Abstract
Recent proteome and transcriptome profiling of Alzheimer's disease (AD)
brains reveals RNA splicing dysfunction and U1 small nuclear
ribonucleoprotein (snRNP) pathology containing U1-70K and its
N-terminal 40-KDa fragment (N40K). Here we present a causative role of
U1 snRNP dysfunction to neurodegeneration in primary neurons and
transgenic mice (N40K-Tg), in which N40K expression exerts a
dominant-negative effect to downregulate full-length U1-70K. N40K-Tg
recapitulates N40K insolubility, erroneous splicing events, neuronal
degeneration and cognitive impairment. Specifically, N40K-Tg shows the
reduction of GABAergic synapse components (e.g., the GABA receptor
subunit of GABRA2), and concomitant postsynaptic hyperexcitability that
is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the
5xFAD amyloidosis model indicates that the RNA splicing defect
synergizes with the amyloid cascade to remodel the brain transcriptome
and proteome, deregulate synaptic proteins, and accelerate cognitive
decline. Thus, our results support the contribution of U1
snRNP-mediated splicing dysfunction to AD pathogenesis.
Introduction
RNA splicing is a fundamental regulatory process in eukaryotic gene
expression, which is particularly important to expand molecular
diversity in the nervous system^[102]1. Mutations of RNA splicing genes
are linked to numerous neurodegenerative disorders^[103]2-[104]4, such
as SMN1 in spinal muscular atrophy^[105]5, TARDBP (TDP-43 protein),
FUS, TAF15, MATR3, TIA1, hnRNPA1, and hnRNPA2B1 in amyotrophic lateral
sclerosis and frontotemporal dementia^[106]6,[107]7. Mutant FUS can
induce neuronal toxicity, which may be mediated by its interaction with
U1 snRNP, an essential complex in RNA splicing^[108]8-[109]10. In
addition, genetic linkage of RNA splicing components to
neurodegeneration has been found in a chemical mutagenesis screen of
mice^[110]11, in which a mutant U2 snRNA gene causes alternative
splicing defects, leading to ataxia and cerebellar neurodegeneration.
Thus, the deregulation of RNA splicing is a key molecular mechanism
linked to neurodegenerative disease.
Dysfunction of RNA splicing has been implicated in the development of
Alzheimer’s disease, as TDP-43 aggregates were detected in up to ~50%
of AD brains^[111]12. While amyloid plaques and neurofibrillary tangles
are the hallmarks of AD pathology^[112]13,[113]14, abnormal synaptic
changes are correlated with progressive cognitive decline, indicating
that synaptic dysfunction is a crucial factor contributing to cognitive
impairment^[114]15,[115]16. More recently, the development of mass
spectrometry (MS) enables comprehensive analysis of proteome directly
from human clinical specimens^[116]17,[117]18. Unbiased profiling of
aggregated brain proteome discovered the deposition of U1 snRNP and
Prp5/DDX46, an RNA helicase connecting the U1 and U2 snRNP
complexes^[118]19. Further profiling of the AD whole proteome and
phosphoproteome identified the alterations of other RNA binding factors
and splicing components^[119]20,[120]21. Global structural profiling of
AD cases also suggested conformational changes of RNA splicing
components, such as the U2 snRNP subunit SF3B, and heterogeneous
nuclear ribonucleoproteins (hnRNP H2, M and U)^[121]22. Consistently,
transcriptomic profiling of human brain tissues confirmed reproducible,
aberrant RNA splicing events in multiple AD cohorts^[122]19,[123]23.
U1 snRNP is highly accumulated in the AD aggregated proteome, only
behind Aβ, tau and complement proteins^[124]19,[125]24,[126]25. The U1
snRNP complex is comprised of U1 snRNA, U1-70K, U1A, U1C, and seven Sm
proteins (B/B’, D1, D2, D3, E, F and G)^[127]26. Immunohistochemical
staining and electron microscopy confirmed that the U1 snRNP forms a
new type of cytoplasmic tangle-like fibril in sporadic and familial AD,
as well as in trisomy 21 cases^[128]19,[129]24,[130]27,[131]28. The
presence of U1 snRNA in the aggregates was also shown by quantitative
PCR and immunofluorescence studies^[132]27. Moreover, The core subunit
U1-70K was found to be cleaved to a N-terminal fragment, ~40 kDa with
~300 amino acids named N40K^[133]29. The aggregation of U1 snRNP occurs
as early as in the stage of mild cognitive impairment^[134]19, and is
correlated with the neuropathological hallmarks of Aβ plaques and tau
tangles during AD progression^[135]24. Interestingly, the U1 snRNP
pathology was not found in other examined neurodegenerative diseases,
including Parkinson’s disease, amyotrophic lateral sclerosis,
frontotemporal lobar degeneration, and corticobasal
degeneration^[136]19. Taken together, these findings strongly support a
unique U1 snRNP pathology in AD. However, the post-mortem human brain
studies only uncover disease-correlated proteins, which do not indicate
causation, especially considering that molecular changes may appear
years earlier than the onset of AD clinical symptoms. So, it is not
clear whether the observed U1 snRNP pathology in human samples plays a
causative role in the development of AD.
In this study, we present a mouse model (N40K-Tg) of U1 snRNP
dysfunction to investigate its role in AD pathogenesis. The function of
U1 snRNP is impaired by the dominant-negative effect of the N40K
fragment, which competes with U1-70K to assemble the U1 snRNP complex,
resulting in a significant loss of full length U1-70K protein and
splicing defects in primary neurons and mice. We have thoroughly
characterized the neuron-specific N40K-Tg mice by multi-omics,
biochemical, histopathological, and electrophysiological approaches,
along with mouse behavior studies. The N40K-Tg mice display protein
insolubility, neurodegeneration and abnormality in memory tests, as
well as splicing defects enriched in synaptic signaling. In particular,
GABAergic synapse deregulation and neuronal hyperexcitability are
identified in the N40K-Tg mice. Finally, the double transgenic model
from N40K-Tg and 5xFAD mice shows a strong synergy between RNA splicing
defects and the amyloid cascade.
Results
U1 snRNP dysfunction elicits excitatory toxicity in neurons
Multiple U1 snRNP components are aggregated in AD brain, which were
previously detected in detergent-insoluble fractions by
semi-quantitative label-free mass spectrometry in two studies (n =
4,216 proteins and 2,711 proteins)^[137]19,[138]24, as aggregated
proteins often have low solubility and are partially resistant to
detergent extraction^[139]30. With the development of the latest
tandem-mass-tag (TMT) strategy, two-dimensional liquid chromatography
(LC/LC) and tandem mass spectrometry (MS/MS) approach^[140]31,[141]32,
we re-profiled 8,917 proteins in the detergent-insoluble proteome from
10 control and 10 AD brains ([142]Supplementary Tables 1, [143]2),
which increased the coverage by more than 2-fold. We found that 365
proteins were significantly accumulated in the AD cases ([144]Fig. 1a,
[145]Extended Data Fig. 1a, [146]1b, FDR < 0.05), including Aβ, MDK,
PTN, NTN1, SMOC1, and U1-70K, as well as other U1 snRNP subunits (U1A,
Sm proteins B/B’, D1, D2, D3, E, F and G, [147]Fig. 1b, [148]Extended
Data Fig. 1c). MDK, PTN, NTN1 and SMOC1 have recently been identified
among the most increased proteins in human AD brains and they are
colocalized in amyloid plaques^[149]18. Spliceosome is the most
enriched pathway and protein-protein interaction module in the AD
detergent-insoluble proteome ([150]Extended Data Fig. 1d, [151]1e, FDR
< 0.05), in agreement with the previous proteomic
finding^[152]19,[153]24.
Figure 1. Tissue proteomics confirms U1 snRNP aggregation and N40K shows
detergent insolubility and dominant negative effects to deplete U1-70K
through proteasomal degradation.
[154]Figure 1.
[155]Open in a new tab
a, Volcano plot of detergent-insoluble proteome by deep TMT-LC/LC-MS/MS
analysis (10 AD and 10 non-dementia control cases). Among 8,917
identified proteins, 365 proteins were accumulated in the AD samples.
Dashed lines indicate the cutoffs (FDR < 0.05, log[2](AD/Ctrl) > 0.30,
equivalent to 2-fold of the standard deviation). b, Diagram of the U1
snRNP complex. c, Diagrams of U1-70K and N40K domains: RRM: RNA
recognition motif, LC: low complexity domain. d, Aggregation analysis
of U1-70K and N40K. Control EGFP (Ctrl), U1-70K-FLAG, and N40K-FLAG
were expressed in mouse cortical neurons (DIV 12) by lentiviruses (MOI
10) and extracted with 1% sarkosyl to produce detergent soluble (Sol)
and insoluble (Insol) fractions for immunoblotting. The experiment was
repeated 4 times and quantified ([156]Extended Data Fig. 1f). e, The
dominant-negative effect of N40K in a time course analysis. Cortical
neurons were infected with lentiviruses for the analysis (DIV 12, MOI
10). The experiment was triplicated with quantification ([157]Extended
Data Fig. 1g). f, Endogenous U1-70K mRNA quantitated by qRT-PCR in the
control and N40K-expressing neurons (3 replicates, mean ± SEM,
Student’s t-test, ns: not significant). g, The model of U1-70K
downregulation induced by N40K expression. h, U1-70K or N40K assembled
in U1 snRNP. Control EGFP (Ctrl), U1-70K-FLAG or N40K-FLAG was
expressed in HEK 293T cells and immunoprecipitated by FLAG Ab for
SDS-PAGE, followed by Ponceau S staining and immunoblotting by U1A and
U1C antibodies. i, U1-70K downregulation is mediated by the
ubiquitin-proteasome pathway. Neurons were infected with the N40K
lentivirus for 2 days (DIV 12, MOI 10), then treated with solvent DMSO
0.1% (Ctrl), the proteasome inhibitor MG132 (10 μM) or the autophagy
inhibitor bafilomycin A (Baf, 10 nM) for 1 or 2 days, and harvested for
immunoblotting. The experiment was repeated 4 times and the U1-70K
levels were quantified (two-way ANOVA and Tukey's multiple comparison
test). Data are shown as mean ± SEM. Full statistical information is in
[158]Source Data Statistics.
