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OUTLINE

1. Highlights
2. Summary
3. Graphical abstract
4. Keywords
5. Introduction
6. Results
7. Discussion
8. STAR★Methods
9. Acknowledgments
10. Data and code availability
11. References

Show full outline

CITED BY (38)

FIGURES (14)

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TABLES (1)

1. Table

CELL

Volume 185, Issue 22, 27 October 2022, Pages 4135-4152.e22

ARTICLE
SYK COORDINATES NEUROPROTECTIVE MICROGLIAL RESPONSES IN NEURODEGENERATIVE
DISEASE

Author links open overlay panelHannah Ennerfelt 1 2 3, Elizabeth L. Frost 1,
Daniel A. Shapiro 1, Coco Holliday 1, Kristine E. Zengeler 1 2 3, Gabrielle
Voithofer 1, Ashley C. Bolte 1 4 5, Catherine R. Lammert 1 2, Joshua A. Kulas 1,
Tyler K. Ulland 6, John R. Lukens 1 2 3 4 5 7
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https://doi.org/10.1016/j.cell.2022.09.030Get rights and content
Under a Creative Commons license
open access
Refers to
TREM2 drives microglia response to amyloid-β via SYK-dependent and -independent
pathways
Cell, Volume 185, Issue 22, 27 October 2022, Pages 4153-4169.e19
Shoutang Wang, Raki Sudan, Vincent Peng, Yingyue Zhou, Siling Du, Carla M.
Yuede, Tingting Lei, Jinchao Hou, Zhangying Cai, Marina Cella, Khai Nguyen,
Pietro L. Poliani, Wandy L. Beatty, Yun Chen, Siyan Cao, Kent Lin, Cecilia
Rodrigues, Ali H. Ellebedy, Susan Gilfillan, Gordon D. Brown, David M. Holtzman,
Simone Brioschi, Marco Colonna
Referred to by
Microglia are SYK of Aβ and cell debris
Cell, Volume 185, Issue 22, 27 October 2022, Pages 4043-4045
Dorothy P. Schafer, Jacob M. Stillman
TREM2 drives microglia response to amyloid-β via SYK-dependent and -independent
pathways
Cell, Volume 185, Issue 22, 27 October 2022, Pages 4153-4169.e19
Shoutang Wang, Raki Sudan, Vincent Peng, Yingyue Zhou, Siling Du, Carla M.
Yuede, Tingting Lei, Jinchao Hou, Zhangying Cai, Marina Cella, Khai Nguyen,
Pietro L. Poliani, Wandy L. Beatty, Yun Chen, Siyan Cao, Kent Lin, Cecilia
Rodrigues, Ali H. Ellebedy, Susan Gilfillan, Gordon D. Brown, David M. Holtzman,
Simone Brioschi, Marco Colonna

HIGHLIGHTS

* •

Disruption of microglial SYK signaling exacerbates disease in models of AD
and MS

* •

Microglial proliferation and association with Aβ plaques is coordinated by
SYK

* •

SYK is a pivotal regulator of microglial activation and AKT/GSK3β-signaling

* •

Phagocytic clearance of neurotoxic material by microglia is dependent on SYK

SUMMARY

Recent studies have begun to reveal critical roles for the brain’s professional
phagocytes, microglia, and their receptors in the control of neurotoxic amyloid
beta (Aβ) and myelin debris accumulation in neurodegenerative disease. However,
the critical intracellular molecules that orchestrate neuroprotective functions
of microglia remain poorly understood. In our studies, we find that targeted
deletion of SYK in microglia leads to exacerbated Aβ deposition, aggravated
neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer’s
disease (AD). Disruption of SYK signaling in this AD model was further shown to
impede the development of disease-associated microglia (DAM), alter
AKT/GSK3β-signaling, and restrict Aβ phagocytosis by microglia. Conversely,
receptor-mediated activation of SYK limits Aβ load. We also found that SYK
critically regulates microglial phagocytosis and DAM acquisition in
demyelinating disease. Collectively, these results broaden our understanding of
the key innate immune signaling molecules that instruct beneficial microglial
functions in response to neurotoxic material.

