Abstract
Microglia and complement can mediate neurodegeneration in Alzheimer’s
disease (AD). By integrative multi-omics analysis, here we show that
astrocytic and microglial proteins are increased in Tau^P301S synapse
fractions with age and in a C1q-dependent manner. In addition to
microglia, we identified that astrocytes contribute substantially to
synapse elimination in Tau^P301S hippocampi. Notably, we found
relatively more excitatory synapse marker proteins in astrocytic
lysosomes, whereas microglial lysosomes contained more inhibitory
synapse material. C1q deletion reduced astrocyte–synapse association
and decreased astrocytic and microglial synapses engulfment in
Tau^P301S mice and rescued synapse density. Finally, in an AD mouse
model that combines β-amyloid and Tau pathologies, deletion of the AD
risk gene Trem2 impaired microglial phagocytosis of synapses, whereas
astrocytes engulfed more inhibitory synapses around plaques. Together,
our data reveal that astrocytes contact and eliminate synapses in a
C1q-dependent manner and thereby contribute to pathological synapse
loss and that astrocytic phagocytosis can compensate for microglial
dysfunction.
Subject terms: Neuroimmunology, Alzheimer's disease, Glial biology,
Ageing
__________________________________________________________________
In this multi-omics study, the authors identified C1q-dependent synapse
elimination by both astrocytes and microglia in Alzheimer’s mouse
models. While astrocytes preferentially removed excitatory synapses,
microglia preferred inhibitory synapses.
Main
Chronic neuroinflammation, manifested by gliosis and elevated levels of
proinflammatory cytokines and synapse loss are hallmarks of
AD^[66]1,[67]2. One pathway that is aberrantly overactivated in mouse
models and brains of patients with AD and drives neuronal damage and
synapse loss is the classical complement pathway (CCP)^[68]2–[69]4. CCP
factors are abnormally elevated in brains and cerebrospinal fluid (CSF)
of patients with AD^[70]3–[71]5. Human genetic association studies
support an involvement of the innate immune response including the
complement pathway, in the pathogenesis of AD^[72]2. Components and
regulators of the complement cascade are also genetically associated
with schizophrenia and age-related macular degeneration^[73]6–[74]9 and
levels of various CCP molecules are increased in brains of patient and
mouse models of AD, multiple sclerosis (MS), frontotemporal dementia
and several other central nervous system (CNS)
disorders^[75]3,[76]4,[77]10–[78]14. This suggests that dysregulation
of the complement pathway could play a role in diverse CNS disorders.
In support of complement’s neurotoxic role, pharmacological or genetic
inhibition of the complement pathway ameliorates neurodegeneration and
synapse loss in mouse models of AD, MS, frontotemporal dementia and
neuro-invasive virus infection^[79]3,[80]4,[81]10–[82]12,[83]15.
During development, microglia refine neuronal circuits by engulfing
excess synapses^[84]16, which is at least in part
CCP-dependent^[85]17–[86]19. The CCP is initiated upon C1q binding to
pathogens, apoptotic cells or other structures, including synapses that
are destined for clearance. Subsequent activation of the CCP results in
proteolytic cleavage of the complement factor C3, leading to microglial
phagocytosis of complement-tagged synapses^[87]17. In addition to
microglia, astrocytes have been shown to remove synapses during
development, in the adult brain and in disease^[88]20–[89]22. In
contrast to microglia, however, synapse eating by astrocytes seems to
be C1q-independent under physiological conditions^[90]21. While a
synaptotoxic role for reactive astrocytes has been identified across
different CNS diseases, including AD, Huntington’s disease, Parkinson’s
disease and MS^[91]22–[92]24, the molecular mechanisms remain largely
unknown.
Here, we show that C1q deletion is neuroprotective in Tau^P301S
transgenic mice (termed P301S hereafter), a mouse model of tauopathy
and AD. Using multi-omics analysis and follow-up experiments, we
unexpectedly found that astrocytes have a major role in the removal of
excitatory synapses and also participate in the removal of inhibitory
synapses in P301S mice in a C1q-dependent manner. In TauPS2APP mice, an
AD mouse model that combines β-amyloid and Tau pathologies, we found
that microglial phagocytosis of synapses near plaques is impaired in
the absence of the AD risk gene Trem2. In TauPS2APP;Trem2^KO brains,
astrocytes compensate for microglial dysfunction around plaques through
increased eating of inhibitory synapses. Our data reveal an unexpected
preference for excitatory versus inhibitory synapse engulfment by
astrocytes versus microglia and support the idea that inhibition of
complement is an attractive strategy to ameliorate neurodegeneration in
AD.
Results
C1q deletion reduces neurodegeneration in P301S mice
To investigate the role of C1q in the progressive neurodegeneration of
P301S mice^[93]4, we genetically ablated C1q and analyzed males using
volumetric brain magnetic resonance imaging (MRI), behavioral and
pathological analysis, as well as transcriptomics and synapse
proteomics (Fig. [94]1a).
Fig. 1. C1q deletion reduces neurodegeneration in P301S mice.
[95]Fig. 1
[96]Open in a new tab
a, Study design. Male P301S and C1q^KO mice were crossed as indicated
and analyzed using longitudinal volumetric brain MRI, behavioral
hyperactivity and pathological analysis, transcriptomics and synapse
proteomics. b, Representative volumetric MRI images in male mice at 6
and 9 months of age. Arrows indicate hippocampal atrophy and ventricle
enlargement in P301S mice. c, Longitudinal volumetric MRI
quantification of whole brain volume changes in indicated mouse
genotypes at 6 and 9 months (normalized to 3 months). d, Whole brain
volume changes in indicated mouse genotypes at 6 and 9 months of age.
P301S transgenic mice were normalized to non-transgenic mice with the
same C1q genotype for comparison. e, Longitudinal volumetric MRI
quantification of hippocampal brain volume changes in indicated mouse
genotypes at 6 and 9 months (normalized to 3 months). f, Hippocampus
volume changes in indicated mouse genotypes at 6 and 9 months of age.
P301S transgenic mice were normalized to non-transgenic mice with the
same C1q genotype for comparison. g, Nine-month-old mice were evaluated
in the open field behavioral test by measuring total beam breaks to
assess for behavioral hyperactivity. Each dot represents the values
from one mouse. n = 8–10 mice per genotype (c–g). One-way ANOVA with
Tukey’s multi-comparisons test (d,f) and one-way ANOVA with Fisher’s
least significant difference test (g). All data are presented as
mean ± s.e.m.
[97]Source data
We monitored brain volume longitudinally at 3, 6 and 9 months of age.
Compared to wild-type (WT) mice, the rate of brain volume growth during
maturation in C1q-deficient mice was reduced in a gene dose-dependent
manner (Fig. [98]1b,c; WT versus C1q^KO; P = 0.04; two-way analysis of
variance (ANOVA) with Dunnett’s test). We did not observe differences
in brain volume changes in C3^KO mice (Extended Data Fig. [99]1a),
suggesting that C1q might have CCP-independent physiological functions
in the brain. In P301S mice, there was a marked decrease in brain
volume between 6 and 9 months, reflecting neurodegeneration (Fig.
[100]1c,d). In P301S;C1q^KO mice, brain volume loss was less severe and
brain volume was not significantly different from C1q^KO brains at 9
months (Fig. [101]1c,d)^[102]4. In the hippocampus, which is the brain
region most severely affected by Tau pathology and gliosis^[103]3, we
observed a reduction in volume even at 6 months and further atrophy
between 6 and 9 months in P301S mice (Fig. [104]1e,f). In P301S;C1q^KO
mice hippocampal volume loss was delayed, with significant protection
at 6 months (Fig. [105]1e,f). In contrast to the protection afforded by
homozygous C1q knockout (KO), P301S;C1q^Het mice resembled P301S mice,
implying that greater than 50% reduction of C1q is needed for
protection against Tau^P301S neurodegeneration.
Extended Data Fig. 1. Immunohistochemical characterization of C1q
experimental cohort.
[106]Extended Data Fig. 1
[107]Open in a new tab
(a) Longitudinal volumetric T2 weighted MRI quantification of whole
brain volume changes in WT and C3^KO mice at 6 and 9 months (normalized
to 3 months). (b) Example C1q immunofluorescence images from hemibrains
of each genotype used in the study. (c) Quantification of C1q
immunofluorescence in the whole brain of 9-month-old mice. (d)
Representative images showing AT8 (pTau), Iba1, GFAP in hemibrains and
Amino Cupric Silver staining in the hippocampus. (e) Quantification of
pTau, Iba1, GFAP and Amino cupric silver positive area in whole brains.
(f) Quantification of the same markers as in E) with analysis
restricted to the hippocampus. (g) Representative images showing NeuN
staining in each genotype with example ROIs for the CA1, CA3, and
dentate gyrus (DG), subfields are illustrated on the first image. (h)
Percentage of NeuN+ area in hippocampal CA1, CA3 and DG subregion
across genotypes. Each dot shows average data from one mouse. 13–14
mice/genotype were used for volumetric MRI experiment in a), 8–10
mice/genotype were analyzed by immunohistochemistry in c-h). One-way
ANOVA with Tukey multiple comparison test was used. All data are
presented as mean ± SEM.
[108]Source data
To test for behavioral consequences of C1q deletion, we measured
locomotor activity in an open field in 9-month-old mice (Fig. [109]1g).
As expected, P301S mice exhibited hyperactivity, which is thought to be
caused by hippocampal damage^[110]3. While there was no effect of C1q
genotype on locomotor activity in the absence of the P301S transgene,
hyperactivity was rescued in P301S;C1q^KO, but not P301S;C1q^Het mice
(Fig. [111]1g).
We then analyzed brain histopathology in 9-month-old mice. C1q
immunoreactivity was strongly increased in P301S brains, compared to a
~50% reduction in P301S;C1q^Het brains and was undetectable in
P301S;C1q^KO brains (Extended Data Fig. [112]1b,c). P301S brains were
characterized by strong phospho-Tau immunoreactivity, microgliosis and
astrogliosis measured by increased Iba1^+ and glial fibrillary acidic
protein (GFAP)^+ area, respectively^[113]3 (Extended Data Fig.
[114]1d–f). There was no difference in these histopathologic readouts
in P301S;C1q^Het versus P301S mice and a slight trend toward reduction
in P301S;C1q^KO compared to P301S mice (Extended Data Fig. [115]1d–f).
We observed trends toward reduced amino cupric staining, which reflects
damaged neurons and increased density of the neuronal marker NeuN in
P301S;C1q^KO hippocampi (Extended Data Fig. [116]1e–h). Bulk RNA-seq of
P301S hippocampi showed upregulation of many genes, including multiple
markers of activated microglia and astrocytes (Extended Data Fig.
[117]2); however, C1q deletion had no effect on these major
transcriptomic changes in P301S hippocampi (Extended Data Fig. [118]2).