Moreover, U1-70K was found to be cleaved to a N-terminal fragment, ~40
kDa with ~300 amino acids named N40K in the AD detergent-insoluble
proteome^[159]19,[160]29. Both U1-70K and N40K contain disordered low
complexity (LC) domains involved in aggregation ([161]Fig.
1c)^[162]33,[163]34. When human U1-70K or N40K is ectopically expressed
in mouse primary cortical neurons and subjected to extraction with 1%
sarkosyl detergent, an anionic surfactant, N40K protein clearly shows
higher insolubility than U1-70K protein ([164]Fig. 1d), suggesting that
N40K is prone to protein aggregation.
In addition to N40K insolubility, we found that N40K exerts a strong
dominant-negative effect to downregulate native U1-70K protein in
neurons. First, in AD brain, N40K appears to be inversely correlated
with full length U1-70K in protein abundance^[165]29. Second, in human
N40K lentivirus infected mouse neurons, endogenous U1-70K protein is
substantially downregulated ([166]Fig. 1d, [167]Extended Data Fig. 1f),
and this downregulation occurs in a time-dependent manner after the
infection of N40K virus ([168]Fig. 1e, [169]Extended Data Fig. 1g). In
contrast, the RNA level of native U1-70K remains unchanged ([170]Fig.
1f), indicating of a posttranscriptional mechanism. As U1-70K
N-terminus interacts with other subunits in the U1 snRNP
complex^[171]35, we propose that N40K can compete with U1-70K to
assemble U1 snRNP, and free U1-70K is then degraded by the proteasome
([172]Fig. 1g). To validate this hypothesis, we performed
immunoprecipitation analysis to indicate that either U1-70K or N40K can
pull down U1A and U1C equally ([173]Fig. 1h). Moreover, when applying
two chemicals to inhibit N40K-induced U1-70K degradation, we found that
U1-70K can be rescued by the treatment of MG132 (a proteasomal
inhibitor) but not bafilomycin A1 (an autophagy inhibitor) ([174]Fig.
1i), supporting that U1-70K is degraded by the ubiquitin-proteasome
system.
We further observed morphological degeneration and cell death caused by
N40K expression in primary neurons ([175]Fig. 2a, [176]2b), like our
previous report of N40K neurotoxicity^[177]29. To investigate the
mechanism of neurotoxicity, we performed deep RNA-seq and MS-based
proteomics experiments ([178]Supplementary Tables 1, [179]3) to profile
15,705 transcripts and 9,987 proteins in N40K-expressing neurons,
identifying N40K-induced changes in RNA splicing, RNA abundance, and
protein levels ([180]Fig. 2c). Consistent with splicing dysfunction in
AD^[181]19, N40K-expressing neurons display a higher global intron read
percentage than controls ([182]Fig. 2d). For individual transcripts, we
computed a splicing deficiency score, the ratio between
length-normalized intron and exon reads^[183]19. A total of 2,874
transcripts exhibit significant splicing changes ([184]Supplementary
Table 4, FDR < 0.05), showing an obvious shift in the distribution of
their splicing deficiency scores in N40K-expressing neurons ([185]Fig.
2e). Subsequently, the abundance of 3,281 transcripts and 2,096
proteins are altered in these neurons ([186]Supplementary Tables 5,
[187]6, FDR < 0.05), in which 468 RNAs/proteins show consistent changes
([188]Fig. 2f). Interestingly, these overlapped RNAs/proteins are
enriched in numerous pathways, including ferroptosis, apoptosis,
glutamatergic synapse, and GABAergic synapse ([189]Fig. 2g,
[190]Supplementary Table 7, FDR < 0.05). Thus, these large-scale
molecular profiling clearly reveals that N40K expression has a
significant impact on RNA splicing, the transcriptome and the proteome
of neurons, especially on the components in synaptic function.
Figure 2. Comprehensive analysis of transcriptome and proteome reveals
synaptic pathway in N40K-induced neuron death.
[191]Figure 2.
[192]Open in a new tab
a, Morphology of representative cultured neurons with lentiviral
expression of control EGFP (Ctrl) and N40K (with EGFP for
visualization). Scale bar: 20 μm. The experiment was repeated three
times independently. b, Neuron survival rate under dose-dependent N40K
expression. Mouse cortical neurons were transduced with the control or
N40K lentivirus at MOI 7, 20, or 60 for 5 days followed by the
CellTiter-Glo luminescent cell viability assay (n=3),). Cortical
neurons were transduced with control or N40K lentivirus at MOI 10 for 4
days and harvested for c-I. c, Transcriptomic and proteomic profiling.
Sample sizes are indicated. To simplify data processing, we analyzed
the major transcripts without considering alternative splicing forms.
d, Mapped intronic reads increase in N40K-expressing neurons. e,
Histograms of splicing deficiency scores of individual transcripts in
N40K-expressing neurons analyzed by Kolmogorov-Smirnov test (left:
comparison of Ctrl1 and Ctrl2 is shown as a null (p = 0.1), right:
comparison of average score of control and N40K neurons (n = 3, p < 2
.2 x 10^−16). f, Overlapping of DE RNAs (FDR < 0.05) and proteins (FDR
< 0.05). g, Pathway enrichment of the overlapped DE
transcripts/proteins (selected from [193]Supplementary Table 7). FDR
was derived from p values (Fisher's exact test) by the BH procedure. h,
Quantitative RT-PCR of Gabra2 mRNA (n=3). i, Western blotting of GABRA2
and N40K in cultured neurons transduced by control and N40K lentivirus.
The analysis was repeated with 4 different wells of neurons. j,
Relative survival rate of pharmacological inhibitors on N40K transduced
neurons. Cortical neurons were transduced with vector (Ctrl) or N40K
lentivirus for 2 days followed with pharmacological treatments for 3
days: 100 μM emricasan (apoptosis inhibitor), 50 μM GSK872 (necrosis
inhibitor), 10 μM MK801 (NMDAR antagonist), or 50 μM muscimol (GABA[A]R
agonist). All replicates were from different wells of neuron culture.
Data presented as means ± SEM, p values as indicated by two-tailed
Student’s t-test (d, h, i) or one-way ANOVA followed by Tukey's
multiple comparison test (b, j). Full statistical information is in
[194]Source Data Statistics.
We then focused on synaptic proteins and found that Gabra2, one of the
alpha subunits in GABA[A] receptors^[195]36, was among the altered
proteins in N40K-expressing neurons ([196]Supplementary Table 6). We
confirmed the decrease of Gabra2 RNA and GABRA2 protein by quantitative
RT-PCR ([197]Fig. 2h) and western blotting ([198]Fig. 2i),
respectively. Since GABA[A] receptors play a central role to inhibit
synaptic activities, GABRA2 downregulation may lead to synaptic
hyperactivity, resulting in N40K-induced cell death. To examine this
hypothesis, we performed a rescue assay by treating the N40K cellular
model with different pathway inhibitors. Indeed, neuronal toxicity can
be markedly reduced by either muscimol (an agonist of inhibitory
GABA[A]receptor) or MK801 (an antagonist of excitatory NMDA receptor)
in the primary culture ([199]Fig. 2j), while the toxicity is partially
rescued by emricasan (a patented apoptosis inhibitor), but not GSK872
(a necroptosis inhibitor). These results suggest that the N40K
neurotoxicity is mediated, at least partially, by elevated synaptic
activities and apoptosis. It should be mentioned that necroptosis,
ferroptosis and excitotoxicity are implicated in AD^[200]37,[201]38.
Our cellular model recapitulates the excitotoxicity but not the
necroptosis, which may be due to its difference from the
neurodegenerative condition in AD brain. Overall, this cellular model
experiments indicate that N40K expression induces U1-70K depletion,
compromises U1 snRNP function, impairs RNA splicing, and alters
synaptic proteins and activities, leading to neurotoxicity.
N40K-Tg displays splicing defects and cognitive impairment
Beyond the N40K cellular model, we generated an N40K-Tg transgenic
mouse model to determine whether N40K accumulation and U1-70K depletion
may be causative of AD symptoms. In the Tg, human N40K gene is
expressed under a neuron-specific CaMKIIα promotor^[202]39 to produce
two lines: Tg396 and Tg318 with the transgene inserted in chromosomes
18 and 10, respectively ([203]Fig. 3a-[204]b, [205]Extended Data Fig.
2a, [206]2b). We fully characterized the Tg396 mouse line and confirmed
major conclusions in the second Tg318 line. In both lines, N40K is
expressed in a brain region-specific pattern at a ~2-fold level of
native U1-70K in wild type (WT) mice ([207]Fig. 3c, and [208]Extended
Data Fig. 2c, [209]2d). As anticipated, N40K expression results in
significant U1-70K reduction in cortex and hippocampus ([210]Fig. 3c).
Immunoprecipitation analysis confirms that N40K competes with U1-70K
for assembling U1 snRNP ([211]Fig. 3d). In protein extraction assay, a
significant portion of N40K is present in the detergent-insoluble
fraction ([212]Fig. 3e). Comprehensive proteomic analysis of the
detergent-insoluble fraction reveals the accumulation of N40K, other U1
snRNP subunits (U1A, U1C, and Sm proteins), and some other RNA binding
proteins ([213]Extended Data Fig. 3a-[214]e and [215]Supplementary
Table 8, FDR < 0.05). The MS analysis profiled a total of 8,509
proteins in the detergent-insoluble fractions, revealing 90 proteins
highly enriched in N40K-Tg, when compared to the WT littermates. These
90 proteins are almost exclusively enriched in RNA related pathways.