GRAPHICAL ABSTRACT

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KEYWORDS

Alzheimer’s disease
microglia
SYK
phagocytosis
amyloid beta
neurodegenerative disease
multiple sclerosis
experimental autoimmune encephalomyelitis
neuroimmunology
disease-associated microglia

INTRODUCTION

Neurodegenerative disorders, such as Alzheimer’s disease (AD), are major public
health issues that are likely to increase in prevalence with the aging
population. In general terms, neurodegenerative diseases are thought to be
driven by the accumulation of neurotoxic material such as amyloid beta (Aβ) or
myelin debris in the central nervous system (CNS) (Nussbaum and Ellis, 2003;
Trapp and Nave, 2008).The buildup of neurotoxic agents is believed to cause
neuronal damage and death, which can ultimately lead to various forms of
neurological dysfunction that include cognitive decline, motor abnormalities,
mental disorders, and loss of inhibition (Chung et al., 2018; Taylor et al.,
2002; Vickers et al., 2009). Mounting evidence suggests that microglia, which
are the professional phagocytes of the CNS, are critically involved in ensuring
the proper containment and removal of neurotoxic material in neurodegenerative
disease pathogenesis (Condello et al., 2015; Hickman et al., 2018; Lampron
et al., 2015). Indeed, human genome-wide association studies (GWAS) have
implicated mutations in microglial receptors in the development of several
neurodegenerative diseases (Cooper-Knock et al., 2017; Efthymiou and Goate,
2017; IMSG, 2019).

Most notably, emerging evidence from both AD patients and neurodegenerative
mouse models has identified key roles for TREM2, CD33, and CD22 in disease
progression (Bemiller et al., 2017; Krasemann et al., 2017; Malik et al., 2013;
Pluvinage et al., 2019; Ulland et al., 2017; Wang et al., 2015). Although there
is growing interest in targeting these receptors to treat neurodegenerative
disease, we currently lack knowledge of the major downstream signaling molecules
and effector mechanisms employed by these receptors to influence disease
pathogenesis. Identification of the intracellular mediators that coordinate
neuroprotective microglial functions will help to uncover novel pathways that
can be targeted to treat neurodegenerative disease, and will also offer new
insights into the pathoetiology underlying neurodegeneration. Moreover,
targeting major shared intracellular signaling pathways may prove more effective
than targeting individual receptors in isolation.

In the studies presented here, we explored whether the intracellular signaling
molecule, spleen tyrosine kinase (SYK), is involved in coordinating
neuroprotective functions in microglia during neurodegenerative disease. SYK is
perhaps best known for the critical role that it plays in mounting protective
antifungal immune responses downstream of C-type lectin (CLEC) receptors
expressed on innate immune cells (Lionakis et al., 2017; Mocsai et al., 2010).
In addition, SYK has been identified as the central kinase that instructs
signaling and effector functions downstream of TREM2, CD33, and CD22 receptors
(Clark and Giltiay, 2018; Wissfeld et al., 2021; Yao et al., 2019). The
activation of SYK in microglia surrounding Aβ and other forms of neurotoxic
material (Schweig et al., 2017) spurred our interest in delineating whether SYK
is a critical regulator of microglial responses in neurodegenerative disease.
Though the ability of SYK to modulate Aβ and tau biology has been explored in
previous in vitro studies using immortalized CNS lines and pharmacological
agents with well-described off-target effects (Lawlor et al., 2018; Paris
et al., 2014), the extent to which SYK influences in vivo microglial responses
and neurodegenerative disease pathogenesis currently remains poorly understood.

Here, we show that microglia-specific deletion of SYK leads to elevated levels
of Aβ deposition, exacerbated neuropathology, and cognitive impairment in the
5xFAD mouse model of AD. We further demonstrate that SYK is critically involved
in both the compaction and phagocytosis of Aβ by microglia as well as the
regulation of AKT/GSK3β-signaling. Interestingly, we identify SYK as a key
intracellular regulator of disease-associated microglia (DAM) phenotype
acquisition and further show that CLEC7A-induced activation of SYK in 5xFAD mice
promotes improved clearance of Aβ. Moreover, we show that the neuroprotective
effects of SYK on microglia are replicated in the context of demyelinating
disease. Collectively, these findings define SYK as a central regulator of
neuroprotective microglial responses in neurodegenerative disease.