Together, these results show that C1q deletion reduces P301S brain
degeneration and normalizes behavior without having a significant
effect on the extent of Tau pathology, gliosis or glial transcriptional
changes. Thus, the protective effect of C1q deletion seems to act
downstream of tauopathy and the overall glial response.
Extended Data Fig. 2. C1q deletion does not impact transcriptional changes in
P301S mice.
[119]Extended Data Fig. 2
[120]Open in a new tab
(a) Heatmap showing Z score of genes that were most highly up-regulated
in P301S vs. WT mice (without respect to C1q genotype). No genes showed
DE when comparison was made between P301S and P301S;C1qKO mice
(adjusted p <0.05) except for C1qc (log2FC = −6.44, p = 0.000256). (b)
Heatmap showing Z score of top 60 DAM genes taken from the list
in^[121]67. (c) Heatmap showing Z score of top astrocytes activated
genes taken from the list in^[122]4.
C1q^KO blunts proteomic changes in P301S synapses
We next examined changes in synaptic protein composition across P301S
and C1q genotypes. We isolated hippocampal postsynaptic density (PSD)
fractions from 6- and 9-month-old male mice to detect changes at an
early and an advanced stage of disease, respectively (Fig. [123]2a).
These fractions are highly enriched in proteins of the PSD and
postsynaptic membrane; they also contain components of the presynaptic
terminal, transsynaptic adhesion molecules and some glia-specific
proteins (that might reflect close interactions of glial cells with
synapses)^[124]3,[125]25,[126]26. Thus, we use the terms PSD and
synapse fraction interchangeably throughout the study. Multiplexed
tandem mass tag (TMT) proteomics detected a total of 7,101 proteins in
PSD fractions from 6-month-old mice and 4,175 proteins in those from
9-month-old mice (Fig. [127]2b and Supplementary Table [128]1). While
our previous analysis of 9-month-old P301S females using label-free
proteomics detected fewer proteins^[129]3, nearly all of them were also
found in the current study (Fig. [130]2b).
Fig. 2. C1q deletion blunts proteomic changes in P301S synapses.
[131]Fig. 2
[132]Open in a new tab
a, Experimental design of hippocampal PSD proteome analysis. Hippocampi
from 6- and 9-month-old male mice were dissected and isolated synapse
fractions were analyzed by TMT multiplex proteomics ([133]Methods). b,
Venn diagram showing the number of identified proteins and overlap
between synapse proteomes in the 6- and 9-month cohort from this study
and synapse proteome from 9-month-old female mice described
previously^[134]3. c, Percentage of DE proteins in the indicated
genotype comparisons at 6 and 9 months. d, A heat map showing z scores
across genotypes for proteins that were DE between C1qKO and WT mice
(regardless of P301S genotype) (P ≤ 0.05; FC ≥ 5). e,f, Volcano plots
showing the comparison between P301S versus WT and P301S;C1q^KO versus
WT synapse proteomes at 6 and 9 months. MSstats was used to calculate
log[2]FC and standard error utilizing a linear mixed-effects model that
considered quantification from each peptide and biological replicate
per protein. P values were then calculated by comparing the model-based
test statistic to a two-sided Student’s t-test distribution.
Significantly up- and downregulated proteins (P < 0.05, log[2]FC ± 0.5)
are shown in blue and red circles, respectively. Selected DE proteins
are labeled with their protein or gene name. g, Selected up- or
downregulated KEGG pathways in P301S versus WT and P301S,C1q^KO versus
WT synapse proteomes at 6 and 9 months. Only DE proteins were included
for pathway analysis.
[135]Source data
Synapse fractions from C1q^KO mice showed only a small number of
differentially expressed (DE) proteins (defined as log[2]fold change
(FC) ± 0.5, nominal P < 0.05) compared to WT at both ages (Fig. [136]2c
and Extended Data Fig. [137]3a,c). In 6-month-old P301S synapse
fractions we found 108 downregulated and 68 upregulated proteins (2.5%
of total proteins; Fig. [138]2c,e). At 9 months, there were 253
downregulated and 434 upregulated proteins, corresponding to 16.5% of
total proteins (Fig. [139]2c,e). By contrast, P301S;C1q^KO synapse
fractions showed only 17 decreased and 19 increased DE proteins (0.5%
of total proteins) at 6 months and 79 decreased and 224 increased DE
proteins (7% of total proteins) at 9 months (Fig. [140]2c,f).
Consistently, we found many DE proteins when comparing P301S;C1q^KO to
P301S synapses at 9 months (Fig. [141]2c and Extended Data Fig.
[142]3b) and reductions in Tau-dependent changes with C1q deletion
(Extended Data Fig. [143]3d). C1q-deficiency did not affect Tau levels
in synapse fractions of P301S brains (Fig. [144]2e,f). Thus, C1q
deletion blunted age-dependent changes induced by Tau pathology even
though C1q deletion had little effect in non-transgenic mice (Fig.
[145]2d and Extended Data Fig. [146]3d). As C1q deletion did not
significantly alter the transcriptomic changes in P301S brains
(Extended Data Fig. [147]2), synapse proteome changes are likely driven
by local protein changes at the synapse.
Extended Data Fig. 3. C1q-dependent synapse proteome changes in P301S mice.
[148]Extended Data Fig. 3
[149]Open in a new tab
(a,b) Volcano plots showing the comparison between A) C1q^KO vs WT and
B) P301S;C1q^KO vs P301S synapse fraction proteomes at 6 and 9 months.
MSStats was used to calculate log2(fold change) and standard error
utilizing a linear mixed-effects model that considered quantification
from each peptide and biological replicate per protein. P values were
then calculated by comparing the model-based test statistic to a
two-sided Student t-test distribution. Significantly up- and
downregulated proteins (p-value < 0.05, log2FC ± 0.5) are shown in blue
and red circles, respectively. Selected differentially expressed
proteins are labeled with their protein or gene name. (c) KEGG pathways
significantly downregulated in synapses from 6 months old C1q^KO mice
and 9 months old P301S;C1q^KO vs P301S mice. (d) Scatterplot comparison
of PSD proteomes from 9 months old P301S vs WT mice (x-axis) and
P301S;C1q^KO vs WT mice (y-axis). Note that protein changes in this
comparison are larger in P301S compared to P301S;C1q^KO synapse
fractions, suggesting that C1q deletion blunts changes in P301S mice.
Red indicates significantly different (p-value <0.05) in P301S vs. WT,
but not significantly different in P301S;C1q^KO vs WT (97 proteins),
green indicates significantly different in P301S;C1q^KO vs WT, but not
significantly different in P301S vs. WT (only C1qC), and blue indicates
significantly different in both comparisons (12 proteins). P-values and
fold changes were calculated by MSStats, as described in methods. (e)
Schematic representation of an excitatory synapse with the localization
of selected proteins grouped by their function. Heatmaps show protein
log2 fold-changes for individual genotype and age comparison. (f) SynGO
analysis of downregulated proteins in P301S vs WT and P301S;C1q^KO vs
WT synapse proteomes at 9 months. (g) Heatmaps of normalized protein
expression of annexins across genotypes in synapses at 6 and 9 months.
The annexin protein set score is shown below. In g) each dot shows data
from one mouse. 2–3 mice/genotype were used. Two-way ANOVA with Šidak’s
test. All data are presented as mean ± SEM.
[150]Source data
We assessed the impact of the P301S transgene and C1q deletion on
various functional classes of proteins in the synapse proteome
(Extended Data Fig. [151]3e). Many core PSD proteins, such as glutamate
receptors, scaffolding proteins, synaptic adhesion molecules and
certain presynaptic active zone proteins tended to be increased in
P301S compared to WT synapses at 6 months but reduced at 9 months
(Extended Data Fig. [152]3e). This could reflect compensatory synaptic
changes at early disease stage that are overcome by synaptic damage at
the later stage. Using the synaptic Gene Ontology tool SynGO^[153]27,
we confirmed that the decreased DE proteins in 9-month-old P301S PSDs
were significantly enriched with synapse organization and canonical
pre- and postsynaptic proteins (Extended Data Fig. [154]3f). While
qualitatively similar synaptic functions were affected in P301S;C1q^KO
synapse fractions, the changes were less significant than in P301S PSDs
(Extended Data Fig. [155]3f).
KEGG pathway analysis of the DE proteins in P301S synapses showed
significant enrichment in ‘metabolic pathways’ (containing mainly
mitochondrial proteins such as Echs1, Maob and Atp8), ‘fatty acid
degradation’ (for example Adh5, Hadha and Hadh), ‘peroxisome’ (for
example Dhrs4, Ehhadh and Abcd1), ‘peroxisome proliferator-activated
receptors (PPAR)’ signaling (for example Cpt1a and Cpt2), ‘Alzheimer’s
disease’ (for example Adam10 and APOE) and ion homeostasis (for example
Slc4a4 and Aqp4), especially prominent at 9 months (Fig. [156]2g).
Increases in these pathways were relatively subdued in P301S;C1q^KO
synapses, with no significantly increased pathways at 6 months and
changes at 9 months resembling alterations seen in 6-month-old P301S
PSDs (Fig. [157]2g). Consistently, most pathways that were increased in
9-month-old P301S synapses, were decreased in the P301S;C1q^KO versus
P301S comparison (Extended Data Fig. [158]3b,c). Notably, ‘metabolic
pathways’, the most significantly increased pathway in 9-month-old
P301S synapses, was not significantly induced in P301S;C1q^KO samples
(Fig. [159]2g). We also noted that annexins were among the most highly
induced proteins in 9-month-old TauP301S synapses and were partly
normalized in P301S;C1q^KO synapses (Fig. [160]2d and Extended Data
Fig. [161]3g).
Analysis of decreased DE proteins in P301S synapses highlighted
pathways including ‘glutamatergic synapse’ (for example Shank1 and
Grin2a), ‘endocytosis’ (for example Vps4a and Rab11Fip2), ‘axon
guidance’ (for example Ntng1 and Smad2), ‘MAPK-’ (for example Mapk1 and
Map4k4), ‘Ras-’ (for example Ksr1 and Syngap1), ‘phosphatidylinositol-’
(for example Dgkb and Dgkq) and ‘Wnt-signaling’ (Apc2 and Dvl3) and
‘AMPK-signaling’ (Ppp2r5c and Prkaa2) (Fig. [162]2g). No pathway was
significantly decreased in 6-month-old P301S;C1q^KO synapses and only
‘glutamatergic synapse’, ‘endocytosis’ and ‘Ras signaling’ were
significantly decreased at 9 months (Fig. [163]2g). Different proteins
of the ‘actin cytoskeleton’ pathway were significantly increased (for
example ezrin and gelsolin) or decreased (for example Baiap2 and Pak6)
in P301S PSD fractions. At least some of the increased actin-regulating
proteins in the synapse fractions are expressed by glial cells that
presumably associate with synapses (see below).
