours
later without shock. A maximum retention latency of 400 seconds was given to mice
that did not enter the dark compartment before that time (Gemini Avoidance
Accelerated rotarod and balance beam. To assess motor coordination and balance, animals were tested on the accelerated
rotarod and balance beams as previously described (42). Animals were tested on 42-cm–long Plexiglas beams (5
mm wide and 11 mm wide). Latency to traverse each beam and hind limb slips were
recorded. The accelerated rotarod was accelerated from 4 to 40 rpm over a period
of 270 seconds, and the time spent on the drum was recorded for each mouse.
Animals were tested 1 trial per day over 8 trials unless otherwise stated.
EEG recordings were performed as previously described (43). Briefly, Teflon-coated silver wires were implanted into the
subdural space over the left and right parietal cortices, and the left frontal cortex
was used as a reference. EEG activity was recorded in freely moving mice with
Harmonie 5.0b software (Stellate). The number of abnormal epileptiform spikes
(sharp-wave discharges with amplitude exceeding 6 times the mean EEG amplitude and
lasting 20–70 ms) was automatically detected by the Gotman spike
detectors (Harmonie; Stellate) over 24 hours of recording. The latency to seize and
the number of seizures were visually quantified.
PTZ (Sigma-Aldrich) was dissolved in PBS. A dose of 30 mg/kg was administered
Transmission electron microscopy
Toluidine blue staining and g ratio analysis. 12-month-old animals were deeply anesthetized and intracardially perfused with 2%
paraformaldehyde and 1.25% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH
7.4 (n = 3 per group for Lmnb1BAC
mice, n = 4 per group for PLP-LMNB1Tg
mice). Brains were postfixed in 2% osmium tetroxide in the same buffer, en block
stained in 2% aqueous uranyl acetate, dehydrated, infiltrated, and embedded in
LX-112 resin (Ladd Research Industries). For light microscopy,1.5 μm
semi-thin sections of pyramidal tracts at the level of the pons near decussation
were cut and stained with 0.5% Toluidine blue O in a Borax solution. Experiments
were performed as previously described with the above modifications (44). The g ratio of axons in the area of
interest was obtained as a ratio of the diameter of an axon over the diameter of
axon plus associated myelin sheath as previously described (45). Approximately 150 to 200 axons for each group of 4
animals were used. Digitized and calibrated images were analyzed using ImageJ
qRT–PCR was performed by the ΔΔCt method
normalized to GAPDH control using ABI Sybr Green/ROX gene-specific primers. Data were
collected and analyzed with an ABI 7900HT machine and software. Total RNA was
isolated from whole brains of 11-week-old, and 12-month-old mice (n
≥ 3/group) using an RNeasy isolation kit (QIAGEN). Total RNA was reverse
transcribed using SuperScriptII Reverse Transcriptase according to the
manufacturer’s instructions (Invitrogen).
Primer sequences for GAPDH (46) and Plp1 (47)
are as follows: Lmnb1 (spanning exons 2–3); forward
sequence, 5′-CAGATTGCCCAGCTAGAAGC-3′; reverse sequence,
Brain lysate and Western blotting
Brain lysates and Western blotting were performed as previously described (48). In brief, flash-frozen hemibrains were
homogenized in RIPA buffer (20 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid,
1% Nonidet P-40, pH 7.4) with protease inhibitors (PI) (Sigma-Aldrich). Supernatants
were supplemented with 1% final SDS and 100 mM DTT, boiled, loaded on 7.5% or 10%
SDS-PAGE gels and transferred to PVDF membrane. The primary antibodies YY1 (1:500,
sc-1703; Santa Cruz Biotechnology Inc.), lamin B1 (1:50, sc-6216; Santa Cruz
Biotechnology Inc.), and α-tubulin (1:5,000, Sigma-Aldrich T9026) were
incubated overnight, followed by species-matched, infrared-labeled (IR-labeled)
secondary anti-mouse or anti-rabbit antibodies (dilution 1:5,000; Li-Cor
Biosciences). Blot membranes were imaged using Odyssey IR Imaging system (Li-Cor
Immunohistochemical analysis was performed as described previously (48). Briefly, hemibrains were fixed in 4%
paraformaldehyde for 24 hours, cryoprotected in 20% sucrose in 0.1 m phosphate buffer
for an additional 24 hours at 4°C, frozen, and stored at
–80°C until use. Free-floating 30-μm serial
sections were processed with primary antisera against GFAP (1:1000, Millipore ab5804)
and Iba1 (1:1000, Wako Chemical, 019-19741). Detection of immunoreactivity was
performed using the Vectastain Elite kit (Vector Laboratories) according to the
manufacturer’s protocol, and sections were developed in ImmPACT DAB
(Vector Laboratories) for visualization. Sections were mounted and dehydrated in
graded alcohols and xylene. Coverslips were affixed with DPX (Electron Microscopy
Sciences). Quantification of GFAP and Iba1 immunoreactivity were performed using NIH
ImageJ software. Particle numbers were quantified with the “analyze
particles” function in “threshold and measure”
functions with the scale standardized to the scale bar and set globally. Experiments
used n = 3 per group for a total of 7–10 sections per
animal and 2 fields of view per section.