RESULTS

SYK SIGNALING IN MICROGLIA LIMITS AΒ ACCUMULATION

To investigate how SYK signaling in microglia impacts Aβ-mediated
neurodegenerative disease, we first generated Sykfl/fl Cx3cr1ERT2Cre mice
(hereafter referred to as SykΔMG mice) as a genetic tool to delete SYK from
microglia. We then crossed SykΔMG mice with 5xFAD mice, an AD mouse model that
develops aggressive Aβ pathology starting at 1.5 months of age (Richard et al.,
2015). Importantly, SYK expression is unchanged between 5xFAD and non-5xFAD
immune cells that have been described to modulate AD pathogenesis (Figures S1A
and S1B). 5xFAD SykΔMG mice were given tamoxifen food for 2 weeks after weaning
to induce deletion of SYK and then returned to normal chow to allow for
peripheral Cx3cr1-expressing immune cells to turn over and regain Syk
expression, while permitting long-lived microglia to remain SYK-deficient
(Figures S1C–S1H). As controls, Cre-negative Sykfl/fl 5xFAD littermates
(hereafter referred to as 5xFAD mice) were similarly fed tamoxifen for 2 weeks
at weaning and then returned to normal chow for the remainder of the experiment.
It is important to note that this genetic targeting strategy may also induce
deletion of SYK in Cx3cr1-expressing CNS border-associated macrophages (BAMs),
which do not undergo the frequent turnover characteristic of Cx3cr1-expressing
peripheral immune cells (Wu et al., 2021).

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Figure S1. Targeted deletion of SYK in tamoxifen-treated Sykfl/fl Cx3cr1Ert2Cre
mice, related to Figure 1

(A-B) Cre-negative 5xFAD Sykfl/fl littermate controls (5xFAD mice) and
Cre-negative Sykfl/fl littermate controls (Sykcon mice) received tamoxifen food
for 2 weeks starting at 3 weeks of age and then mice were returned to regular
food for the remainder of the experiment. Spleens and brains were harvested at
5 months of age to evaluate SYK expression in T cells, monocytes, and microglia.
T cells, monocytes, and microglia were sorted from single-cell suspensions using
respective anti-CD90.2+cells), anti-CD11b+, and anti-CD11b+(microglia) -coated
magnetic beads and magnetic column sorting. (A) MACS-sorted splenic T cells and
monocytes and MACS-sorted brain microglia were harvested from 5-month-old Sykcon
and 5xFAD mice, and levels of SYK protein (top panel) and Actin or
Ponceau-stained protein (bottom panel) were measured by Western blotting. (B)
Quantification of intensity of SYK protein bands normalized to total Actin in
sorted splenic T cells and monocytes, and intensity of SYK protein bands
normalized to Ponceau staining in brain microglia from Sykcon and 5xFAD mice.
(C-F) Sykfl/fl Cx3cr1ERT2Cre (SykΔMG mice) and Cre-negative Sykfl/fl littermate
controls (Sykcon mice) received tamoxifen food for 2 weeks starting at 3 weeks
of age and then mice were returned to regular food for the remainder of the
experiment. Brains and spinal cords were later harvested to evaluate SYK
deletion. Microglia were sorted from single-cell suspensions using
anti-CD11b+-coated magnetic beads and magnetic column sorting. (C)
Representative flow cytometry gating strategy used to validate purity of
MACS-sorted microglia from naive Sykcon and SykΔMG combined brain and spinal
cord samples. (D) Expression levels of Sykb mRNA from MACS-sorted microglia
quantified by qPCR. (E) Levels of SYK protein (top panel) and total protein
loaded (bottom panel) from MACS-sorted microglia determined by Western blotting
and SDS-PAGE with a stain-free gel, respectively. (F) Quantification of
intensity of SYK protein bands normalized to intensity of bands from total
protein loaded. (G-H) Sykcon and SykΔMG mice received tamoxifen food for 2 weeks
starting at 3 weeks of age and then mice were returned to regular food for the
remainder of the experiment. Spleens were harvested at 5 months of age to
evaluate SYK expression in T cells and monocytes. (G) Levels of SYK protein (top
panel) and Actin protein (bottom panel) from MACS-sorted splenic T cells and
monocytes determined by Western blotting. (H) Quantification of intensity of SYK
protein bands normalized to total Actin in sorted splenic T cells and monocytes
from Sykcon and SykΔMG mice. Statistical significance between experimental
groups was calculated by unpaired Student’s t test (B, D, F, H). ns = not
significant, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars represent mean ± SEM and
each data point represents an individual mouse.