Furthermore, U1-70K immunostaining validates its dramatic
neuron-specific reduction in cortical and hippocampal regions in
N40K-Tg compared with WT littermates ([216]Fig. 3f, [217]3g). Together,
the N40K-Tg model exhibits N40K insolubility and U1-70K downregulation.
Figure 3. Biochemical and cellular characterization of the N40K-Tg mouse
model.
[218]Figure 3.
[219]Open in a new tab
a, Tg construct expressing N40K specifically in neurons, including
CaMKIIα (CaMKII) promoter, a woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE) and long terminal repeats
(LTR). b, N40K mRNA expression by in situ hybridization. Tg brain
slides were stained with RNAscope probe specific for human N40K (Red)
and hematoxylin for nuclei (blue). Scale bar: 1 mm. c, Western blotting
to confirm N40K expression and loss of U1-70K specifically in the
brain. Ctx: cortex; Hipp: hippocampus. The experiments were performed
with three replicates and the loss of U1-70K by western blotting was
quantified (mean ± SEM, Student’s t-test, two-tailed). d,
N40K-assembled U1 snRNP complex in the Tg brain occludes the binding of
U1-70K. Immunoprecipitation of U1C Ab was followed by immunoblotting (3
replicates). e, Aggregation analysis of N40K in the Tg brain. The mouse
brain tissues were differentially extracted to generate detergent
soluble (Sol) and insoluble (Insol) fractions, followed by
immunoblotting (3 replicates). f, Immunostaining of WT and Tg brain
tissue with U1-70K C-terminal Ab (brown) that recognized only full
length U1-70K but not N40K. Slides were counter-stained with
hematoxylin for nuclei (blue). DG: dentate gyrus. Scale bar, 100 μm
(upper), 10 μm (lower) (n = 3 replicates). g, Confocal images of
immunofluorescence staining (hippocampal DG region, green: U1-70K and
N40K detected by U1-70K N-terminal Ab, red: full length U1-70K detected
by U1-70K C-terminal Ab. Scale bar, 10 μm. In b, f, g, each assay was
repeated from three mice. Full statistical information is in
[220]Source Data Statistics.
Next, we examined whether N40K-Tg displays neurodegeneration in the
brain. Compared with WT littermates, the Tg mice show no visible change
in whole body weight ([221]Extended Data Fig. 4a) but display a small
decrease (<10%) in brain weight and volume in 3- and 12-month-old mice
([222]Extended Data Fig. 4b, [223]4c). Magnetic resonance imaging (MRI)
shows a comparable decrease of cortical and hippocampal volumes in
N40K-Tg ([224]Fig. 4a and [225]Extended Data Fig. 4d, both Tg lines).
Counting of neuronal marker NeuN-stained cells indicates a significant
decrease (~30%) in neuronal density within cerebral cortex and
hippocampus ([226]Fig. 4b). These experiments support age-dependent
neuronal loss in the N40-Tg mice.
Figure 4. N40K-Tg mice display neuron loss and cognitive impairment.
[227]Figure 4.
[228]Open in a new tab
Tg396 was used in these studies. a, Volume change of mouse whole brain,
cortex and hippocampus (Hipp) measured by MRI at different ages
(1-month-old: WT n = 11, Tg n = 8; 3-month-old: WT n = 14, Tg n = 11;
12-month-old: WT n = 17, Tg n = 20; two-way ANOVA and Sidak's multiple
comparison test). b, Quantification of NeuN+ neurons in 12-month-old
mouse cortex, CA1 and DG of hippocampus (WT n = 3-4, Tg n = 3-4,
Student's t-test, two-tailed). c, Novel object recognition test
(3-month-old: WT n = 14, Tg n = 11; 12-month-old: WT n = 17, Tg n = 10,
Student's t-test, two-tailed). d, Percent of correct choice in Y-maze
(3-month-old: WT n = 12, Tg n = 12; 12-month-old: WT n = 15, Tg n =12,
Student’s t-test, two-tailed). e, Morris water maze task (12-month-old:
WT n = 18, Tg n = 14, two-way ANOVA and Sidak's multiple comparison
test). f, Morris water maze probe test. At day 6, the hidden platform
was removed to measure distance traveled in the target quadrant
(12-month-old: WT n = 14, Tg n = 12, two-way ANOVA and Sidak's multiple
comparison test). g, Visual test of 12-month-old WT and N40K-Tg mice
(12-month-old: WT n = 9, Tg n =9, Student's t-test, two-tailed). h,
Open field test. Left: total traveling distance. Right: center/total
ratio (12-month-old: WT n = 11, Tg n = 12, Student's t-test,
two-tailed). Data are shown as mean ± SEM. Full statistical information
is in [229]Source Data Statistics.
We then asked whether N40K expression perturbs brain cognitive
function. Compared with WT, N40K-Tg mice (12-month-old) display a
decline in novel object recognition ([230]Fig. 4c), and significant
defects of working memory in Y-maze test ([231]Fig. 4d) and spatial
memory in Morris water maze ([232]Fig. 4e, [233]4f, and [234]Extended
Data Fig. 4e, both Tg lines). The deficit appears to be limited to
memory tasks, as the Tg animals display normal performance in swimming
speed in Morris water maze ([235]Extended Data Fig. 4f), visual acuity
test ([236]Fig. 4g) and locomotor activity in open field test
([237]Fig. 4h). These results demonstrate impairments of recognition
and spatial memory in the aged N40K-Tg mice.
GABAergic synapses mediate hyperexcitability in N40K-Tg
To investigate how N40K expression contributes to cognitive impairment,
we performed RNA-seq analysis of WT and N40K-Tg mouse brain at 3 and 12
months of age (n = 15,705 transcripts, [238]Supplementary Tables 3,
[239]9). Compared with WT, N40K-Tg mice display an increase in intron
reads ([240]Fig. 5a and [241]Supplementary Table 3). While 416
transcripts show statistically significant splicing changes in the
3-month-old Tg mice, the number increases to 4, 428 in the 12-month-old
Tg mice ([242]Supplementary Table 9, FDR < 0.05), illustrated by a
shift of the distribution of splicing deficiency scores ([243]Fig. 5a).
We also re-analyzed RNA transcripts (n = 18,957) in human brain tissues
previously reported^[244]23 (ROS/MAP cohort, filtered by RNA integrity
number (RIN) of at least 8, accepting 18 control and 35 AD cases), and
found higher intron reads and splicing deficiency of 2,673 transcripts
in AD ([245]Fig. 5b, [246]Supplementary Tables 3, [247]10, FDR < 0.05).
Cross-species comparison reveals 984 overlapped transcripts ([248]Fig.
5c and [249]Supplementary Table 11), significantly enriched in multiple
pathways, such as glutamatergic, GABAergic, cholinergic, dopaminergic,
and serotonergic synapses, long-term potential (LTP), and autophagy
([250]Fig. 5d, [251]Supplementary Table 12, FDR < 0.05). The
transcripts/proteins were further analyzed by protein-protein
interaction (PPI) network^[252]21, also revealing PPI modules enriched
in glutamatergic synapse, GABAergic synapse, LTP, and autophagy
([253]Fig. 5e, [254]Supplementary Table 13, FDR < 0.05). Strikingly,
these PPI modules contain 15 transcripts/proteins identified in the
inhibitory postsynaptic density (iPSD), including GABRA2, GABRA3,
GABRA5, GABRB2, and GABRB3 ([255]Fig. 5e)^[256]40. It should be aware
of that these iPSD proteins can locate on the surface of excitatory
neurons, which is expected from the cell type specific expression of
N40K under the CaMKIIα promotor.
Figure 5. N40K-Tg mice exhibit AD-related splicing defects enriched in
synaptic function.
[257]Figure 5.
[258]Open in a new tab
a, Age-dependent increase of mapped intronic reads and histogram of
splicing deficiency score of individual transcripts in Tg mice (Tg396,
3-month-old: WT n = 3, and Tg n = 3, 12-month-old: WT n = 3, and Tg n =
3 biologically independent mouse samples). We quantified the percentage
of intron reads (Student's t-test, two-tailed) and transcript splicing
deficiency (Kolmogorov–Smirnov test). b, Increase of mapped intron
reads and histograms of splicing deficiency scores of individual
transcripts in ROS/MAP human AD cases^[259]23 (Ctrl n = 18, AD n = 35,
left: Student's t-test, two-tailed; right: Kolmogorov–Smirnov test).
Each point represents data of individual human cases c, Venn diagram of
mouse and human splicing-defective transcripts. d, Pathway enrichment
analysis of shared splicing-defective transcripts in the Tg and AD,
showing selected pathways from [260]Supplementary Table 12. FDR was
derived from p values (Fisher's exact test) by the BH procedure. e,
Four examples of enriched PPI modules ([261]Supplementary Table 13),
with the proteins in iPSD highlighted in red. Each dot represents a
protein, whereas the interaction is indicated by connected lines. f,
Validation of intron retention in selected transcripts by the density
of RNA reads (ex: exon; in: intron) and RT-PCR. Red boxes show genomic
regions selected for quantifying RNA-seq reads (Student's t-test,
two-tailed). Scale bar, 10 kb. g, qRT-PCR analysis of Gabra2
transcripts (Student's t-test). Data are shown as mean ± SEM. in a, f,
g. each point represents a data point of one mouse (WT n = 3, and Tg n
= 3). Full statistical information is in [262]Source Data Statistics.