Quantitative ChIP PCR
ChIP assays were performed with the SimpleChIP-Enzymatic Chromatin IP Kit (Cell
Signaling) according to the manufacturer’s instructions. Brain lysates
were prepared in PBS with PIs. Samples of input DNA were prepared in the same manner.
Cell lysates were sonicated and immunoprecipitated with normal IgG or specific
antibody against YY1 (sc-1703; Santa Cruz Biotechnology Inc.). The DNA thus obtained
was analyzed by real-time PCR with a set of primers directed against the
Plp1 promoter region containing the YY1 consensus site (18). Plp1 3′ UTR was
used as a negative control. PCR reactions were performed in triplicate in the
presence of SYBR Green (Roche Diagnostics).
iTRAQ analysis was performed as previously described (49). Isolated hindbrain protein powders were dissolved in 50
μl of iTRAQ dissolution buffer (20-fold diluted, final concentration = 25
mM, pH 8.5), and protein concentrations were measured using a protein assay kit
(Bio-Rad). 40 μg of protein for each sample was labeled for each iTRAQ
experiment (Applied Biosystems) following the manufacturer’s
instructions. The final mixture of labeled samples was desalted and purified using an
MCX cartridge (Mixed-mode Cation Exchange, Oasis solid phase extraction, 30
μm, 3 cc, 60 mg). They were then subjected to HPLC fractionation and mass
OPC purification. Purification of oligodendrocyte progenitor cells (OPCs) from P7-8
Lmnb1BAC mice or WT was carried out as described
previously (50). In brief, OPCs were
cultured in proliferation medium (+PDGF10 ng/ml +NT3, 1 ng/ml) for 6 days followed
by differentiation medium (40 ng/ml triiodothyronine) for 4 days. OPCs were fixed
with 1% formaldehyde solution and frozen immediately at
–80°C before the ChIP-Seq using the RNA polymerase II
antibody (Active Motif).
Data analysis. Analysis of Pol II–binding sites on a genome-wide scale from
oligodendrocytes purified from Lmnb1BAC and WT control
mice were normalized by the total number of reads between the transgenic and the
control samples. For each mouse gene defined by Ensembl, including 2-kb upstream
and 6-kb downstream regions, we counted the number of reads pulled down by Pol II
in ChIP-Seq. The count was then divided by the genomic length (in unit of kb) of
the gene. The reads per kb of gene length were used as an estimate of the strength
Comparisons of different groups (between genotypes) were performed on experiments
requiring nonrepeated trials using 2-tailed independent t tests and
regression analysis. Repeated measures t test with between-subject
factor for genotype and repeated measures for training were performed on experiments
for behavioral tests requiring repeated trials. There were no interactions observed
between genotype and training. If there was no effect from repeated training, trials
were collapsed, and a 2-tailed t test was performed. Paired
t test was performed when animals were subjected to different
behavioral conditions in a given paradigm. All t tests and
P values presented were performed from 2-tailed tests. All data
are presented as mean ± SEM. P < 0.05 was
considered to be statistically significant. The Huynh-Feldt adjustment was used in
correcting for the violation of sphericity when necessary to adjust nonuniform
variance across days or groups. Data were analyzed with IBM SPSS 20.0.
All procedures were conducted in strict compliance with the Guide for the Care and
Use of Laboratory Animals as adopted by the NIH and were approved by the University
of California San Francisco.
View Supplemental data
The authors thank Jinny Wong and Junli Zhang from the Electron Microscopy and Transgenic
Gene Targeting Cores, respectively, at the J. David Gladstone Institutes. The authors
also recognize the Center for Mass Spectrometry and Proteomics at the University of
Minnesota and various supporting agencies, including the National Science Foundation for
Major Research Instrumentation grants 9871237 and NSF-DBI-0215759 used to purchase the
instruments described in this study. This work was supported by NIH grants F32
NS066722-01A1 to M.Y. Heng, and a fellowship from the A.P. Giannini Foundation to M.Y.
Heng; NIH NS062733 to Y.H. Fu, and the Sandler Neurogenetics Fund to Y.H. Fu and L.J.
Ptáček. L.J. Ptáček is an investigator
of the Howard Hughes Medical Institute.
Conflict of interest: The authors have declared that no conflict of
Citation for this article:J Clin Invest. 2013;123(6):2719–2729.
Quasar S. Padiath’s present address is: Department of Human Genetics,
University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Ying Tong’s present address is: College of Life Sciences, Sichuan
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