Using these newly generated transgenic mouse lines, we found that SYK-deletion
in 5xFAD SykΔMG mice leads to significantly elevated accumulation of Aβ in the
cortex, hippocampus, and thalamus at 5 months of age (Figures 1A and 1B).
Microglia help to limit the spread of pathological Aβ in the brain parenchyma by
forming a barrier around Aβ deposits and promoting the physical compaction of Aβ
into dense spherical plaques, which ultimately decreases Aβ interaction with
susceptible neurons (Condello et al., 2015). Therefore, the lack of Aβ plaque
sphericity is often used to identify potentially neurotoxic Aβ aggregates and to
provide insights into the efficacy of microglial compaction (Condello et al.,
2018; Wang et al., 2016). In addition to higher amounts of Aβ load, we observed
that Aβ plaques in the cortex and hippocampus of 5xFAD SykΔMG mice exhibited
lower sphericity than the plaques found in 5xFAD littermate controls
(Figures 1C,1D, S2A, and S2B). Importantly, delayed deletion of SYK in
4-month-old 5xFAD mice yields similarly increased plaque load in the hippocampus
and decreased plaque sphericity in the cortex when harvested at 8 months of age
(Figures S2H–S2K). This suggests that microglial SYK remains influential in
attenuating pathology after disease onset in 5xFAD mice. In further support of
there being less-efficient compaction of Aβ into plaques by SYK-deficient
microglia, we observed that Aβ aggregates in 5xFAD SykΔMG mice were more
filamentous (increased 6E10 antibody labeling that detects filamentous Aβ; Lee
et al., 2018) and less compact (decreased staining of Thioflavin S [ThioS] that
detects inert plaques; Lee et al., 2018) than the Aβ deposits found in 5xFAD
littermate controls (Figures 1E and 1F). Taken together, these findings suggest
that SYK signaling in microglia plays a critical role in the control of Aβ
accumulation and compaction.

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Figure 1. Deletion of Syk in microglia leads to increased Aβ burden and altered
plaque composition in 5xFAD mice

5xFAD Sykfl/flCx3cr1ERT2Cre (5xFAD SykΔMG mice) and Cre-negative 5xFAD Sykfl/fl
littermate controls (5xFAD mice) received tamoxifen food for 2 weeks starting at
3 weeks of age and then mice were returned to regular food for the remainder of
the experiment. Mice were later harvested at 5 months of age to evaluate amyloid
beta (Aβ) load in the brain.

(A and B) Immunofluorescence staining of Aβ (D54D2, red; DAPI, blue) was
performed on sagittal sections and the percent area covered by Aβ was
quantified.

(D) Quantification of sphericity with 1.00 being the most spherical, combined
data from a total of 50–100 plaques from 3 matching brain sections per mouse.

(E) Representative images of Aβ plaque composition labeling 6E10 (purple) and
ThioS (blue).

(F) Quantification represents the percent volume of the 6E10/ThioS ratio per
field of view (FOV) from a total of 10–15 plaques from 3 brain sections per
mouse.

(G–I) Soluble and insoluble fractions of Aβ1-40 and Aβ1-42 measured by ELISA.

Statistical significance between experimental groups was calculated by unpaired
Student’s t test (B), (D), and (F)–(I). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001,
∗∗∗∗p < 0.0001. Error bars represent mean ± SEM and each data point represents
an individual mouse.

See also Figure S6.