To verify the splicing deficiency of individual transcripts in N40K-Tg
mice, we used the RNA-seq data to define intron retention events and
analyzed specific intron retention by RT-PCR. We focused on 4
transcripts involved in synaptic regulation, including Gabra2 (introns
4 and 6), Gng7 (intron 2, a G protein subunit that modulate
neurotransmission^[263]41,[264]42), Kcnh1 (intron 9, potassium channel
involved in epilepsy^[265]43) and Camk1d (intron 5, the neurosignaling
kinase linked to AD^[266]44) ([267]Fig. 5f, [268]Extended Data Fig. 5a,
[269]5b). In addition, splicing deficiency may lead to downregulation
of mRNA level, as exemplified by Gabra2 through quantitative RT-PCR
([270]Fig. 5g). Thus, our data support that the N40K-Tg mice display
splicing defects throughout the transcriptome, especially in the
transcripts associated with synaptic function, consistent with splicing
defects identified in human AD cases^[271]19,[272]23.
Considering that a large number of iPSD transcripts exhibit splicing
deficiency in N40K-Tg and in human AD cases, we next analyzed the level
of GABRA2 protein in the Tg mice and human AD brains, as well as the
relevant synaptic activity. The GABRA2 protein level dramatically
decreases in the Tg, as early as in 3-month-old mice ([273]Fig. 6a).
The GABRA2 protein also decreases in human AD brain tissues ([274]Fig.
6b), reminiscent of a previous report of reduced GABA[A] receptor
inhibitory function in AD^[275]45. As GABRA2 reduction is likely to
de-repress synaptic activity, we performed electrophysiological
experiments in the perforant pathway ([276]Fig. 6c) which plays a
central role in learning and is altered early and severely by
neuropathology in AD^[277]46,[278]47. Indeed, N40K-Tg mice show
significant impairments in long-term potentiation in the perforant
pathway ([279]Fig. 6d). We then examined the presynaptic and
postsynaptic activities. Although the frequency of presynaptic
neurotransmitter release was normal ([280]Fig. 6e), excessive
postsynaptic strength was observed in response to stimuli, and this
postsynaptic hyperexcitability was largely rescued by the
administration of the GABA[A] receptor agonist muscimol ([281]Fig. 6f,
[282]6g). Thus, our results strongly support that N40K-induced splicing
defects lead to downregulation of GABAergic synapse components and
neuronal hyperexcitability, contributing to the deterioration of memory
and cognition in mice.
Figure 6. N40K-Tg mice show GABRA2 reduction, post-synaptic hyperexcitation
and LTP impairment.
[283]Figure 6.
[284]Open in a new tab
a, GABRA2 protein reduction in N40K-Tg by western blotting (n = 5 mice
in each group, Student’s t-test). b, GABRA2 protein in AD cases by
western blotting (Ctrl n = 8, and AD n = 8, Student’s t-test). c,
Synaptic activity assay in perforant pathway, stimulating from
entorhinal cortex and recording on dentate gyrus (DG). d, LTP was shown
as field excitatory postsynaptic potential (fEPSP) slope versus
recording time, showing impairment in Tg396 mice. Representative traces
before and after Theta-burst stimulation (TBS) were shown (WT animals n
= 5, slices n = 7; Tg animals n = 5, Tg slices n = 11). The data of the
last 20 min were used for statistical analysis (two-way ANOVA). e,
Paired-pulse ratios that quantify presynaptic function, showing no
significant difference between WT and Tg mice. two-way ANOVA). f-g, The
plot of fEPSP slope against stimulus intensity shows a larger
excitability in Tg compared with WT, which were alleviated upon the
treatment with 2.5 μM muscimol (an agonist of GABAA receptor). Data are
shown as mean ± SEM. Statistical significance was analyzed by two-way
ANOVA: all four conditions together (vertical line), or any of the two
conditions. (WT animals n = 5, slices n = 7; Tg animals n = 5, Tg
slices n = 11, WT + muscimol animals n = 5, slices n = 5; Tg + muscimol
animals n = 5, slices n = 5 in d, e, f, g). Full statistical
information is in [285]Source Data Statistics.
Splicing dysfunction synergizes with the amyloid cascade
One central question is whether splicing dysfunction interacts with the
amyloid cascade in AD pathogenesis, as the U1 snRNP and plaque
pathologies are correlated but distinct in the brain of AD
patients^[286]19,[287]24. To address this question, we crossed N40K-Tg
with 5xFAD, an AD model of amyloidosis, to generate double transgenic
mouse model (dTg) that presents both pathologies ([288]Extended Data
Fig. 6a). To dissect the interaction between splicing dysfunction and
amyloid cascade in molecular level, we profiled 15,705 transcripts and
10,256 proteins in the four genotypes (WT, N40K-Tg, 5xFAD and dTg,
6-month-old, [289]Fig. 7a and [290]Extended Data Fig. 6b). Compared
with WT by splicing deficiency scores, dTg and N40K-Tg show the same
severity of splicing defect, while 5xFAD has no detectable splicing
defect ([291]Extended Data Fig. 6c, and [292]Supplementary Table 14).
Conversely, the N40K-Tg in the 5xFAD background can induce U1-70K loss
but does not significantly affect the Aβ level ([293]Extended Data Fig.
6d).
Figure 7. Synergistic effects of human N40K and amyloid pathway on synaptic
deregulation and cognitive deficiency.
[294]Figure 7.
[295]Open in a new tab
a, N40K-Tg mice bred with 5xFAD mice to obtain WT, N40K (N40K-Tg,
Tg396), FAD (5xFAD), and double transgenic mice (dTg) to profile
transcriptome (3 mice/genotype, 6-month-old) and proteome (5
mice/genotype except 4 mice for FAD, 6-month-old). To simplify data
processing, we only analyzed the major transcripts without considering
alternative splicing forms. b, Numbers of transcripts and proteins
differentially expressed (DE, FDR < 0.05) in N40K, FAD or dTg mice
alone and in any combinations of these three genotypes. c, Heatmaps of
selected DE proteins to show synergistic effects. d, Pathway enrichment
of the DE proteins in dTg (selected from [296]Supplementary Table 17).
FDR was derived from p values (Fisher's exact test) by the BH
procedure. e, Expression levels of GABRA2, MDK, NTN1, GPHN, CBLN4, and
NCALD in 6-month-old WT, N40K, FAD, and dTg mice (3 mice/genotype) were
analyzed by western blotting and quantified. Data are shown as mean ±
SEM. Statistical significance was analyzed by one-way ANOVA followed by
Tukey's multiple comparison test. The ANOVA overall p value is
significant for each protein. dTg vs FAD or dTg vs N40K comparisons: f,
The diagram of synergistic effect of splicing dysfunction and amyloid
pathway. g, Morris water maze test of 4-genotyped mice at three
different ages (3-month-old: WT n = 18, N40K n = 19, FAD n = 19, dTg n
= 19; 6-month-old: WT n = 18, N40K n = 19, FAD n = 19, dTg n = 18;
12-month-old: WT n = 18, N40K n = 16, FAD n = 15, dTg n = 14).
Statistical significance at each age was analyzed by two-way ANOVA. The
effect of genotype in all three ages is significant, and the F value of
the genotype increases with ages, consistent with larger difference at
the older ages. The comparison of FAD and dTg at each training day
using Tukey's multiple comparison test, the difference was not
significant at 3-month-old, but became significant at 6-month-old (3
and 5 days) and 12-month-old (all five individual days). Full
statistical information is in [297]Source Data Statistics.
We further analyzed differentially expressed (DE) transcripts/proteins
in three Tg genotypes versus WT. In N40K-Tg, 5xFAD and dTg mice, 191
transcripts/172 proteins, 173 transcripts/211 proteins, and 820
transcripts/979 proteins are significantly changed, respectively
([298]Fig. 7b, [299]Supplementary Tables 15, [300]16, FDR < 0.05). As
expected, N40K-Tg and 5xFAD mice share only a few DE components, but
both genotypes share their majority of DE components with dTg.
Strikingly, the dTg mice displays more than 50% unique DE components
(508 transcripts/666 proteins, [301]Fig. 7b) that are not present in
either N40K-Tg or 5xFAD. This synergy is shown in both upregulated and
downregulated proteins ([302]Fig. 7c). The DE proteins in dTg mice are
enriched in diverse pathways ([303]Fig. 7d, [304]Supplementary Table
17, FDR < 0.05); for example, the downregulated proteins are highly
enriched in synaptic function. The dTg DE list also contains a large
number of cell type specific proteins, including 163 in neurons, 70 in
astrocytes, 18 in oligodendrocytes, 150 in microglia, and 24 in
endothelia, implicating the contribution of different cell types
([305]Extended Data Fig. 6e, [306]Supplementary Table 18). We validated
some DE proteins in the mouse brain by western blotting, including
GABRA2, MDK/midkine and NTN1/netrin 1 (two Aβ-corrected proteins in
human AD brain^[307]21), GPHN/gephyrin (a scaffold at inhibitory
synapses to cluster glycine and GABA receptors^[308]48),
CBLN4/cerebellin 4 (an inhibitory transmission regulator reducing Aβ
toxicity^[309]49), and NCALD/neurocalcin-delta (a neuronal calcium
sensor related to SMA^[310]50) ([311]Fig. 7e). Additionally, we
performed deep RNA-seq analysis of a tau mouse model (P301S)^[312]51,
and did not find difference in the percentage of total intron reads,
but a slight defect in splicing deficiency, compared with wild type
littermates ([313]Extended Data Fig. 6g-[314]I, [315]Supplementary
Table 19). We did not cross N40K-Tg and P301S mice in this study.