As mutations in synaptic proteins cause a variety of neurological and
neuropsychiatric diseases, we investigated whether DE proteins were
enriched for genetic signals in genome-wide association studies (GWAS)
of relevant traits and disorders^[164]28–[165]34. For the 1,000 most
up- and downregulated proteins in P301S versus WT synapses, we tested
for polygenic signal in 752 traits primarily from the UK Biobank and
selected GWAS studies using stratified linkage disequilibrium
(LD)-score regression ([166]Methods). After grouping traits into 23
categories or domains, we found that the downregulated proteins in
9-month-old P301S and P301S;C1q^KO synapses had significant enrichments
in cognitive, psychiatric and activities domains, which included
educational attainment, fluid intelligence score, cognitive performance
and concept interpolation (Extended Data Fig. [167]4a). In contrast
there was limited enrichment for these traits in the upregulated
proteins in 9-month-old P301S PSD fractions or in DE proteins at 6
months (Extended Data Fig. [168]4b). This suggests that proteins
downregulated in 9-month-old P301S and P301S;C1q^KO synapses have
relevance in human cognitive function and behavior.
Extended Data Fig. 4. Heritability enrichment analyses in differentially
abundant proteins identified in P301S and P301S;C1q^KO synapse fractions.
[169]Extended Data Fig. 4
[170]Open in a new tab
(a) Manhattan plots of per-SNP enrichment p-values for the top 1000
down-regulated proteins in 9-month P301S and P301S;C1q^KO PSDs for 752
traits across 23 of the 24 UK Biobank Domains. The dotted red line
corresponds to the Bonferroni threshold at 0.05 correcting for 752
traits (6.65 ×10^−5). (b) Per-SNP heritability coefficients and 95%
confidence intervals of seven select cognitive and psychiatric traits
for the top 1000 up- or down-regulated proteins in C1qKO, P301S and
P301S;C1q^KO PSDs at 6 and 9 months. The GWAS for the seven cognitive
and psychiatric traits have sample sizes between 46,350 to 1.1 million
individuals (see methods). Dots with p-value < 0.001 were labeled in
the graph. All P values are one-sided and calculated using s-LDSC.
C1q-dependent elevation of glial proteins at P301S synapses
We noticed that a number of canonical astrocyte-specific proteins, such
as Aqp4, Mlc1 and Slc1a4 were increased in 9-month-old P301S synapse
fractions in a C1q-dependent manner (Fig. [171]2d). While contamination
with astrocyte proteins is a possibility, we also considered whether
the copurification of astrocytic proteins with synaptic preparations
might result from the close interaction of astrocyte processes with
synapses^[172]25. We generated pseudobulk single-cell RNA-sequencing
(scRNA-seq) data from P301S hippocampi and found that the majority of
the 55 most highly upregulated proteins were predominantly expressed by
glial cells, rather than by excitatory neurons (Fig. [173]3a). In
contrast, the most highly decreased proteins were mainly produced by
excitatory neurons (Extended Data Fig. [174]5a). Besides Aqp4 and Mlc1,
many other upregulated DE proteins were selectively or predominantly
expressed by astrocytes (for example, clusterin, Slc1a3, Sdc4, AHNAK,
ezrin, GFAP and Thbs4) (Fig. [175]3a). A smaller number of upregulated
proteins were expressed predominantly by microglia, (for example, Gpnmb
and Myo1f) (Fig. [176]3a). We hypothesized that the surge in glial
proteins in 9-month-old P301S synapse fractions might reflect an
increase in their secretion and subsequent accumulation at synapses
and/or an increase in contact of glial processes with damaged synapses.
Consistently, many of the increased glial proteins are either localized
at the plasma membrane (for example, Slc16a1 and Aqp4), cytoskeleton
(for example, ezrin) or are extracellular/secreted (clusterin and
Thbs4; Extended Data Fig. [177]5b) and are known to be present in
astrocyte processes^[178]35,[179]36. Notably, the increase in glial
proteins in P301S synaptic fractions was C1q-dependent (Fig. [180]3b).
This argues against an indiscriminate contamination of PSD preps with
glial-derived proteins. The relative reduction in glial proteins in
P301S;C1q^KO synapses seems to be due to specific changes in
association of glial proteins with synaptic fractions, rather than
overall abundance of glial proteins, as the expression of the
corresponding genes was not significantly different in P301S versus
P301S;C1q^KO brains (Extended Data Fig. [181]5c).
Fig. 3. Glial proteins are elevated at P301S synapses and normalized by
C1q^KO.
[182]Fig. 3
[183]Open in a new tab
a, Cell-type-specific expression of genes that encode the most highly
increased proteins in P301S synapses at 9 months. Percentage of gene
expression in the major brain cell types (excitatory (exc.) neurons,
astrocytes, microglia and oligodendrocytes (oligo)) was calculated
based on pseudobulk analysis of scRNA-seq data from P301S mice. b, Heat
maps showing z scores for normalized levels of glial proteins across
genotypes in synapses at 6 and 9 months. Genes from a were defined as
glial if the percentage of gene expression in excitatory neuron was
<4%. Dotted lines indicate the cell type(s) that mainly express the
corresponding gene. A, astrocyte; M, microglia; O, oligodendrocyte.
Glia protein set score (right). c, Representative immunoEM images of
EAAT2 in DG. Presynapses are pseudo-colored in red, postsynapses in
green. EAAT2^+ astrocyte processes are shown in blue. The synapse
perimeter is outlined in orange and the astrocytic plasma membrane that
is in contact with the synapse is in yellow. Scale bar, 200 nm. d,
Length of astrocyte plasma membrane in association with the synapse in
WT and P301S mice. e, Quantification of synapse perimeter in WT and
P301S mice. f, Two-tailed Pearson’s correlation of astrocyte–synapse
association and percentage of C1q-labeled presynapses, which was
quantified previously in the same mice^[184]3. g, Representative images
showing the raw confocal immunofluorescence and the corresponding
Imaris-processed image of GFAP (blue) and Homer1 (yellow) from a P301S
brain. Inset shows three-dimensional (3D)-reconstructed
GFAP^+ astrocyte processes in the P301S brains and representative
images from WT and P301S;C1q^KO brains. Only Homer1 puncta that
associate with astrocytes are shown (pink dots). h, Fraction of Homer1
puncta associated with astrocytes. Data were analyzed by two-way ANOVA
with Tukey’s multi-comparisons test (b); two-tailed unpaired Student’s
t-test (d,e) (10–16 astrocyte-synapses were quantified per mouse) and
one-way ANOVA with Dunnett’s multiple comparisons test (h). Each dot
shows average data from one mouse; n = 2–3 mice per genotype (b); n = 3
mice per genotype (d,e) and n = 7–10 mice per genotype (h). All data
are presented as mean ± s.e.m.
[185]Source data
Extended Data Fig. 5. Abundance of glial proteins at synapses and expression
of their corresponding genes.
[186]Extended Data Fig. 5
[187]Open in a new tab
(a) Cell-type-specific expression of genes that encode the most highly
decreased proteins in P301S synapses at 9 months. Percentage of gene
expression was calculated based on pseudobulk analysis of scRNAseq data
from P301S mice. (b) Volcano plot comparing P301S and WT synapse
proteomes highlighting increased proteins that are selectively
expressed by glial cells (<5% gene expression by neurons) and their
annotated subcellular localization. MSStats was used to calculate
log2(fold change) and standard error utilizing a linear mixed-effects
model that considered quantification from each peptide and biological
replicate per protein. P values were then calculated by comparing the
model-based test statistic to a two-sided Student t-test distribution.
(c) Heatmap showing z-scores from bulk RNAseq across genotypes for
astrocyte and microglia specific genes encoding proteins in Fig
[188]3c. The glial gene set score is shown on the right. (d)
Mitochondrial proteins (blue dots) highlighted in volcano plots
comparing 9 months old P301S vs WT synapse proteomes. Statistical tests
were done as in panel B. The most highly up- or downregulated
mitochondrial proteins are labeled using their gene or protein name.
(e) Gene set score for mitochondrial proteins that are significantly
increased in P301S synapses are shown across genotypes and age as
indicated. (f) Cell-type-specific expression of genes encoding
mitochondrial proteins that are up- or down-regulated in P301S PSDs at
9 months. Percentage of gene expression was calculated based on
pseudobulk analysis of scRNAseq data from P301S mice. (g) Gene set
score for peroxisome proteins that are significantly increased in P301S
synapses are shown across genotypes and age as indicated. (h)
Cell-type-specific expression of genes encoding peroxisome proteins
that are up- or down-regulated in P301S PSDs at 9 months. Each dot
shows data from one mouse. 2–5 mice/genotype were used. In c one-way
ANOVA with Tukey multiple comparison test and in e, g two-way ANOVA
with Tukey multiple comparison test was used. Percentage of gene
expression in a, f and h is based on scRNAseq data from P301S mice.
[189]Source data
Among the most highly increased proteins in P301S synapse fractions
were astrocyte-specific mitochondrial proteins Echdc3 and Sfxn5 (Fig.
[190]3a,b). Many mitochondrial proteins were increased in P301S synapse
fractions in an age- and C1q-dependent manner (Extended Data Fig.
[191]5d,e). Energy metabolism differs between CNS cell types^[192]37
and ‘metabolic pathways’ were strongly elevated in P301S but not
P301S;C1q^KO synapses at 9 months (Fig. [193]2g). Although mitochondria
can be transferred from astrocytes to neurons under pathological
conditions^[194]38, we reasoned that mitochondria in synaptic fractions
might originate from glial processes that were in close physical
contact with synapses. Consistently, the 20 most highly increased
mitochondrial proteins in P301S synapses are predominantly expressed by
astrocytes (for example, Maob, Cpt1a and Tst, Slc25a18), whereas
significantly decreased mitochondrial proteins had a broad expression
pattern, including stronger neuronal production (for example, Wasf1)
(Extended Data Fig. [195]5f). Similarly, the peroxisomal proteins that
were increased in 9-month-old P301S synapse fractions in a
C1q-dependent manner (Extended Data Fig. [196]5g) were also
predominantly expressed by astrocytes (Extended Data Fig. [197]5h).
We next used immuno-electron microscopy (IEM) to determine whether
there was altered physical association of astrocytes with synapses in
P301S mice. We identified astrocyte processes by immunolabeling for the
astrocyte-specific glutamate transporter EAAT2/Glt1 and quantified the
length of astrocyte processes that are in contact with synapses in the
hippocampus dentate gyrus (DG) and CA1 region (Fig. [198]3c and
Extended Data Fig. [199]6a). Compared to WT mice, the length of
astrocytic processes that associated with synapses was increased
~twofold in the DG and CA1 region in P301S mice (Fig. [200]3d and
Extended Data Fig. [201]6b). The average synaptic perimeter was
unchanged in P301S mice, implying that astrocytes contact a larger
fraction of synaptic membrane (Fig. [202]3e and Extended Data Fig.
[203]6c). Notably, the extent of astrocyte–synapse association
correlated significantly with the percentage of C1q-labeled
presynapses^[204]3 (Fig. [205]3f).
Extended Data Fig. 6. IEM and IHC analysis of astrocyte–synapse interaction.