The inability to phagocytose myelin debris in the cuprizone model is known to
obstruct aspects of oligodendrocyte biology, including the differentiation of
oligodendrocyte precursor cells (OPCs) into myelin-producing oligodendrocytes
(Back et al., 2005). Therefore, we hypothesized that any phagocytic deficits
seen in SykΔMG mice in the cuprizone model would subsequently manifest as
impaired OPC proliferation and/or differentiation into mature oligodendrocytes
during the remyelination phase that follows cuprizone cessation. Consistent with
this hypothesis, we found that SYK deficiency in cuprizone-treated SykΔMG mice
leads to greatly reduced numbers of OPCs (Olig2+Pdgfrα+ cells) and
oligodendrocytes (Olig2+CC1+cells) during the remyelination phase of the
cuprizone model (Figures 7C–7E). We also noted that OPCs in cuprizone-treated
SykΔMG mice had severely impaired proliferation during demyelination
(Figures S6I and S6J), which likely accounts for the decreased numbers of OPCs
and oligodendrocytes seen in cuprizone-treated SykΔMG mice. In comparison, we
observed comparable numbers of OPCs and oligodendrocytes in SykΔMG mice and
Sykcon littermate controls that were fed normal chow (Figures 7C–7E), suggesting
that SYK deficiency in microglia does not appreciably affect
oligodendrocyte-lineage cell numbers under homeostatic conditions. Our findings
indicate that disruption of SYK signaling in microglia causes prominent defects
in the clearance of damaged myelin in the cuprizone model of demyelinating
disease. Moreover, they suggest that the lack of neuroprotective functions in
SYK-deficient microglia can ultimately lead to impaired oligodendrocyte
generation during remyelination. Altogether, these cuprizone data support our
5xFAD and EAE findings that define a critical role for SYK in promoting
protective microglial responses that limit neurodegenerative disease
progression.

DISCUSSION

In the studies presented here, we have identified SYK as a pivotal intracellular
regulator of neuroprotective microglial responses in mouse models of both AD and
MS. SYK is perhaps best known for its essential roles in the generation of
protective immunity against many fungal infections as well as in the regulation
of T cell and B cell receptor signaling (Cornall et al., 2000; Latour et al.,
1997; Malik et al., 2018). However, in recent years there has been growing
appreciation for the critical involvement of SYK in models of sterile
inflammation (Chung et al., 2019). For instance, the SYK homolog in Drosophila,
known as Shark, was shown to be critical for glial phagocytosis of axonal debris
(Ziegenfuss et al., 2008). In addition, there have been a handful of recent
studies that have used pharmacological inhibitors and in vitro cell culture
systems to explore how SYK affects CNS biology (Paris et al., 2014). Beyond
these initial studies, we lack more integrated knowledge concerning how SYK
signaling affects neurological health and disease.

The role of SYK in demyelinating neuroinflammatory disease also currently
remains poorly described. This is surprising given that mutations in various
SYK-related molecules have been identified as prominent MS genetic risk factors
(Ramagopalan et al., 2010). For instance, mutations in multiple upstream
activators of SYK, including CD37, TREM2, numerous CLEC receptors (e.g.,
CLEC16A, CLECL1, and CLEC2D), and Fc receptor-like proteins (i.e., FCRL2 and
FCRL3) have been strongly linked to MS in GWAS studies (IMSG, 2019; IMSG, 2011).
Likewise, mutations in downstream molecules involved in SYK signaling, including
BCL10 and MALT1, have also been associated with MS risk (Mc Guire et al., 2013;
Molinero et al., 2012).

In summary, although pivotal roles for microglia have recently been uncovered in
AD, MS, and many other neurodegenerative disorders (Krasemann et al., 2017), the
key signaling pathways that microglia leverage to instruct neuroprotective
functions remain poorly defined. Through the studies presented here, we have
identified SYK as an instrumental regulator of neuroprotective microglial
responses in mouse models of AD and MS. Moreover, our studies suggest that
targeting SYK may offer novel strategies to boost microglial protective
responses, including phagocytosis of neurotoxic material, to treat
neurodegenerative disease.

LIMITATIONS OF THE STUDY

Future studies are needed to fully define all of the key microglial receptors
that rely on SYK to influence neurodegenerative disease progression. Based on
receptor structure alone, there are a wide range of microglial receptors that
could potentially leverage SYK to coordinate their effects on neuropathogenesis.
Most notably, and of relevance to neurodegenerative disease research, this list
includes TREM2, CLEC7A, CD33, CD22, FcγR, and complement receptor 3 (CR3) (Clark
and Giltiay, 2018; Hadas et al., 2012; Pluvinage et al., 2019; Wissfeld et al.,
2021; Yao et al., 2019; Ye et al., 2020). However, various other CLEC receptors,
SIGLEC receptors, and integrin receptors could also potentially leverage SYK
signaling to influence neurological disease (Mocsai et al., 2010).