Overall, the transcriptomic and proteomic data raise a new concept that
the splicing and amyloid pathways can synergize to amplify molecular
alterations in the dTg mouse brain ([316]Fig. 7f).
In addition, we compared the DE proteins in the three mouse models
(N40K-Tg, 5xFAD and dTg) with the human AD proteome, motivated by
recent transcriptomic comparison between AD human brains and mouse
models^[317]52. A meta-analysis of seven deep AD proteomic datasets
revealed 2,698 DE proteins in the brain^[318]18, in which 2,381
proteins have orthologues in mice. When overlapping the 2,381-protein
list with the 172 (N40K-Tg), 211 (5xFAD) and 979 (dTg) DE proteins, 15,
80 and 296 proteins displayed consistent changes, respectively
([319]Extended Data Fig. 7a, [320]7b). These overlapped proteins are
enriched in numerous pathways and protein-protein interaction modules,
such as RNA splicing, amyloid cascade, cytoskeleton and extracellular
matrix, endocytosis and exocytosis, growth and development,
inflammation, lipid metabolism, protein degradation and synaptic
function ([321]Extended Data Fig. 7c, [322]7d, [323]Supplementary
Tables 20, [324]21). Importantly, during this mouse-human comparison,
the synergistic effect in the dTg is observed in almost all pathways
listed above, and the enriched pathways in the dTg are consistent with
those identified in AD proteomic studies^[325]21,[326]53.
To investigate whether splicing dysfunction can synergize with the
amyloid cascade in cognition impairment, we performed Morris water maze
with littermate animals of the four genotypes (WT, N40K-Tg, 5xFAD, and
dTg) at three different ages (3-, 6- and 12-month-old) ([327]Fig. 7g).
Compared with N40K-Tg or 5xFAD, dTg mice show a trend of incapability
at 3-month-old, statistically significant memory impairment at
6-month-old, and much more severe impairment at 12-month-old, although
swimming speed in the test is similar for all mice ([328]Extended Data
Fig. 6f). These age-dependent results suggest that the synergistic
interaction of the splicing and amyloid pathways accelerates the
development of cognitive impairment.
In addition, as TDP-43 inclusions display in up to half of AD
cases^[329]12, we examined the TDP-43 pathology (stage 0 to 3) in the
ROS/MAP cases^[330]23 ([331]Supplementary Tables 3, RIN > 8, n = 53),
and found a weak correlation between TDP-43 stages and intron read
percentage in the RNA-seq analysis ([332]Extended Data Fig. 8a, r =
0.12). Although direct intron read comparison between different TDP-43
stages did not yield results of statistical significance ([333]Extended
Data Fig. 8b), the splicing deficiency scores of individual transcripts
revealed expected splicing alteration in the cases with high TDP-43
pathology ([334]Extended Data Fig. 8c), implicating a role of TDP-43 in
splicing dysfunction, which might co-exist with the impact of U1 snRNP
pathology in some AD cases. We further explored if TDP-43 proteinopathy
is induced in the mouse models of N40K-Tg, 5xFAD and tau P301S mice,
but found negative results in these models ([335]Extended Data Fig.
8d).
Discussion
A mouse model to recapitulate RNA splicing dysfunction in AD
U1 snRNP proteinopathy has recently been identified by proteomics and
validated by immunohistochemistry in AD patient
brain^[336]19,[337]24,[338]27-[339]29, together with widespread RNA
splicing dysfunction detected by transcriptomics^[340]19,[341]23, but
its contribution to AD progression was not well studied due to the
challenge of recapitulating mRNA splicing dysfunction in AD mouse
models (e.g. the 5xFAD amyloidosis model). Since U1 snRNP is central in
the function of RNA splicing, U1 snRNP genes are essential for
viability of human cell lines in a genome-wide CRISPR screen^[342]54,
and knocking out U1-70K gene in Drosophila also results in
lethality^[343]55. During our study of N40K toxicity, unexpectedly, we
found a potent dominant-negative effect of N40K to reduce the U1-70K
protein level in neurons. We then utilized this effect to impair U1
snRNP-mediated splicing function in a Tg mouse model. This model
provides a general approach to perturb RNA splicing mechanisms in the
mouse brain.
In N40K-Tg mice, neuron-specific expression of N40K is driven by the
CaMKIIα promotor^[344]39, and N40K impacts neuronal function by
potential loss- and gain-of-function mechanisms. Clearly, N40K
expression causes a loss-of-function effect by depleting full length
U1-70K protein. The depletion is dependent on the expression level of
N40K, which is approximately 2-fold higher than that of native U1-70K
in the WT mice. As a result, the majority of U1-70K in the cortex and
hippocampus is depleted to perturb the normal process of RNA splicing.
Moreover, N40K aggregation might exhibit a role of gain-of-function.
The disordered low complexity (LC) domains in N40K are liable to
protein aggregation^[345]33,[346]34, possibly through a process of
liquid-liquid phase separation^[347]4,[348]56. Although we did not find
obvious tangle formation in N40K-Tg, we detected evident accumulation
of N40K in detergent-insoluble fraction of mouse brain by western
blotting and by large-scale mass spectrometry. In addition, other
RNA-binding components (e.g. UT14A, and RRP1) are also sequestrated in
the detergent-insoluble fraction, which may restrict their
physiological functions in neurons, similar to the toxic roles of many
known misfolded proteins in neurodegeneration^[349]57. In addition,
protein aggregation might lead to the activation of unfolded protein
response and ER stress^[350]58,[351]59. We then examined the known
genes/proteins activated by the ER stress in the transcriptomic and
proteomic analyses of WT and N40K-Tg mice. Although we detected the
expression of six genes/proteins in three ER stress pathways (i.e.,
XBP1/JNK, PERK/EIF2A/ATF4, ATF6), none were significantly changed in
N40K-Tg, suggesting that the ER stress may not contribute to the
defects observed in N40K-Tg, in accordance with the marginal
overexpression of N40K. Based on our collected data, although we cannot
completely rule out the role of other pleiotropic effects of genetic
perturbation, RNA splicing dysfunction is believed to be the major
event detected in the N40K-Tg mice.
How can the N40K-Tg mice survive with the loss of the majority of
U1-70K in neurons? We believe that the truncated N40K protein replaces
U1-70K in spliceosome and can partially function in mRNA splicing.
Structural studies of human U1 snRNP complex reveal that the N-terminus
and RRM domain in N40K are indispensable for U1 snRNP assembly^[352]35,
supporting that N40K has ability to stabilize the complex of U1 snRNP.
A genetic experiment in Drosophila strongly supports this partially
functional hypothesis of N40K, in which expressing fly N40K homologous
portion rescues the embryonic lethality of the U1-70K knockout
fly^[353]55. However, without the C-terminus of U1-70K, the function of
the N40K-assemblied U1snRNP complex is compromised, demonstrated by
impaired splicing efficiency in the cellular and mouse models.
More excitingly, the N40K-Tg model mimics U1 snRNP proteinopathy, RNA
splicing defects and neuronal loss observed in AD brain. The N40K-Tg
results are highly similar in two independent mouse lines (Tg396 and
Tg318) that have different gene integration sites in mouse chromosomes.
Remarkably, splicing dysfunction in N40K-Tg mice alters a variety of
synaptic components, which may contribute to the imbalance between
excitatory and inhibitory pathways^[354]60. In particular, GABRA2
decreases significantly in the N40K-Tg, which is also confirmed in
examined human AD cases by western blotting and is supported by recent
meta-analysis of AD proteomics^[355]18, and single-cell RNA profiling
of human AD samples^[356]61. The downregulation of GABAergic pathway
proteins is in agreement with synaptic hyperexcitability and LTP
impairment, demonstrated by our electrophysiological study. The
synaptic hyperexcitability may also be attributed to neuronal loss
observed in the N40K-Tg mouse brain.
Interaction of U1 snRNP, amyloid, tau and TDP-43 pathologies
We provide clear evidence that U1 snRNP dysfunction synergizes with the
amyloid pathway to accelerate cognitive impairment, although Aβ and U1
snRNP pathologies appear to be independent in mice. It should be noted
that the human mutated APP was expressed as a recombinant protein in
5xFAD mice, bypassing the processing of RNA splicing. Therefore, the
5xFAD model is not suitable for studying the direct impact of splicing
dysfunction on human APP transcripts. Nevertheless, our double
transgenic mice offer a model to study the synergistic effect
downstream of Aβ and U1 snRNP pathologies. Through temporal spatial
memory test by water maze, we determined the synergetic effect in
cognitive decline as early as in 6-month-old mice. Moreover, proteomics
profiling of the same age mice identified aggravated alterations of
numerous synaptic components (e.g., Gabra2 and gephyrin) in double Tg
mice, compared with N40K-Tg or 5xFAD mice. The results suggest the
synergetic effect can occur to reduce inhibitory neurotransmission,
which orchestrates neuronal networks underlying the cognitive
process^[357]48, although the synergy is also observed in other
pathways. The result is consistent with the known function of
Aβ-induced synaptic hyperexcitability^[358]60, leading to cognitive
impairment by abnormal patterns of neuronal activity^[359]62. Together,
Aβ may exacerbate synaptic hyperexcitability together with
dysfunctional U1 snRNP, providing a possible explanation to the
synergistic effect.
Unlike amyloid plaques, tau tangles appears to be partially overlapped
with aggregated U1-70K by immunohistochemistry in human AD
brain^[360]19, and U1 snRNP complex and/or other RNA splicing proteins
(e.g. TIA1 and SRRM2) were detected to interact with tau in AD brain
tissues and/or tau mouse models^[361]63-[362]65. The U1 snRNP
pathology, however, has not been identified in the human cases of
non-AD tauopathies, such as corticobasal degeneration and
frontotemporal lobar degeneration (FTLD)-tau^[363]19, implicating that
tau pathology alone cannot induce U1-70K aggregation. Furthermore, our
RNA-seq comparison of a tau mouse model (P301S) and wild type did not
show statistically significant difference in the percentage of total
intron reads, although a minor change of splicing deficiency was
observed. Therefore, the N40K-Tg model is the only mouse model
currently recapitulating widespread splicing defects in AD.