[206]Extended Data Fig. 6
[207]Open in a new tab
(a) Representative immunoEM images of EAAT2 in hippocampal CA1 region.
Presynapses are pseudo-colored in red, postsynapses in green. EAAT2^+
astrocyte processes are shown in blue. The synapse perimeter is
outlined in orange and the astrocytic plasma membrane that is in
contact with the synapse in yellow. Scale bar = 200 nm. (b) Length of
the astrocytic plasma membrane associated with the synapse in CA1
region from WT and P301S mice. (c) Quantification of synapse perimeter
in CA1 region from WT and P301S mice. (d) Representative confocal image
and Imaris 3D reconstructions of immunostained S100B (green) and Homer1
(red). In the 3D reconstructions only S100B-associated Homer1 puncta
are shown. Scale bar = 5µm. (e) Percentage of Homer1 puncta that
associated with S100B^+ astrocytes. B, c Unpaired two-tailed t-test; e
One-way ANOVA with Dunnett multi comparison test. Each dot shows
average data from one mouse, in b,c) n = 3 mice/genotype, in e) n =
8–10 mice/genotype. All data are presented as mean ± SEM.
[208]Source data
As an orthogonal measurement of astrocyte–synapse interaction across
all genotypes, we quantified the spatial contact of excitatory synapses
(Homer1 puncta) with the surface of GFAP^+ astrocytes in the
hippocampal CA1 region using confocal microscopy (Fig. [209]3g). As
loss of one copy of C1q had no impact on pathology in P301S mice (Fig.
[210]1 and Extended Data Fig. [211]1), we grouped P301S and
P301S;C1q^Het brains in this analysis to increase statistical power.
While there was no difference between C1q^KO versus WT hippocampi, we
observed a significant increase in surface GFAP–Homer1 association in
P301S hippocampi, which was significantly reduced in P301S;C1q^KO
versus P301S hippocampi (Fig. [212]3h). Using the cytoplasmic astrocyte
marker protein S100b to render astrocyte volume confirmed the
C1q-dependent increase in astrocyte–Homer1 association in P301S mice
(Extended Data Fig. [213]6d,e). Together, our analysis of synapse
proteomics data, IEM and immunohistochemistry (IHC) measurements
suggests that at a stage of disease with synapse engulfment and loss,
astrocytes can increase their physical interaction with synapses in a
C1q-dependent manner.
Glial proteins are elevated in synapses of Alzheimer’s brain
We wondered whether glial proteins are also elevated in synaptic
fractions from patients with AD. Comparison with a recently published
synaptoneurosome proteome from the superior temporal gyrus (BA 41/42)
in patients with AD^[214]39 revealed a notable positive correlation
between changes in human AD versus control and 9-month-old P301S versus
WT mice synapse proteomes (Fig. [215]4a). Notably, glial proteins that
were elevated in P301S synapse fractions, including complement factors
C1q and C4, astrocytic marker proteins MLC1 and GFAP, microglial GPNMB
and AHNAK and annexins were among the most highly increased proteins in
AD synaptoneurosomes (Fig. [216]4a).
Fig. 4. Glial proteins are increased in human AD synapse fractions and C4 is
elevated in AD CSF.
[217]Fig. 4
[218]Open in a new tab
a, Scatter-plot comparison of synapse proteomes from 9 months old P301S
versus WT mice (x axis) and AD versus control patients (y
axis)^[219]39. Only orthologous protein pairs that were present in both
datasets are shown. Of the 315 proteins that were significantly
increased in P301S versus WT mice (P < 0.05, FC > 2) regardless of C1q
genotype), 175 were increased in patients with AD versus controls,
including the labeled proteins. Overall correlation of 0.34. NC, no
change. b, Levels of total and processed C4 and Factor B and processed
Bb fragment in CSF from controls and patients wth AD. Each dot
represents the values from one individual. CSF samples from 15 controls
and 14 patients with AD were analyzed (the same patients identified in
previous works^[220]4; cohort 1). Data were analyzed by two-tailed
unpaired Student’s t-test. All data are presented as mean ± s.e.m.
[221]Source data
We reasoned that elevated levels of C4 and activation of the CCP could
be detectable in CSF from patients^[222]4, which might be a useful
biomarker of complement activation. Indeed, total and processed
(cleaved and activated), C4 concentrations were significantly increased
in CSF from patients with AD (Fig. [223]4b). Similar results, with a
trend toward elevated total C4 and significantly increased processed
C4, was also seen in CSF from an independent patient cohort (Extended
Data Fig. [224]7). By comparison, complement Factor B, a component of
the alternative complement pathway, was not robustly changed in AD CSF
(Fig. [225]4b and Extended Data Fig. [226]7). Levels of activated
subunit Bb were very low in control and AD CSF but did show trends
toward increases in AD CSF (Fig. [227]4b and Extended Data Fig.
[228]7). Thus, the upregulation of glial proteins in the P301S synapse
proteome is also present in AD and might be relevant to disease
pathophysiology^[229]3,[230]4. The elevated levels of C4 (and C3 (ref.
^[231]4)) in CSF from patients with AD are consistent with a role for
the CCP in Alzheimer’s neurodegeneration.
Extended Data Fig. 7. Complement C4 and Factor B concentrations in AD CSF.
Extended Data Fig. 7
[232]Open in a new tab
Levels of total and processed C4 and Factor B and processed Bb fragment
in CSF from controls and AD patients. Each dot represents the values
from one individual. CSF samples from 10 controls and 10 AD patients
were analyzed (the same patients from^[233]4, cohort 2). Unpaired
two-tailed t-test. All data are presented as mean ± SEM.
[234]Source data
Glial C1q-dependent synapse elimination in P301S mice
Because of our proteomics, IEM and IHC data, we hypothesized that
astrocytes might be interacting with synapses in a C1q-dependent
fashion during the process of synapse engulfment. To analyze synapse
engulfment by astrocytes and microglia, we immunostained GFAP^+
astrocytes, Iba1^+ microglia, Lamp1^+ lysosomes along with the
excitatory postsynapse marker Homer1 (Fig. [235]5a). As inhibitory
synapses are also affected in AD^[236]40, we additionally immunolabeled
the inhibitory postsynaptic marker gephyrin. By confocal microscopy and
3D reconstruction of the hippocampal CA1 area, we measured the amount
of Homer1 and gephyrin puncta inside microglial and astrocytic
lysosomes within the same image (Fig. [237]5a). Of note, using GFAP or
S100B we identified essentially the same population of astrocytic
Lamp1^+ structures (Extended Data Fig. [238]8a). As expected from
previous studies^[239]3,[240]4, microglial lysosomes in P301S
hippocampi contained excitatory synapses and showed a ~tenfold increase
in Homer1 puncta compared to WT controls (Fig. [241]5b). Compared to
P301S, microglial phagocytosis of Homer1 was significantly decreased in
P301S;C1q^KO brains (Fig. [242]5b). Notably, we also found a
considerable fraction of Homer1 puncta inside astrocytic lysosomes,
which was increased ~five- to tenfold in P301S hippocampi (Fig.
[243]5c). The fraction of Homer1 puncta inside astrocyte lysosomes was
significantly reduced in P310S;C1q^KO brains, indicating that
astrocytic eating of excitatory structures in P301S mice was at least
in part C1q-dependent (Fig. [244]5c). Gephyrin puncta were also present
in microglial and astrocytic lysosomes (Fig. [245]5d,e). As with
Homer1, eating of gephyrin by microglia and astrocytes was elevated in
P301S hippocampi and was partly C1q-dependent (Fig. [246]5d,e). In
healthy brains, however, engulfment of excitatory and inhibitory
synapses was unaffected by loss of C1q, as WT and C1q^KO hippocampi had
the same low amount of Homer1 and gephyrin in glial lysosomes (Fig.
[247]5b–e). The amount of phagocytosed synapse puncta corresponded with
changes in the volume of astrocytic and microglial lysosomes across
genotypes (Extended Data Fig. [248]8b).
Fig. 5. Astrocytes and microglia eliminate excitatory and inhibitory synapses
in P301S mice in a complement-dependent manner.
[249]Fig. 5
[250]Open in a new tab
a, Representative images of a confocal z-stack and Imaris 3D
reconstructions of mouse brain sections immunostained for GFAP (blue),
Iba1 (white), Lamp1 (green), Homer1 (yellow) and gephyrin (red).
LAMP1^+ lysosomes within GFAP^+ or Iba1^+ volumes were classified as
astrocytic or microglial lysosomes, respectively. Scale bar in the raw
image, 10 µm; scale bar in the Imaris 3D-rendered image, 2 µm. b,c,
Fraction of Homer1 puncta identified inside astrocytic or microglial
lysosomes across genotypes. d,e, Fraction of total gephyrin puncta
identified inside astrocytic or microglial lysosomes across genotypes.
f, Normalized number of Homer1 puncta engulfed by astrocytes or
microglia, respectively (left). Ratio of Homer1 puncta within
astrocytic/microglial lysosomes (right). g, Normalized number of
gephyrin puncta engulfed by astrocytes or microglia, respectively
(left) and ratio of gephyrin puncta within astrocytic/microglial
lysosomes (right). Dotted line in f and g at a ratio of 1 indicates
that astrocytic and microglial lysosomes contained the same number of
synaptic puncta, ratio of >1 means that more synaptic puncta were
localized within astrocytic lysosomes and <1 indicates that microglial
lysosomes contained more synaptic puncta. Connected dots in the left of
f and g show astrocytic and microglial Homer1 or gephyrin engulfment
from the same mouse. h, Excitatory and inhibitory synapse density
across genotypes as measured by number of identified Homer1 and
gephyrin puncta per field of view (FOV). i, Representative confocal
images of immunostained Homer1 (green) and C3 (red) in the CA1 region
of WT, P301S and P301S;C1q^KO brains. Colocalized Homer1 and C3 puncta
are indicated by circles. Scale bar, 2 µm. j, Graph shows percentage of
C3-labeled Homer1^+ synapses. k, Total number of C3 puncta per FOV.
Data were analyzed by one-way ANOVA with Dunnett’s post hoc test
(b–e,h,j,k) and a two-tailed paired Student’s t-test (f,g). Each dot
shows average data from one mouse; 7–10 mice per genotype were
analyzed. All data are presented as mean ± s.e.m.
[251]Source data
Extended Data Fig. 8. Complement-dependent engulfment of excitatory and
inhibitory synapses by astrocytes and microglia in P301S mice.
[252]Extended Data Fig. 8
[253]Open in a new tab
(a) Representative image and Imaris 3D rendering of immunostained S100B
(green), GFAP (red) and Lamp1 (white). 3D reconstructions show
lysosomes (Lamp1 structures) within S100B^+ astrocytes (white) or
GFAP^+ astrocytes (blue). Note that lysosome structures segmented
within S100B^+ and GFAP^+ astrocytes are almost identical. Scale bar =
10µm. (b) Volume of LAMP1+ lysosomes inside astrocytes and microglia,
respectively, in hippocampi from WT, C1q^KO, P301S and P301S;C1q^KO.
(c) Representative images of WT, P301S and P301S;C1q^KO brains
immunostained for Homer1 (green), Gephyrin (red), LAMP1 (white), Iba1
(yellow) and GFAP (blue). Images on the right show 3D reconstructed
GFAP+ astrocytes and Iba1+ microglia together with the raw
immunofluorescence from Homer1, Gephyrin and LAMP1. Arrows highlight
Gephyrin immunoreactivity accumulated in microglial lysosomes. Note
that the neighboring astrocytic lysosomes do contain accumulated
Gephyrin. (d,e) Fraction of Homer1 and Gephyrin puncta inside
astrocytic or microglial lysosomes across genotypes. (f,g) Fraction of
Gephyrin puncta inside astrocytic or microglial lysosomes across
genotypes. (h) Normalized number of Homer1 puncta inside astrocytes or
microglia, respectively (left graph) and ratio of Homer1 puncta within
astrocytic/microglial lysosomes. (i) as in H) but showing engulfment
data for Gephyrin. Dotted line at a ratio of 1 indicates that
astrocytic and microglial lysosomes contained the same number of
synaptic puncta, ratio of >1 means that more synaptic puncta were
localized within astrocytic lysosomes and <1 indicates that microglial
lysosomes contained more synaptic puncta. One-way ANOVA with Dunnett’s
post hoc test (B, D-G) or paired two-tailed t-test (H, I). Each dot
shows average data from one mouse. In b) 7–10 mice/genotype and in d-i)
9–10 mice/genotype were used. All data are presented as mean ± SEM.
[254]Source data
Notably, we consistently found more Homer1 puncta inside astrocytic
versus microglial lysosomes in all genotypes (Fig. [255]5f).
Conversely, gephyrin puncta were less abundant in astrocytic versus
microglial lysosomes in P301S hippocampi (regardless of C1q genotype)
and similar in astrocytes and microglia in non-transgenic WT animals
(Fig. [256]5g). This microglial proclivity for engulfing gephyrin
puncta was particularly notable in P301S brains, where strong
accumulation of gephyrin immunoreactivity was often observed in
microglial but not astrocytic lysosomes (Extended Data Fig. [257]8c).
One possibility is that this strong immunoreactivity could reflect
removal of dendritic segments containing many inhibitory synapses. In
line with reduced engulfment of synapses in P301S;C1q^KO brains,
excitatory and inhibitory synapse loss was ameliorated in C1q-deficient
P301S mice (Fig. [258]5h).
Finally, we tested whether astrocytic eating of synaptic structures
requires C3, a central complement component downstream of C1q. The
significant increase of Homer1 and gephyrin engulfment by microglia and
astrocytes in P301S mice was partially reduced on average in
P301S;C3^KO mice (Extended Data Fig. [259]8d–g). However, only the
reduction of gephyrin puncta in astrocytic lysosomes reached
statistical significance in P301S;C3^KO versus P301S mice (Extended
Data Fig. [260]8g), possibly due to high inter-animal variability. Like
in the C1q experimental cohort, we found substantially more Homer1
puncta inside astrocytic versus microglial lysosomes in every brain
that we analyzed, whereas gephyrin puncta were preferentially found in
microglial lysosomes in P301S mice in this C3 experimental cohort
(Extended Data Fig. [261]8h,i).
Next we analyzed C3-labeling of excitatory synapses in the P301S;C1q^KO
cohort. The percentage of C3^+ Homer1 puncta was significantly
increased in P301S versus WT hippocampi, whereas P301S;C1q^KO was
comparable to WT hippocampi and significantly decreased compared to
P301S brains (Fig. [262]5i,j). There was no significant difference in
the total number of C3 puncta in P301S versus P301S;C1q^KO brains (Fig.
[263]5k), indicating that C1q deletion specifically affects C3
deposition at synapses. Overall, the data are consistent with C3 acting
downstream of C1q activation and the CCP facilitating synapse
elimination by astrocytes and microglia.
Astrocytes compensate for impaired microglial phagocytosis
Our findings led us to ask, what happens when phagocytic activity is
impaired in one of the cell types? Loss-of-function mutations in the
microglia-specific TREM2 strongly increase AD risk^[264]41,[265]42 and
loss of Trem2 in AD mouse models has a profound effect on microglial
function and impairs their activation, migration to Aβ plaques and
phagocytic activity^[266]43–[267]47.
To examine whether dysfunctional microglia might result in altered
synapse handling by astrocytes, we analyzed the effects of Trem2
deletion in an AD mouse model that combines β-amyloid and Tau
pathologies (TauPS2APP)^[268]46. At 17 months of age, when plaques,
phospho-Tau, dystrophic axons and gliosis are present^[269]46, we
immunostained TauPS2APP and TauPS2APP;Trem2^KO brain sections using the
previously established protocol and imaged hippocampal CA1 regions with
and without amyloid plaques (identified by the presence of Lamp1^+
dystrophic axons) (Fig. [270]6a). In TauPS2APP brains, microglia and
astrocytes phagocytosed more Homer1 and gephyrin puncta near plaques
compared to plaque-free areas (Fig. [271]6b–e). In TauPS2APP;Trem2^KO
brains, plaque-proximal microglial synapse eating was significantly
reduced compared to TauPS2APP mice (Fig. [272]6b,c), whereas astrocytic
eating of Homer1 puncta was unaffected by the lack of Trem2 (Fig.
[273]6d). Notably, astrocytic phagocytosis of gephyrin puncta in the
vicinity of plaques was significantly increased in TauPS2APP;Trem2^KO
versus TauPS2APP brains (Fig. [274]6e). As in P301S mice, astrocytic
lysosomes contained more Homer1 puncta compared to microglial lysosomes
(Fig. [275]6f), whereas gephyrin puncta were more abundant in
microglial lysosomes in TauPS2APP mice (Fig. [276]6g). The overall
effect of Trem2-deficiency in TauPS2APP mice was the increase in ratios
of both Homer1 and gephyrin inside astrocytic versus microglial
lysosomes (Fig. [277]6f,g). Thus, Trem2 is necessary for efficient
synapse engulfment by microglia near plaques and astrocytes can, at
least in part, compensate for impaired microglial phagocytosis of
inhibitory synapses.
Fig. 6. Astrocytes compensate for impaired microglial phagocytosis of
inhibitory synapses in Trem2-deficient TauPS2APP mice.
[278]Fig. 6
[279]Open in a new tab
a, Representative images of confocal z-stack and Imaris 3D
reconstructions of mouse brain sections immunostained for GFAP (blue),
Iba1 (white), Lamp1 (green), Homer1 (yellow) and gephyrin (red).
LAMP1^+ lysosomes within GFAP^+ or Iba1^+ volumes were classified as
astrocytic or microglial lysosomes. Plaques were identified indirectly
by the presence of large clusters of Lamp1 accumulation (outside of
glial cell bodies), which labels dystrophic axons. In the 3D
reconstructions (right hand image of each pair), only Lamp1 structures
within GFAP or Iba1 volume are rendered. Scale bars, 5 µm. b,c,
Fraction of Homer1 (b) or gephyrin (c) puncta identified within
microglial lysosomes. d,e, Fraction of Homer1 (d) or gephyrin (e)
puncta identified within astrocytic lysosomes. f,g, Ratio of Homer1 (f)
and gephyrin (g) puncta within astrocytic/microglial lysosomes. Dotted
line at a ratio of 1 indicates that astrocytic and microglial lysosomes
contained the same number of synaptic puncta, ratio of >1 means that
more synaptic puncta were localized within astrocytic lysosomes and <1
indicates that microglial lysosomes contained more synaptic puncta.
Images containing dystrophic axons (plaques), were considered as ‘near
plaque’ and images without any dystrophic axons were defined as ‘away
plaque’. Data were analyzed by one-way ANOVA with Dunnett’s multiple
comparisons test. Each dot shows average data from one mouse; n = 10–12
mice per genotype. Note that due to increased plaque load, in some
TauPS2APP;Trem2^KO mice we were not able to image plaque-free areas.
All data are presented as mean ± s.e.m.
[280]Source data
Discussion
Multiple lines of evidence support the idea that overactivation of the
CCP contributes to synapse loss and neuronal damage in
AD^[281]3,[282]4,[283]15,[284]17. Here, we found that C1q deletion was
protective against neurodegeneration without preventing gliosis, Tau
pathology or gross transcriptomic changes, implying C1q and CCP act
downstream of Tau pathology and gliosis. We provide a deep proteomic
dataset that catalogs synaptic protein changes as well as altered glial
protein association with synapses. Our data show a new role for
astrocytes in complement-dependent removal of excitatory and inhibitory
synapses. These follow-up experiments also indicated
concomitant/coordinated roles for astrocytes and microglia in synapse
engulfment during pathophysiology.
Although functional benefits upon C1q deletion in P301S mice (and C3
deletion in P301S and PS2APP mice^[285]4) suggest complement-dependent
glial elimination of functional synapses, one limitation of our study
is that by immunostaining and analysis of fixed brains, we could not
distinguish between glial pruning mechanisms and cleaning of debris.
That technical caveat noted, we found that while astrocytic lysosomes
contained more Homer1 puncta, gephyrin immunoreactivity was
preferentially found in microglial lysosomes, revealing an unexpected
‘division of labor’ between astrocytes and microglia^[286]20. In
TauPS2APP;Trem2^KO mice where microglial synaptic engulfment was
impaired, astrocyte phagocytosis partially compensated for the
engulfment of gephyrin puncta, possibly because inhibitory synapses are
normally predominantly engulfed by microglia. Together with a recent
study that identified astrocytic removal of synapses in the adult brain
using fluorescent phagocytosis reporters^[287]20, our study identifies
that astrocytes are a key phagocyte of synapses.
In contrast to the C1q-dependent synapse engulfment in P301S mice, we
find that the basal levels of astrocytic (and microglial) synapse
engulfment in healthy brains is C1q-independent. One explanation for
this difference could be the low expression of complement genes^[288]4
(and low basal complement activity) in the healthy adult brain.
Alternatively, disease-associated astrocytes might induce the
expression of (yet unknown) complement receptor(s). Microglial removal
of synapses is mediated by microglial CR3 recognition of C3b deposited
on synapses^[289]15,[290]18; however, astrocytes do not express CR3 as
microglia do. Phosphatidylserine (PS) has been identified as an
‘eat-me’ signal that labels synapses for microglial removal during
development as well as plaques in AD models^[291]48,[292]49.
Potentially, the increase in annexin family proteins in human AD and
P301S synapse fractions might reflect their binding to damaged,
PS-exposing synaptic membranes, whereas their reduction in P301S;C1q^KO
synapses may reflect less damaged synapses in C1q-deficient brains.
MERTK and Megf10 are astrocyte-expressed phagocytosis receptors that
bind exposed PS via its ligands Gas6 and Protein S and mediate
engulfment of synapses under physiological conditions^[293]20,[294]21.