Nevertheless, it is still highly possible that U1 snRNP complex may
co-aggregate with tau in neurodegenerative cases, reminiscent of
enhanced fibrillization of tau and alpha-synuclein in the inclusion
formation^[364]66.
In addition, TDP-43 inclusions have been reported in a significant
portion (up to ~50%) of AD cases^[365]12, and are emphasized by a
recently proposed disease entity of limbic-predominant age-related
TDP-43 encephalopathy (LATE)^[366]67. Our correlation analysis of RNA
splicing and TDP-43 pathology suggest that TDP-43 and U1 snRNP
pathologies may occur simultaneously to affect RNA splicing in some AD
cases, but the TDP-43 proteinopathy cannot be induced in N40K-Tg, 5xFAD
and tau P301S mouse models. The data reiterate the limitation of the
mouse models, none of which recapitulates the full spectrum of complex
pathologies in human AD brains. Each mouse model, however, allows the
detailed investigation of selected mechanism, and different models
(e.g., N40K-Tg and 5xFAD) may be crossed to dissect the interaction of
multiple pathways.
The N40K-Tg model has some limitations. First, the expression of human
N40K in the mice is controlled under a short CaMKIIα promotor^[367]39
due to the package capacity of recombinant Lentivirus. It is possible
that N40K-induced splicing defects might perturb brain development. For
instance, downregulation of GABRA2 protein is detected in young N40K-Tg
mice (i.e., 3-month-old). This caveat may be alleviated by N40K
inducible mouse models in the future. Moreover, N40K expression in
neurons results in the depletion of full length U1-70K
(loss-of-function) and the co-aggregation of N40K and other proteins
(gain-of-function), increasing the complexity for data interpretation.
Finally, some of the splicing-related molecular changes in N40K-Tg mice
are not expected to occur in human cases, because of the species
difference in genomic intron-exon architecture. Future development of
human iPSC models would partially address this concern.
In summary, we thoroughly studied the toxic mechanism of AD-associated
N40K and splicing defects in primary cultured neurons and mouse models.
Our findings lead to critical mechanistic insights into understanding
how RNA splicing dysfunction causes neural hyperexcitation, cognitive
abnormalities, and neurodegeneration in these disease models. Our
studies strongly suggest that U1 snRNP proteinopathy and splicing
disruption may play an unexpected role in AD pathogenesis, implicating
a novel pathway for therapeutic targeting.
Methods
All experiments conducted in this study comply with all relevant
ethical regulations. All animal experiments were approved by the
Institutional Animal Care and Use Committee (IACUC) at St Jude
Children’s Research Hospital. Human postmortem brain tissue samples
(frontal gyrus) were provided by the Brain and Body Donation Program at
Banner Sun Health Research Institute, in which the studies were
approved and written consent for sample collection was obtained.
Clinical and pathological diagnoses were based on the established
criteria^[368]68.
Statistics and reproducibility.
No statistical methods were used to predetermine sample sizes, but the
sample sizes were similar to those reported in previous
publications^[369]69-[370]71. Data distribution was assumed to be
normal, but this was not formally tested. Statistical methods were
applied to estimate p values followed by the analysis of false
discovery rate. No animals were excluded from the study. The
experiments were not randomized, in which animals were used when
available. The investigators were blinded to the genotype information
during data acquisition for experiments of MRI, stereology,
electrophysiology and behavior tests. In small-scale analyses,
two-tailed unpaired Student’s t-test was used for two-sample
comparisons, while one-way or two-way ANOVA (Graphpad Prism 8.0.2) was
used for group comparisons followed by Tukey's or Sidak’s post hoc
correction. The Kolmogorov-Smirnov test was used to analyze statistical
significance of splicing deficiency of the comparison between mouse
models and human cases.
Primary mouse neuronal culture.
Mouse cortical neurons were isolated from mouse embryos (C57BL/6,
Charles River) at E18, and cultured in completed Neurobasal A (Life
Technologies, Cat# 10888022) supplemented with B27 complex (Thermo
Fisher, Cat# 17504044) and GlutaMAX (Thermo Fisher, Cat# 35050-061).
Half of the medium was replaced by new medium every two days.
Lentiviral infection was performed at DIV 7-12. For time-dependent
assay: neurons were infected at MOI 10 for 2, 4, and 6 days. For
inhibition of protein degradation, after neuronal infection with N40K
lentivirus (MOI 10) for 2 days, MG132 or Bafilomycin A1 were added for
1-2 days, followed by harvest and immunoblotting.
Mouse model.
All mice (C57BL/6J) were housed under a 12 hour: 12 hour (light: dark)
cycle at 22-25 °C and 40% - 60% humidity. N40K-Tg mouse lines were
generated in St Jude Children’s Research Hospital by lentiviral
transgenic method as described^[371]72. N40K lentiviral vector
(FCK1.3GW-N40K, 1x10^8 titer) was microinjected into perivitelline
space of fertilized eggs from C57BL/6J. Genetic background was
confirmed by C57BL/6 substrain characterization panel (The Jackson
Laboratory). The founder line (Tg396) and another line (Tg318) showed
high N40K expression and was backcrossed to C57BL/6J for at least two
generations. Hemizygous Tg mice and non-transgenic wild type
littermates were used in experiments. 5xFAD mice were purchased from
The Jackson Laboratory (MMRRC_034848-JAX). We crossed N40K-Tg with
5xFAD, to generate double transgenic mouse model (dTg). In all mouse
experiments, similar numbers of male and female animals were used.
Lentiviral vectors.
Human U1-70K or N40K coding sequences were cloned into BamHI/EcoRI
cites of FCK(1.3)GW vectors (Addgene, plasmid #27230), driven by a
modified mouse CaMKIIα promoter which shows strong expression in
pyramidal neurons^[372]39.
Cell culture and immunoprecipitation (IP).
HEK 293T cells (ATCC, CRL-3216) were cultured in DMEM media (Thermo
Fisher Scientific, Cat# 11995-065) supplemented with 10% FBS (Thermo
Fisher Scientific, Cat# 16140-071). Cells were transfected with
plasmids of pCDNA3.1, pCNDA3.1-U1-70K-FLAG, or pCDNA3.1-N40K-FLAG using
X-tremeGENE 9 DNA Transfection Reagent (Millipore Sigma, Cat# XTG9-RO),
following the manual with 1:3 ratio (DNA/transfection reagent). After
two days, transfected cells were harvested and extracted with the lysis
buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, 1 mM
EGTA, and 1x protease inhibitor cocktail (Roche, Cat# 11836170001). IP
was performed with anti-FLAG M2 magnetic beads (Millipore Sigma, Cat#
M8823) following the manufacturer’s protocol.
In the IP experiment from mouse brain, the cortex of 6-month-old WT or
N40K-Tg mice was homogenized in the hypotonic buffer (10 mM HEPES, pH
8.0, 0.2 mM PMSF, 0.5 mM DTT, phosphatase inhibitor, protease inhibitor
cocktail and RNAse inhibitor) at 5:1 ratio (w/w) in Dounce homogenizer,
followed by the addition of other components to adjust the final
concentrations to 10 mM HEPES, pH 8.0, 1.5 mM MgCl[2], 600 mM NaCl, and
0.5% Triton X-100. The homogenates were further lysed by glass bead
beating in Bullet Blender at 4 C for 5 min at speed 6. The lysates were
centrifuged at 21,000 x g for 15 min to remove debris. The supernatants
were dialyzed against the low salt buffer containing 10 mM HEPES, pH
8.0, 1.5 mM MgCl[2], 150 mM NaCl and 0.5% Triton X-100. IP was
performed with rabbit anti-U1C Ab (Santa Cruz, Cat# sc-374428) coupled
to Dynabeads (Thermo Fisher Scientific, Cat# 10003D) overnight at 4 C.
Then the beads were washed three times with the cold low salt buffer
and spliceosome complexes were eluted by the sample loading buffer for
SDS-PAGE and western blotting.
Neuron survival assay.
Neuron survival/death was determined by Cell-Titer Glo assay (Promega,
Cat# G7572) for viability. The cells were equilibrated to 21 °C for 20
min, and then an equal volume of Cell-Titer Glo was added to each well.
After incubation for 30 min, luminescence was detected with an Envision
2102 Multilabel Plate Reader (PerkinElmer Life Sciences). For
dose-dependent toxicity assay of N40K, mouse cortical neurons at DIV 7
were infected with lentiviral particles at MOI 7, 20, 60 for 5 days.
For pharmacological inhibition assay, neuron infected with lentivirus
at MOI 20 for 2 days, and then inhibitor compounds were added for 3
more days.
Detergent extraction.
The analysis was adapted from a previously published protocol^[373]19.
Human brains or primary neurons were extracted with 1% sarkosyl
(N-lauroyl-sarcosine) in Lysis Buffer (10 μM Tris, pH 7.5, 5 mM EDTA, 1
mM DTT, 10% sucrose, and protease inhibitor cocktail). Primary neurons
(DIV 12) were infected with lentiviral vector expressing EGFP,
U1-70K-FLAG, and N40K-FLAG by CaMKIIα promoter (MOI 10) for 4 days.