Megf10 has been shown to bind C1q and thereby mediate clearance of
apoptotic cells by astrocytes^[295]50 and hence might be involved in
astrocytic synapse eating; however, none of the known glial phagocytic
receptors was identified in our synapse fractions. Future experiments
will need to determine whether specific astrocytic receptors directly
detect complement deposition on neurons or whether there may be
indirect complement-dependent signaling that triggers local activation
of astrocyte processes around synapses.
Microglia have previously been shown to prune inhibitory synapses in
physiological and pathological conditions in a PS- and
complement-dependent manner, respectively^[296]11,[297]51. GABA[B]
receptors expressed in a subset of microglia facilitate microglial
pruning of inhibitory synapses during circuit development^[298]52 and
GABA-receptive microglia might also engulf inhibitory synapses in a
disease context. Given that astrocytes and microglia eliminate synapses
in a C1q/complement-dependent manner in P301S mice it is possible that
they ‘compete’ for the same synapses that are destined for removal or
phagocytosis of synapses might be orchestrated between astrocytes and
microglia, like the removal of apoptotic cells^[299]53. In ischemia,
phagocytic activity of astrocytes and microglia show spatiotemporal
differences^[300]54, suggesting that there might be astrocyte–microglia
coordination in remodeling of damaged tissue. In this context, it is
notable that in MS mouse models, microglia but not astrocytes eliminate
synapses through the alternative complement pathway^[301]12. Thus, it
is possible that astrocytes and microglia sense different complement
molecules at the synapse.
Overall, our results greatly advance our understanding of the
mechanisms underlying complement-mediated synapse elimination and
neuronal damage, identify an unexpected division of labor of inhibitory
versus excitatory synapse phagocytosis between microglia and astrocytes
and open new avenues into potential therapeutic approaches for AD.
Methods
Mice
P301S mice (expressing human Tau with the P301S mutation, driven by the
PrP promoter^[302]55), were crossed to C1qC KO mice (Jax, 029409). All
P301S mice were hemizygous for the TauP301S transgene and cohorts were
produced with all genotypes as littermates. The TauPS2APP model was
generated as previously described by crossing PS2APP mice with mice
expressing the P301L mutant human Tau protein and all experimental
animals were homozygous for PS2APP and hemizygous for the P301L
transgene^[303]46. As previously described, TauPS2APP mice were crossed
with mice carrying the Trem2tm1(KOMP)Vlcg null allele^[304]46.
Throughout the study we analyzed male mice. All animal studies were
authorized and approved by the Genentech Institutional Animal Care and
Use Committee. Mice were group-housed up to five mice per cage in
individually ventilated cages within animal rooms maintained on a
14:10-h, light–dark cycle. Animal rooms were temperature and
humidity-controlled, between 20–26 °C and 30–70%, respectively, with
10–15 room air exchanges per hour. Mice had ad libitum access to water
and food. All testing occurred during the light phase.
Human CSF samples
CSF from patients with AD and healthy controls was obtained from Folio
Biosciences and Precision Medicine with ethics committee approval and
written informed consent. The patient cohorts were described
previously^[305]4.
Volumetric brain MRI
MRI was performed on a 9.4T Bruker system with a four-channel
receive-only cryogen-cooled surface coil and a volume transmit coil
(Bruker). T2-weighted images were acquired with a multi-spin echo
sequence: TR 5,100 ms; TE 10, 20, 30, 40, 50, 60, 70 and 80 ms; 56
contiguous axial slices of 0.3 mm thickness; FOV 19.2 mm × 19.2 mm;
matrix size 256 × 128, 1 average, with a scan time of 11 min per mouse.
During imaging, anesthesia was maintained at 1.5% isoflurane and body
temperature was maintained at 37 ± 1 °C using a feedback system with
warm air (SA Instruments). The regional and voxel differences in the
brain structure were evaluated by registration-based region of interest
analysis. In brief, multiple echo images were averaged and corrected
for field inhomogeneity to maximize the contrast-to-noise ratio and
images were analyzed based on a 20-region predefined in vivo mouse
atlas ([306]https://github.com/dmac-lab/mouse-brain-atlas) that was
co-registered to a study template and warped to individual mouse
datasets. All the co-registration steps were performed in SPM8
(Wellcome Trust Centre for Neuroimaging, UCL).
Behavior/open field
Spontaneous locomotor activity was measured with an automated Photobeam
Activity System-Open Field (San Diego Instruments). Mice were placed
individually in a clear plastic chamber (41 cm length × 41 cm
width × 38 cm height) and their horizontal and vertical movements were
monitored for 15 min per session with two 16 × 16 photobeam arrays.
Histology and analysis
Mice were deeply anesthetized and transcardially perfused with
phosphate-buffered saline (PBS). Hemi-brains were drop-fixed for 48 h
at 4 °C in 4% paraformaldehyde. After being cryoprotected and frozen,
up to 40 hemi-brains were embedded per block in a solid matrix and
sectioned coronally at 30 μm (MultiBrain processing by NeuroScience
Associates) before being mounted onto slides.
Immunohistochemistry
Brain sections were stained for AT8 (Thermo Scientific MN1020B, 1:5,000
dilution), GFAP (Dako Z0334, 1:20,000 dilution), Iba1 (Abcam ab178846,
1:100,000 dilution), NeuN (Millipore MAB377B, 1:1,500 dilution) and
Amino Cupric (using established protocols as described
previously^[307]4). Brightfield slides processed by NeuroScience
Associates were imaged on the Leica SCN400 whole-slide acquisition
system (Leica Microsystems) at ×200 magnification. Quantification of
chromogenic staining area was performed using grayscale and color
thresholds followed by morphological operations. Positive stain area
was normalized to the whole brain section or the manually marked up
hippocampal area.
Immunofluorescence measurement of C1q levels
Free-floating sections in PBS with 0.1% Triton X-100 (PBST), were
blocked with 5% normal donkey serum in PBST (NDST) and incubated
overnight at 4 °C with primary antibody in 1% NDST. Secondary
antibodies in 1% NDST were incubated for 2–3 h at room temperature,
washed in PBST and PBS and mounted using NeuroScience Associates
Mounting Solution pH 6.0 (NeuroScience Associates). Slides were
cover-slipped with ProLong Diamond Anti-fade Mountant with DAPI.
Primary antibody was C1q (1:1,000 dilution clone 4.8, rabbit
monoclonal, Abcam ab182451). Alexa Fluor secondary antibody goat
anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa
Fluor 594 (Thermo Fisher, A11012) was used at 1:500 dilution.
Immunofluorescent slides were imaged at ×200 magnification using the
Nanozoomer-XR (Hamamatsu) whole-slide scanner equipped with a
fluorescent imaging module and standard filter wheel. All whole-slide
image analysis was performed in a blinded manner using MATLAB v.9.4
(Mathworks). Total tissue area was detected by thresholding on the DAPI
and Alexa-594 signal and merging and processing of the binary masks by
morphological operations. Hippocampal regions of interest were marked
up manually. Pixel intensity was evaluated in 8-bit grayscale and the
C1q integrated pixel intensity in the whole-brain section or
hippocampus was normalized to the whole tissue or hippocampal area,
respectively. Data were averaged from two sections per animal.
CSF total and processed complement assays
C4 and processed C4, Factor B and processed Factor B were measured in
human CSF using custom single molecule array (Simoa) assays
(Quanterix). For the C4 assay, the main reagents consisted of
paramagnetic carboxylated beads (Quanterix) coated with a rabbit
anti-C4 antibody (abx102219, Abbexa) and a biotinylated mouse anti-C4c
detection antibody (A211, Quidel). For processed C4, which measures the
C4c protein fragment, the main reagents consisted of paramagnetic
carboxylated beads (Quanterix) coated with a mouse anti-C4c antibody
(C7850-18B1, US Biological) and a biotinylated mouse anti-human C4
(LS-C128299, LSBio). Conjugations were performed using the standard
recommended concentrations and challenge ratios from Quanterix. For the
Factor B (FB) assay, the main reagents consisted of paramagnetic
carboxylated beads (Quanterix) coated with a mouse anti-FB antibody
(ab17927, Abcam) and a biotinylated anti-Bb detection antibody
(Genentech). For processed Factor B, which measures the Bb protein
fragment, the main reagents consisted of paramagnetic carboxylated
beads (Quanterix) coated with an anti-Bb antibody (Genentech; same
antibody for FB capture) and a biotinylated mouse anti-human Bb (A252,
Quidel). Conjugations were performed using the standard recommended
concentrations and challenge ratios from Quanterix.
Assays were run using one of the standard protocols for the Simoa HD-1
instrument from Quanterix^[308]4. In the protocol, 25 μl of
capture-coated beads were incubated for 30 min with 25 μl of diluted
sample. After washing, immunocomplexes were incubated for 5 min with
100 μl of the biotinylated detection antibody. Washed immunocomplexes
were incubated for 5 min with 100 μl of streptavidin-conjugated
β-galactosidase (Quanterix). After a last round of washes, the beads
were resuspended in resorufin β-d-galactopyranoside (Quanterix) and the
mixture was then applied to Simoa disks. The HD-1 analyzer was used to
read the resulting fluorescent signal and calculate the average number
of enzymes per bead (AEB) for tested samples. The reported AEB values
were analyzed against a calibrator curve constructed by AEB
measurements on native human C4 (A105, Complement Technology, C4 assay)
or C4c (32R-AC050, Fitzgerald, PC4 assay) or Factor B (A135, Complement
Technology, FB assay) or Bb (A155, Complement Technology, Bb assay)
protein serially diluted in assay diluent. Samples were analyzed using
a single batch of reagents and testing across three runs. For each of
the three runs, the PC4 and C4 or FB and Bb assays were run together
with calibrators and controls for each assay and an approximately equal
number of samples from healthy individuals and patients with AD samples
were tested at the chosen dilutions.
Bulk RNA-seq
Ten-month-old WT (n = 4), C1q^KO (n = 4), P301S (n = 4) or P301S;C1q^KO
mice were perfused with cold PBS and the hippocampi were immediately
sub-dissected and preserved in RNAlater. RNA was extracted from samples
using QIAGEN RNeasy Plus Mini kit. The concentration of RNA samples was
determined using a NanoDrop 8000 (Thermo Scientific) and RNA integrity
was determined by Fragment Analyzer (Advanced Analytical Technologies).
Then, 0.5 μg of total RNA was used as an input material for library
preparation using TruSeq RNA Sample Preparation kit v2 (Illumina).
Library size was confirmed using a Fragment Analyzer (Advanced
Analytical Technologies). Library concentrations were determined by
qPCR-based using a Library quantification kit (KAPA). The libraries
were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to
generate 30 M of single-end 50-bp reads per library^[309]56.
The fastq sequence files for all RNA-seq samples were filtered for read
quality (keeping reads where at least 70% of the cycles had Phred
scores ≥23) and ribosomal RNA contamination. The remaining reads were
aligned to the mouse reference genome (GRCm38) using the GSNAP
alignment tool^[310]57. These steps and the downstream processing of
the resulting alignments to obtain read counts were implemented in the
Bioconductor package HTSeqGenie
([311]https://bioconductor.org/packages/release/bioc/html/HTSeqGenie.ht
ml). Only uniquely mapped reads were used for further analysis.
Differential gene expression analysis was performed with
voom + limma^[312]58 (only C1qc was significant in the KO versus WT
comparison, so further results are not shown in the text).
For heat maps (Extended Data Figs. [313]2 and [314]5c), gene expression
data were first normalized to nRPKM statistic as described^[315]58,
then transformed to a log[2] scale. Any values less than −40 were then
replaced by −40 and a standard z score calculation was performed (for
each gene, subtracting mean and dividing by s.d.) and then used for
visualization.
Single-cell RNA-seq
Nine-month-old WT (n = 3) or P301S^het (n = 6) mice were perfused with
cold PBS and the hippocampi were immediately sub-dissected. Single-cell
suspensions were prepared from the hippocampi as described
elsewhere^[316]46. Briefly, hippocampi were chopped into small pieces
and dissociated with enzyme mixes in a Neural Tissue Dissociation kit
(P) (Miltenyi, 130-092-628) in the presence of actinomycin D. After
dissociation, cells were resuspended in Hibernate A Low Fluorescence
medium (Brainbits) containing 5% FBS, with Calcein Violet AM (Thermo
Fisher, [317]C34858) and propidium iodide (Thermo Fisher, P1304MP).
Flow cytometry was used to sort and collect live single-cell
suspensions for the scRNA-seq study.
Sample processing and library preparation was carried out using the
Chromium Single Cell 3′ Library and Gel Bead kit v3 (10x Genomics)
according to the manufacturer’s instructions. Cell-RT mix was prepared
to aim for 10,000 cells per sample and applied to Chromium Controller
for gel bead-in-emulsion generation and barcoding. Libraries were
sequenced with HiSeq 4000 (Illumina). scRNA-seq data were processed
with an in-house analysis pipeline as described
previously^[318]46,[319]59. Reads were demultiplexed based on perfect
matches to expected cell barcodes. Transcript reads were aligned to the
mouse reference genome (GRCm38) using GSNAP (2013-10-10)^[320]57. Only
uniquely mapped reads were considered for downstream analysis.
Transcript counts for a given gene were based on the number of unique
molecular identifiers (UMIs) (up to one mismatch) for reads overlapping
exons in sense orientation. Cell barcodes from empty droplets were
filtered by requiring a minimum number of detected transcripts. Sample
quality was further assessed based on the distribution of per-cell
statistics, such as total number of reads, percentage of reads mapping
uniquely to the reference genome, percentage of mapped reads
overlapping exons, number of detected transcripts (UMIs), number of
detected genes and percentage of mitochondrial transcripts. After this
primary analysis step, cells with less than 1,000 total UMIs or greater
than 10% mitochondrial UMIs were discarded. UMI normalization was
performed by dividing each gene expression value for a cell by a factor
proportional to the total number of transcripts in that cell. Letting
n[c] represent the total number of UMIs for cell c, then the
normalization factor f[c] for that cell was given by
[MATH:
fc=<
/mo>ncmedianc′(nc′) :MATH]
(with c′ going over all cells) and the ‘normalized UMIs’ for gene g and
cell c given by nUMI[g,c] = n[c] / f[c]^[321]60.
Pseudobulk microglial, astrocyte, oligodendrocyte and neuron expression
profiles were derived from single-cell datasets first by aggregating
each sample’s data for each cell type as described^[322]46. A single
‘raw count’ expression profile was created for each pseudobulk simply
by adding the total number of UMIs for each gene across all each cell
of that type from that sample. This gave a gene-by-pseudobulk count
matrix, which was then normalized to a normalizedCount statistic using
the estimateSizeFactors function from DESeq2 (ref. ^[323]61), used for
calculating gene set scores and visualizing gene expression and for
normalization factors for DE analysis. DE was performed on pseudobulk
datasets using voom + limma methods for bulk RNA-seq. To put this into
more formal notation, let n[ij] be the raw UMI number of gene i in each
cell type j. Let s[j] indicate the sample of cell j. The pseudobulk
count matrix B, with rows indexed by genes and columns indexed by
samples (instead of cells) is defined as
[MATH:
Bis=∑j:
sj=snij :MATH]
The matrix is then size-factor normalized and analyzed using the
standard methods of bulk RNA-seq, including DE analysis using
voom + limma^[324]58. Finally, the normalized pseudobulk expression
matrix was then used to construct Fig. [325]3A and Extended Data Fig.
[326]5a,f,h by calculating the average percentage of gene expression in
each cell type (excitatory neurons, astrocytes, microglia and
oligodendrocytes) in P301S mice.
PSD/synapse fraction isolation and mass spectrometry analysis
Synapse fractions were isolated as previously described with minor
modifications^[327]3. Briefly, dissected hippocampi in ice were
homogenized in cold buffer (5 mM HEPES (pH 7.4), 1 mM MgCl[2], 0.5 mM
CaCl[2] supplemented with phosphatase and protease inhibitors) with a
Teflon homogenizer. After 1,400g, 10 min centrifugation at 4 °C the
supernatant was pelleted by centrifugation (13,800g, 10 min at 4 °C).
The pellet was resuspended in 0.32 M Tris-buffered sucrose and
ultra-centrifuged into a 1.2, 1 and 0.85 M sucrose gradient at 82,500g
for 2 h at 4 °C. The synaptosome fraction between the 1 M and 1.2 M
sucrose interface was carefully collected, the same volume of 1% Triton
X-100 was added, then mixed and incubated on ice for 15 min. The final
synapse fraction was pelleted at 32,800g for 20 min at 4 °C. For each
age, a cohort of 11 samples was analyzed. For the 6-month-old group of
mice, we isolated synapse fractions from three WT, three P301S, two
C1q^KO and three P301S;C1q^KO hippocampi. Due to hippocampal atrophy
and to reduce inter-animal variability, we pooled hippocampi in the
9-month-old cohort. Each pool contained samples from two mice (WT,
C1qKO and P301S;C1q^KO) or four mice (P301S), respectively.
Enriched PSD samples were adjusted to a pH of 8.5 before reduction
(5 mM dithiothreitol, 45 min at 37 °C), alkylation (15 mM IAA, 30 min
at room temperature in the dark) and capping (5 mM dithiothreitol,
15 min at room temperature in the dark). Proteins were digested by LysC
(1:50 ratio of enzyme to substrate) for 3 h at 37 °C before digestion
with trypsin (1:50 ratio of enzyme to substrate) O/N at room
temperature while shaking. Peptides were acidified, desalted using the
Phoenix peptide cleanup kit (PreOmics) and dried before quantification
with a peptide BCA kit (Thermo Fisher). Peptides were labeled with TMT
multiplexing reagents (Thermo Fisher) according to the manufacturer’s
instructions. Following labeling with isobaric tags, the samples were
mixed, dried and desalted before fractionation. For young mouse
samples, peptides were separated by offline high-pH reversed-phase
fractionation using an ammonium formate-based buffer system delivered
by an 1100 HPLC system (Agilent). Peptides were separated over a
2.1 × 150 mm, 3.5 µm 300Extend-C18 Zorbax column (Agilent) and
separated over a 75-min gradient from 5% ACN to 85% ACN into 96
fractions. The fractions were then pooled into 24 tubes, of which 12
were analyzed. For old mouse samples, peptides were fractionated using
a high-pH spin cartridge (Pierce Thermo Fisher) where 16 fractions were
collected and concatenated into eight final fractions, all of which
were analyzed. Following fractionation, peptides were dried and
desalted a final time by stage tip.
Samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer
(Thermo Fisher) coupled to an Ultimate 3000 RSLCnano ProFlow HPLC
system (Thermo Fisher). Peptides were separated over a 100 µm × 250 mm
PicoFrit column (New Objective) packed with 1.7 µm BEH-130 C18 (Waters)
at a flow rate of 450 nl min^−1 or over a 25-cm IonOpticks Aurora
column (IonOpticks) at 300 nl min^−1 for a total run time of 180 min.
The gradient started at 2 or 5% B (98% ACN and 1% FA) and ended at 30%
B over 140 min and then to 50% B at 160 min. Orbitrap MS1 survey scans
(120,000 resolution, AGC = 1 × 106 and maxIT = 50 ms) were used to
select the top ten most intense precursors, ensuring that only one
charge state per precursor (±10 ppm) was selected once every 45 s.
Selected peptides were fragmented by CAD (normalized collision
energy = 35) and analyzed in the ion trap (AGC = 2 × 104,
maxIT = 100 ms) for identification. For quantification, the eight most
intense peaks from the MS2 were selected for SPS-MS3 analysis where
peptide fragments were re-isolated and fragmented (higher-energy
collisional dissociation and normalized collision energy = 55) and
analyzed in the Orbitrap (50,000 resolution, AGC = 2.2 × 105 or
2.5 × 105, maxIT = 150 ms or 200 ms).
Mass spectral data were assigned to peptides using a concatenated
target-decoy database consisting of mouse sequences and common
laboratory contaminants from UniProt (v.2016-06) using Mascot (Matrix
Science) with a 25-ppm precursor ion mass tolerance, 0.8-Da fragment
ion tolerance, a fixed proprionamide modification on all cysteines, a
fixed modification of the TMT six-plex reagent on lysine and peptide
amino termini, a variable modification of the TMT six-plex reagent on
tyrosine and a variable oxidation modification on methionine, full
tryptic specificity and a maximum of one missed cleavage.
Peptide-spectral matches were filtered to a false discovery rate (FDR)
of 5% at the peptide level using a linear discriminant approach and
subsequently filtered to a 2% protein FDR.
Quantification and statistical testing of TMT proteomics data was
performed using MSstats v.3.14.1 (ref. ^[328]62). Before MSstats
analysis, peptide-spectral matches (PSMs) were filtered to remove
matches from decoy proteins; peptides with length less than 7;
isolation specificity <50%; reporter ion intensity <256; and summed
reporter ion intensity (across all channels) <30,000. In the case of
redundant PSMs (multiple PSMs in one MS run that map to the same
peptide), PSMs were summarized by the maximum reporter ion intensity
per peptide and channel and median equalized. In the case of redundant
PSMs across fractions (redundant matching PSMs being found in multiple
fractionated runs), PSMs were summarized by selecting the fraction with
the maximum reporter ion intensity for each PSM. Protein level
summarization was performed using a Tukey median polish approach.
Differential abundance analyses between conditions were performed in
MSstats based on a linear mixed-effects model per protein.
Pathway analysis and protein subcellular location
Pathway enrichment analysis was performed using ShinyGO^[329]63. Only
up- and downregulated DE proteins were included in the analysis.
Enriched KEGG pathways with an FDR < 0.05 were considered statistically
significant and selected KEGG pathways are represented with their
respective FDR values.