Mouse hippocampi were extracted with RIPA buffer (10 mM Tris pH 7.5,
100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% Triton X-100, 0.5%
sodium deoxycholate, 10% glycerol, and protease inhibitor cocktail, 10
μl buffer per mg tissue). The insoluble pellet was resuspended in 8 M
Urea with 2% SDS for Western blotting or proteome profiling.
Mass spectrometry-based proteomics.
We used an optimized protocol of TMT-LC/LC-MS/MS for deep proteome
profiling^[374]31,[375]32. Protein samples were lysed by homogenization
in the lysis buffer (50 mM HEPES, pH 8.5, 8 M urea, and 0.5% sodium
deoxycholate), and their concentrations were measured by the BCA assay
(Thermo Fisher, Cat# 23227) and confirmed by Coomassie-stained short
SDS gels ^[376]73. Quantified protein samples (~0.1 mg per TMT channel)
were digested with Lys-C (Wako, distributor 121-05063, 1:100 w/w) for 2
h at 21 °C, followed by dilution to decrease urea to 2 M and trypsin
digestion (Promega, Cat# V5113, 1:50 w/w) overnight at 21 °C. Cys
residues were reduced and alkylated by iodoacetamide. The proteolysis
was terminated by adding trifluoroacetic acid to 1%. The resulting
peptides were desalted with the Sep-Pak C18 cartridges (Waters),
TMT-labeled (Thermo Fisher, Cat# A34808 and [377]A44520), and pooled
equally. The pooled peptides were resolved by basic pH reverse phase LC
on an XBridge C18 column (3.5 μm beads, 4.6 mm x 25 cm, Waters; buffer
A: 10 mM ammonium formate, pH 8.0; buffer B: 95% acetonitrile, 10 mM
ammonium formate, pH 8.0, ~2 h gradient, 40-80 concatenated fractions
collected)^[378]74. Each fraction was analyzed by acidic pH LC-MS/MS
(75 μm x ~20 cm, 1.9 μm C18 resin from Dr. Maisch GmbH, buffer A: 0.2%
formic acid, 5% DMSO; buffer B: buffer A plus 65% acetonitrile, ~1.5 h
gradient). The settings of Q Exactive HF Orbitrap MS (Thermo Fisher)
included the MS1 scan (~410-1600 m/z, 60,000 resolution, 1 x 10^6 AGC
and 50 ms maximal ion time) and 20 data-dependent MS2 scans (fixed
first mass of 120 m/z, 60,000 resolution, 1 x 10^5 AGC, ~110 ms maximal
ion time, HCD, 32-35% normalized collision energy, ~1.0 m/z isolation
window with 0.3 m/z offset, and ~15 s dynamic exclusion)^[379]32.
The raw MS data were searched against protein database by the JUMP
software (v1.13.4)^[380]75, which utilizes both pattern matching and de
novo tag scoring to improve the sensitivity and specificity. A
composite target/decoy database was used to evaluate FDR in peptide
identification^[381]76,[382]77. The protein target database combined
downloaded Swiss-Prot, TrEMBL, and UCSC databases (human: 83,955
entries; mouse: 59,423 entries). Search parameters included
precursor/product ion mass tolerance (± 10 ppm), full trypticity,
static mass shift (TMT tags of 229.16293 and Cys carbamidomethylation
of 57.02146 on cysteine), dynamic mass shift (Met oxidation of
15.99491), two maximal miscleavage sites, and three maximal
modification sites. Peptide-spectrum matches (PSMs) were filtered by
matching scores and mass accuracy to limit protein FDR below 1%.
Proteins were quantified from TMT reporter ions based on our published
method^[383]78. Briefly, TMT reporter ion intensities were extracted
for each PSM, corrected by isotopic distribution of TMT reagents,
filtered to remove poor PSMs (e.g., minimum intensity of 1,000), and
adjusted to eliminate sample pooling bias. The relative protein
intensities were averaged from all assigned PSMs after removing
outliers (e.g., Dixon’s Q-test or generalized extreme Studentized
deviate test). Finally, the absolute protein intensities were derived
by multiplying the relative intensities by the grand mean of top three
abundant PSMs.
The analysis of differential expressed proteins in proteomic datasets
essentially followed the limma R package (v3.48.3)^[384]79 in multiple
steps: (i) obtain the protein quantification data from MS as described
above; (ii) perform a log transformation of the data^[385]80; (iii)
calculate the p values by moderated t-test, and FDR values by the
Benjamini-Hochberg procedure, using limma R package; (iv) calculate the
mean for each protein under different conditions and derive log[2](fold
change); and (v) fit log[2](fold change) data of all proteins to
Gaussian distribution to generate a “global” standard deviation value.
Statistically significant changes were usually based on FDR cutoff
(0.05) and log[2](fold change) cutoff (two standard deviations).
RNA-seq analysis.
For primary neurons, mouse cortical neurons (DIV7) were infected with
lentiviral vectors expressing EGFP or N40K driven by CaMKIIα promoter
at MOI 20 for 3 days. Total RNA was extracted by RNAeasy Mini kit
(Qiagen, Cat# 74104). For mouse hippocampus or cortex, total RNA was
extracted by RNeasy Lipid Tissue Mini Kit (Qiagen, Cat# 74804) as
described in the manual, including with DNAse I digestion to remove
contaminated DNA. Total RNA was eluted with RNase-free water and stored
at −80°C. RNA quality was examined by 2100 Bioanalyzer RNA 6000 Nano
assay (Agilent, Cat# 5067-1511). RNA sequencing libraries were prepared
with the TruSeq Stranded mRNA Library Prep Kit (Illumina, Cat#
20020594), including poly(A) RNA purification. Libraries were then
quantified using the Quant-iT PicoGreen dsDNA assay (Thermo Fisher,
Cat# [386]P11496) and MiSeq Reagent Kit Nano v2 (Illumina, Cat#
MS-102-2001). One hundred cycle paired-end sequencing (100 bp) was
performed on an Illumina HiSeq 2500. Library preparation and RNA-seq
were performed at the Genome Sequencing Core at the St Jude Children’s
Research Hospital. An average of 130 million reads were obtained for
each sample. For human cortex samples, we obtained the RNA-seq raw
reads from the ROS/MAP study^[387]23. Individual cases were first
filtered by RNA integrity (RIN > 8) and then categorized by the
cognitive COGDX score and the neuritic plaque CERAD score, resulting in
18 control samples (COGDX = 1 and CERAD = 4) and 35 AD samples (COGDX ≥
4 and CERAD of 1-3).
Reads from RNA-seq were mapped by our in-house mapping pipeline
‘Strongarm’ (v1.0)^[388]81. Reads were aligned to the following four
database files using BWA (v0.5.5) aligner: (i) the (mouse mm9) human
hg19 reference sequence; (ii) RefSeq; (iii) a sequence file
representing all possible combinations of non-sequential pairs in
RefSeq exons; and (iv) AceView database flat file downloaded from UCSC
representing transcripts constructed from ESTs. The mapping results
were aligned to the reference genome coordinates and the final BAM file
was constructed by selecting the best alignment in the four databases.
The quantification of mapped transcripts was estimated with
htseq-count^[389]82.
To extract the reads mapped to whole genes, including exons and
introns, we converted the BAM to BED files using bedtools
([390]http://code.google.com/p/bedtools, bamToBed, v2.16.1), and used
Table Browser program ([391]http://genome.ucsc.edu) to generate three
BED files: (i) Reference Sequence Genes (Whole Gene), (ii) Reference
Sequence Genes (Exons) and (iii) Reference Sequence Genes (Introns).
Finally, we used intersectBed (v2.16.1) to define the intersections
between mapped reads and the three RefSeq BED files.
Differential gene expression in transcriptomic datasets was performed
using moderated t-test in the limma R package and voom^[392]83 (inside
limma v3.48.3) with the raw read counts. The p values were converted to
FDR values (cutoff of 0.05) by the Benjamini-Hochberg procedure.
Splicing deficiency analysis.
The global splicing deficiency was first evaluated by the percentage of
reads mapped to introns, after normalized to the total reads mapped to
whole genes^[393]19. Moreover, we defined a splicing deficiency score
for each gene as the ratio of length-corrected read counts aligned to
introns and exons:
[MATH: Splicing deficiency
score=(#intronic reads∕total intronic length)(#exonic reads∕total exonic length) :MATH]
A high splicing deficiency score indicates low splicing efficiency. For
comparative analysis, p values were calculated by moderated t-test in
the limma R package and followed by the Benjamini-Hochberg procedure to
derive FDR values (cutoff of 0.05).
RT-PCR for validating intron retention.
RT-PCR was performed by a two-step method. Total RNA from each sample
was converted to cDNA using random primers [High-Capacity Reverse
Transcription Kit (Thermo Fisher, Cat# 4374966). The products are then
subjected to PCR analysis using primers ([394]Supplementary Table 23).
RT-PCR products were resolved by gel electrophoresis followed by SYBR
Safe DNA Gel Stain. Gel bands were quantified by Image J. Intron
retention was calculated by
[MATH: Intron retention%=(Exon-Intron junction PCR band
intensity)(Exon-Intron PCR band
intensity+Exon-Exon PCR band
intensity)
:MATH]
Quantitated RT-PCR.
mRNA levels of mouse endogenous U1-70K and human N40K levels were
quantitated with TaqMan assay (Mouse U1-70K Mm0120544, Human N40K
Hs01091623, Thermo Fisher Scientific) Relative expression levels were
calculated by ΔΔCT method with using 18S as the reference gene.
Pathway enrichment by KEGG and gene ontology databases.
Pathway enrichment analysis was carried out by the JUMPn software
(v1.13.0)^[395]84 to identify the biological functions of dysregulated
genes/proteins in a given dataset. The analysis was performed using
Fisher’s exact test against the Gene Ontology (GO) biological process,
molecular function, and cellular component annotations, and KEGG
pathway database, separately. The homologous genes between human and
mouse were used as the background. The p values derived from Fisher’s
exact test were further adjusted into FDR using the Benjamini-Hochberg
procedure for multiple testing. Enriched pathways with FDR values <
0.05 were considered statistically significant.