UniProt annotation and GO cellular component was used to define protein
subcellular location^[330]64. Primary literature search was used to
validate the Uniprot-annotated subcellular location of selected
proteins.
LD-score regression
We applied stratified LD-score regression (S-LDSC)^[331]30 to evaluate
polygenic enrichment in differentially abundant proteins in the P301S,
P301S;C1q^KO and C1q^KO PSDs. First, we mapped proteins from the mouse
proteome to their corresponding genes using Uniprot Knowledgebase
([332]https://www.uniprot.org/id-mapping) and converted the mouse genes
to their human orthologs using the NCBI HomoloGene database. S-LDSC had
primarily been used to analyze enrichment of large gene sets and it was
shown that a Type I error is not always controlled in the analysis of
small annotations or gene sets^[333]65. Therefore, we defined two
protein sets of the top 1,000 upregulated and 1,000 downregulated
proteins in each PSD proteome. In brief, S-LDSC tested whether the
heritability explained by SNPs near a set of genes or a specific
genomic annotation was significantly greater than expectation. The
model estimated significance after correcting for LD structure and
controlling for genomic properties that include epigenetic marks,
evolutionary conservation and protein-coding regions (as defined in the
S-LDSC baseline model). We focused our analysis on HapMap
single-nucleotide polymorphisms (SNPs) that were 100 kb up- and
downstream of each protein in the protein set. We tested for enrichment
using the GWAS summary statistics from 752 traits (629 from the UK
Biobank traits and 123 traits from other genetic
studies)^[334]28–[335]34. The UK Biobank traits selected were
demonstrated to have a significant, non-zero heritability as estimated
using LD-score regression. We used Bonferroni correction across 752
traits at α = 0.05 (multiple corrections adjusted P threshold 0.05 /
752 = 0.000066) to define significantly enriched traits. As performed
in other analyses of UK Biobank phenotypes, we assigned each trait to
one of 24 domains to identify enrichment trends among similar
phenotypes^[336]66.
Immuno-electron microscopy and quantitative analysis
IEM analysis was performed on hippocampus sections that were prepared
previously^[337]3. Briefly, mice were anesthetized and perfused with
PBS followed by 4% PFA fixative. The brains were then cut in 1-mm thick
sagittal sections and post-fixed overnight in 4% PFA. The tissue slices
were rinsed in PBS and PBS with 0.15% glycin, embedded in flat slabs of
12% gelatin in 0.1 M phosphate buffer (PB) and cryoprotected with 2.3 M
sucrose in 0.1 M PB. The hippocampus was excised from the brain slices
and cut in an anterior and posterior half, each ~1 mm^3 in size. Each
block was mounted on an aluminum pin such that sagittal hippocampus
sections could be cut with known ventral–dorsal orientation and frozen
in liquid nitrogen. From these blocks, ultrathin cryosections were cut
at −120 °C on cryo-ultramicrotomes Leica EM UC6 and UC7 with attached
cryo-chamber FC6 and FC7 (Leica Microsystems), thawed and placed on
copper carrier grids. The grids with sections were sequentially
incubated in PBS at 37 °C to dissolve gelatin, then at room temperature
with guinea pig anti-EAAT2/GLT-1 antibody (Millipore, AB1783, 1:300
dilution), followed by 10 nm Protein A-gold particles (Cell Microscopy
Core, University Medical Center Utrecht), both in blocking solution.
The blocking solution contained 0.5% fish skin gelatin (Sigma, G7765),
0.1% acetylated BSA (Aurion, 900.099) and 1% BSA (Sigma, A4503) in PBS.
Final staining of the sections was performed with uranyl acetate
(SPI-Chem, 02624-AB) followed by a uranyl acetate-methylcellulose
(Sigma, M-6385) mixture. To retrace the CA1 and DG regions in the
sections of the posterior half of the hippocampus in electron
microscopy, serially sectioned semi-thin cryosections deposited on
glass slides were stained sequentially with toluidine blue
(Sigma-Aldrich, T3260, 1% in distilled water) and methylene
blue-borax-azur(II) (Merck, 101283, 106308 and 109211, respectively,
each 0.5% in distilled water). The light microscopy and electron
microscopy images were then correlated.
For the quantitative analysis of synapses and their association with
astrocytes, EAAT2/GLT-1-labeled sections were examined in a JEM-1011
transmission electron microscope (JEOL) equipped with a Veleta Megaview
G2 CCD camera with Radius software (EMSIS). Images of synapses at
×60,000 magnification were collected in a systematic random way on
sections from three WT and three P301S mice, separately in the CA1 and
DG regions. From each synapse profile, recognizable by a synaptic
cleft, a PSD in the spine and synaptic vesicles in the axon terminal,
the perimeter length was measured. In addition, the length of all
GLT-1-positive astrocyte plasma membrane segments directly facing the
plasma membrane of each synapse was measured. Membrane lengths were
quantified using Fiji software. Average values for 10–17 synapses per
hippocampal region for each mouse were calculated.
Astrocyte–Homer1 association, synapse engulfment imaging and analysis
Synapse engulfment analysis
Free-floating sections were incubated with PBS with 0.2% Triton X-100
(PBST) and 10% normal goat serum for 1 h at room temperature. After
blocking, sections were incubated with primary antibodies in PBST at
4 °C for 16–24 h. The following primary antibodies were used: mouse
anti-GFAP (1:1,000 dilution, clone ASTRO6, Thermo Fisher), rabbit
anti-Iba1 (1:1,000 dilution, polyclonal, Wako), rat anti-LAMP1 (1:250
dilution, BioLegend), chicken anti-Homer1 (1:1,000 dilution, Synaptic
Systems) and guinea pig anti-gephyrin (1:750 dilution, Synaptic
Systems). After three washes with PBST, sections were incubated with a
secondary antibody cocktail consisting of goat anti-mouse IgG (H+L)
Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 405 (Thermo
Fisher, A48225), goat anti-rat IgG H&L (Alexa Fluor 488) preadsorbed
(Abcam, ab150165), goat anti-chicken IgY H&L (Alexa Fluor 555)
preadsorbed (Abcam, ab150174), goat anti-guinea pig IgG (H+L) Highly
Cross-Adsorbed Secondary Antibody, Alexa Fluor 633 (Thermo Fisher,
A21105) and goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 680 (Thermo Fisher, A21109), all 1:1,000 dilution
in PBST for 1 h at room temperature. After a wash with the second
antibody, sections were mounted with anti-fade reagent (ProLong Diamond
Invitrogen). Digital images were acquired using a ×100 (NA 1.4)
oil-immersion objective on a Leica SP8 laser scanning confocal
microscope using 405 nm, 488 nm, 555 nm, 633 nm and 680 nm excitation
wavelengths for collecting corresponding Alexa fluorescence signals.
Synapse engulfment analysis was performed as previously
described^[338]4. First, Iba1^+ microglia and GFAP^+ astrocytes were
3D-reconstructed using the surface-rendering function. Next Lamp1^+
lysosomes within microglia and astrocytes, respectively, were segmented
using the surface-rendering function. Homer1 and gephyrin puncta were
identified using the spots function and classified a lysosomal versus
non-lysosomal using the minimal distance function. In TauPS2APP mice,
plaques were identified indirectly by the presence of Lamp1
accumulation, which labels dystrophic axons. Z-stacks with xy
dimensions of 93.1 × 93.1 µm containing dystrophic axons (plaques),
were considered as ‘near plaque’ and images without any dystrophic
axons were defined as ‘away plaque’. Fraction of lysosomal Homer1 and
gephyrin puncta were calculated by dividing lysosomal
puncta/non-lysosomal puncta. A total of 4–5 images containing multiple
microglia and astrocytes in the CA1 region were analyzed per mouse.
Astrocyte–Homer1 association
For the astrocyte–Homer1 association, free-floating brain sections were
immunostained as described above. The following primary antibodies were
used: mouse anti-GFAP (1:1,000 dilution, clone ASTRO6, Thermo Fisher),
mouse anti-S100B (1:750 dilution, Abcam) and chicken anti-Homer1
(1:1,000 dilution, Synaptic System), followed by incubation with
Alexa-conjugated secondary antibodies. Digital images were acquired
using a ×100 oil-immersion objective on a Leica SP8 laser scanning
confocal microscope or a ×60 oil-immersion objective on an Andor
DragonFly spinning disk confocal microscope. Confocal stacks were
analyzed using Imaris 9.6.1. For astrocyte–Homer1 associations, the
GFAP or S100B channel was subjected to Gaussian filtering and
background subtraction. GFAP^+ or S100B^+ astrocytes were
3D-reconstructed using the surface-rendering function. Homer1 puncta
were reconstructed using the spots function and their total number was
calculated. Next, the number of Homer1 puncta located up to 0.3 µm from
the astrocyte surface was identified and considered as Homer1 puncta
associated with astrocytes. Percentage of astrocyte-associated Homer1
puncta was calculated by dividing it with the total number of Homer1
puncta in each image.
Homer1–C3 colocalization
Free-floating brain sections were immunostained as described above with
the following primary antibodies: rabbit anti-C3 (1:750 dilution,
Dako/Agilent A063) and chicken anti-Homer1 (1:1,200 dilution, Synaptic
System). Images were acquired using a ×60 oil-immersion objective on an
Andor DragonFly spinning disk confocal microscope. Colocalization was
calculated using the Fiji ComDet plugin
([339]https://github.com/ekatrukha/ComDet). Briefly, images were
convoluted with a Gaussian Mexican hat filter using an approximate
puncta size of 3 pixels (1 pixel = 100 nm). Puncta were identified
using an intensity threshold of 3 × s.d. for Homer1 and 6 × s.d. for
C3. Puncta were considered as colocalized if the max distance between
the spots’ centers was <3 pixels (300 nm).
Statistics and reproducibility
Experimenters were blind to genotype for all behavioral measurements,
microscopic and histological analyses. No specific methods were used to
randomly allocate samples to groups. No statistical method was used to
predetermine sample size, but our samples sizes are similar to those
reported in previous publications^[340]3,[341]4. No data were excluded
from the analyses. Statistical analyses were performed with GraphPad
Prism software v.9 (GraphPad Software). All parameters were expressed
as mean ± s.e.m., unless otherwise stated. Data distribution was
assumed to be normal but this was not formally tested. Two-by-two group
comparisons were analyzed using two-way ANOVA followed by post hoc
tests (stated in the figure legends). For comparison of two groups, a
two-tailed Student’s t-test was used. For multiple groups, one-way
ANOVA followed by a post hoc test (listed in the figure legends) was
used.
Reporting summary
Further information on research design is available in the [342]Nature
Research Reporting Summary linked to this article.
Supplementary information
[343]Reporting Summary.^ (3MB, pdf)
[344]Supplementary Table 1^ (1.7MB, xlsx)
Quantitative proteomics data of the hippocampal synapse proteomes from
6- and 9-month-old mice.
Acknowledgements