Protein-protein interaction (PPI) network analysis.
The analysis was performed based on our previously published
protocol^[396]85 with modifications. DE genes/proteins were
superimposed onto a composite PPI database by combining STRING
(v11)^[397]86, BioPlex (v3.0)^[398]87, and InWeb_IM
(v2016_09_12)^[399]88. The BioPlex database was developed by the method
of affinity purification and mass spectrometry, whereas the STRING and
InWeb contain information from various sources. Due to this
heterogeneity of PPI interactions, the STRING and InWeb databases were
further filtered by the edge score to ensure high quality, with the
following rules: (i) only edges with evidence of physical interactions
(e.g. through co-IP or yeast two-hybrid) were considered; (ii) edges of
high confidence, as filtered by the edge score, with the cutoff
determined by best fitting the log-log degree distribution using the
scale free criteria^[400]89. The finally accepted STRING and InWeb
databases were combined with BioPlex and the inhibitory postsynaptic
density interactome^[401]40 to construct a composite PPI database,
which includes 20,485 proteins and 1,152,607 PPI connections. PPI
modules were then defined by a three-step procedure: (i) extracting a
subnetwork by retaining PPI between two proteins if both were from the
DE protein list; (ii) calculating a topologically overlapping measure
(TOM)^[402]90 between each pair of proteins for the resulting PPI
subnetwork, and (iii) dividing this network into individual modules
based on the TOM clustering using the hybrid dynamic tree-cutting
method^[403]91. The biological functions of each PPI module were
further obtained using the proteins in each module as the input to
perform the pathway enrichment analysis as described above.
Fluorescence in situ hybridization of mouse chromosomes.
Purified FCK(1.3)GW-U170K plasmid DNA was labeled with a red-dUTP
(Alexa Fluor 594, Thermo Fisher, Cat# [404]C11400) by nick translation.
The labeled transgene probe was combined with sheared mouse cot DNA and
hybridized to metaphase and interphase nuclei derived from the
transgenic mouse lung fibroblast culture. Specific chromosome control
probes labeled with green-dUTP (Alexa Fluor 488, Thermo Fisher, Cat#
[405]C11397) were used to confirm the localization. Chromosome 18
control probe (RP24-255J24) for Tg396 and chromosome 10 control probe
(RP24-360A19/10A1)) for Tg318 were used. The chromosomes were stained
with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Cat#
[406]D21490) and analyzed.
mRNA in situ hybridization.
In situ hybridization for human N40K mRNA in formalin-fixed paraffin
embedded (FFPE) tissues^[407]92 was completed on a Discovery Ultra
automation system platform (Ventana Medical Systems) using RNAscope^®
VS Reagent Kit – RED (Advanced Cell Diagnostics). An oligoprobe set
specific for N40K (not for U1-70K) (Advanced Cell Diagnostics, Cat#
760-234) was applied according to the manufacturer’s instruction.
Hybridization signals were detected by chromogenic development with
Fast Red, followed by counterstaining with hematoxylin. Each sample was
quality controlled for RNA integrity with RNAscope oligoprobes for PPIB
RNA, and for non-specific background staining with oligoprobes for
bacterial dapB RNA. The specific RNA staining signal was identified as
intracellular red punctate dots.
Immunohistochemistry.
The staining was performed essentially as previously reported^[408]21.
Briefly, brain tissue samples were fixed with 10% formalin, and
embedded in paraffin. 5-10 μm sections were deparaffinized, rehydrated
and rinsed with water. Antigen retrieval was performed with 10 mM
citric buffer (pH 6.0) with boiled water bath (20 min) and cool down to
room temperature. Endogenous peroxidase activity is blocked by 1%
H[2]O[2] in PBS buffer (10 min). After rinsed by PBS, sections were
then blocked with 5% donkey serum in PBS with 0.3% Triton X-100 (PBST)
for 30 min at room temperature followed with primary antibodies diluted
with PBST plus 2% BSA and 0.2% skim milk for overnight at 4°C. Resource
and dilution of primary antibodies are listed in [409]Supplementary
Table 22. After washing with PBS, sections were then incubated with
secondary antibodies (Jackson ImmunoResearch Laboratories) with 1:500
dilution, 1 hour at room temperature. For fluorescence staining,
fluorescent dye-conjugated secondary antibodies were used with nuclear
counter stain by DAPI (Thermo Fisher Scientific, Cat# [410]D21490) or
followed with amyloid plaque stain by 0.02% thioflavine S
(Sigma-Aldrich, Cat# T1892) for 5 min. For chromogenic immunodetection,
biotin-conjugated secondary antibodies were used followed with ABC
reaction by Vectastain ABC kit (Vector Laboratories, Cat# PK-4000) and
developed by 3,3-diaminobenzidine solution (Vector Laboratories, Cat#
SK-4100). Images were captured by Zeiss LSM 780 confocal microscopy or
Zeiss Axioscan.Z1.
Quantitation of brain cells by stereology.
Design-based stereology (thick section, 3-dimensional) was carried out
in an investigator blinded manner. Brain tissue was cryopreserved
followed by serially sagittal cryosectioning at 40 μm. Every 5th
section was immunostained with anti-NeuN antibodies followed with
chromogenic development of DAB. NeuN+ neurons were counted in the
cortical regions using the optical fractionator probe
(StereoInvestigator). Counts of NeuN+ cells were estimated using
Microbrightfield Stereo Investigator (MBF Biosciences, Williston, VT)
and the optical fractionator method^[411]93 using a Olympus BX-51
microscope and 40 X objective. The density of cortical NeuN+ neurons
were calculated by total number of NeuN+ neurons divided by the
estimated volume.
In vivo magnetic resonance imaging (MRI) and brain volume analysis.
Preclinical MRI studies were performed using a 7 T Bruker ClinScan
system (Bruker BioSpin MRI GmbH) equipped with a 12 S gradient coil. A
mouse head volume coil (Bruker BioSpin) was used for high resolution
brain imaging. Animals were anesthetized and maintained with 1.5-2%
isoflurane during MRI sessions. Transverse T2-weighted turbo spin echo
images were acquired for volume measurements (TR/TE = 3840/50 ms, FOV =
25 x 25, matrix = 320 x 320, NEX = 1, Thickness = 0.4 mm, scan time =
11.5 min). Volume measurements (brain, hippocampus and cortex) were
obtained by manually segmenting the regions and computing volumes using
OsiriX (v5.7, Pixmeo, Switzerland).
Behavior studies.
Spatial working memory was conducted by “Y-maze” with delayed
non-matching to position task^[412]94. Y-maze apparatus consisted of a
start arm (47 x 10 x 10 cm) leading to two goal arms (34 x 10 x 10 cm).
Each mouse was first familiarized individually, with running in the
maze and presentation with a positive reinforcer of a drop of sweet
milk. Habituation was performed on the first three days of testing, in
which mice were released from the start location to freely investigate
the maze, with the positive reinforcer placed in the stem and in the
reward wells. After habituation and demonstration of consumption of
milk reward, the experiment trials were initiated. Each trial consisted
of a run from a start arm to a sample arm, a return to the start arm
with 15 s interval, and subsequent free choice of goal arm. Each mouse
choosing the non-sample arm was counted as correct response, and the
mouse was rewarded. Sample arm was determined randomly, with an upper
limit of three consecutive same sample arms. A total of 12 trials were
given over a period of 3 days. Water restriction was performed through
the study, in which mice were allowed 2 h of free access to water per
day after testing.
General locomotor and exploratory activities were measured by “open
field test”. The apparatus consisted of a clear square arena with blue
plastic floor measuring 16 inches × 16 inches with walls 16 inches tall
(San Diego Instruments). Testing was performed under white light
condition. Mice were brought into the testing room and allowed to
habituate for 1 h before the test. The activity of the mice was video
recorded and scored using visual tracking software (CleverSys, TopScan
Suite v3). Locomotor activity was determined by allowing the mice to
freely investigate the testing arena for 15 min. The surfaces of the
field were wiped between each animal with 70% alcohol to avoid
olfactory cues.
Long-term recognition memory was measured by “novel object recognition
test (NOR)”^[413]95. NOR was conducted in the square locomotion chamber
as open field under normal light. Each mouse was habituated to the
testing arena for 10 min and returned to home cage for 5-10 min. On the
day of testing, the mice were habituated to the testing arena for 3-10
min and then returned to home cages for 5-10 min. Next the mice were
placed back in the testing arena for 8 min with two identical objects
(T1). The objects were plastic, custom fabricated (4cm in height, 4cm
in diameter). The mice were then placed back into home cages. 1 h
later, each mouse was returned to the testing arena for 8 min and
exposed to one familiar object (T1) and one novel object (T2). The
activity of the mice was video recorded and scored using visual
tracking software (CleverSys, TopScan Suite v3). The time each mouse
spent intentionally touching the object and the time the mouse was
within 1 cm of the object facing it were recorded as touch time. NOR%
was calculated as the percent of time each animal explored the novel
object relative to all objects. The surfaces of the field were cleaned
with 70% alcohol after each trial.
Long-term spatial memory was measured by “Morris water maze”^[414]96.
The maze consisted of a 122-cm-diameter pool filled with water clouded
with white, nontoxic, water-based paint. The maze was virtually divided
into four quadrants, with one containing a hidden platform located 0.8
cm below the water surface. Each mouse was trained to find the
platform, orienting by extra maze cues placed asymmetrically as spatial
references. Individually, the mouse was placed into the water of a