Though previous in vitro studies have shown that TREM2 activation can provoke
SYK signaling (Yao et al., 2019), it still remains to be seen whether TREM2
relies exclusively on SYK to coordinate its effects on in vivo
Alzheimer’s-related disease progression. Therefore, additional in vivo studies
in relevant disease models are needed to address this. Likewise, even though our
studies provide promising early evidence that promoting SYK activation via
CLEC7A engagement can boost the clearance of Aβ, future studies are needed to
better characterize the role of CLEC7A in AD-related disease. In particular, it
still remains to be seen why CLEC7A is so highly expressed by microglia in
response to neurodegenerative disease pathology in mice (Deczkowska et al.,
2018; Keren-Shaul et al., 2017; Krasemann et al., 2017). Therefore, although our
early studies suggest the SYK is a major regulator of neuroprotective microglial
responses in models of AD and MS, future studies are needed to better
characterize both the upstream and downstream players that coordinate the
effects of SYK on neurodegenerative disease progression.

It is also important to note that replacing one allele of Cx3cr1 with
Cre-recombinase in Cx3cr1ERT2cre mice may potentially affect aspects of
microglial biology. However, in a recent study it was shown that targeting
microglia with this strategy actually leads to improved clearance of Aβ and
ameliorated disease progression in the APP/PS1 mouse model of AD (Hickman
et al., 2019). Therefore, our observation of worsened disease status in 5xFAD
SykΔMG is likely not explained by this variable. In addition, future studies are
also needed to ascertain whether SYK deletion in long-lived BAMs contributes at
any level to the phenotypes seen in our SykΔMG studies.

STAR★METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesSYK (D3Z1E) XP Rabbit AntibodyCell
Signaling TechnologyCat# 13198
RRID: AB_2687924StarBright Blue 700 Goat Anti-Rabbit IgGBio-RadCat# 12004161
RRID: AB_2721073β-actin (D6A8) Rabbit mAb (HRP Conjugate)Cell Signaling
TechnologyCat# 12620
RRID: AB_2797972β-Amyloid (D54D2) XP Rabbit mAbCell Signaling TechnologyCat#
8243
RRID: AB_2797642Purified (azide-free) anti-beta-Amyloid, 1-16BioLegendCat#
803004
RRID: AB_2715854Iba1 antibodyAbcamCat# ab5076
RRID: AB_2224402Ki-67 Monoclonal Antibody (SolA15), Alexa Fluor 600,
eBioscienceThermo Fisher ScientificCat# 606-5698-80
RRID: AB_2896285Anti-mDectin-1-IgGInvivoGenCat# mabg-mdect
RRID: AB_2753143Anti-TMEM119 antibody [29-3] – Microglial markerAbcamCat#
ab209064
RRID: AB_2800343Rat Anti-Mouse CD68 Monoclonal antibody, Unconjugated, Clone
FA-11Bio-RadCat# MCA1957
RRID: AB_322219Amyloid beta precursor protein antibody [Y188]AbcamCat# ab32136
RRID: AB_2289606Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)Thermo
Fisher ScientificCat# MN1020
RRID: AB_223647Anti-NeuNMilliporeCat# MAB377
RRID: AB_2298772Mouse PDGF R alpha AntibodyR and D SystemsCat# AF1062
RRID: AB_2236897Anti-APC (Ab-7) Mouse mAb (CC-1)MilliporeCat# OP80
RRID: AB_2057371Anti-Olig-2 AntibodyMilliporeCat# AB9610
RRID: AB_570666Anti-Myelin Basic ProteinMilliporeCat# AB5864
RRID: AB_2140351Rat Anti-Myelin Basic Protein Monoclonal Antibody, Unconjugated,
Clone 12AbcamCat# ab7349
RRID: AB_305869Donkey anti-Rabbit IgG (H + L) Antibody, Alexa Fluor 488
ConjugatedThermo Fisher ScientificCat# A-21206
RRID: AB_2535792Donkey anti-Rabbit IgG (H + L) Antibody, Alexa Fluor 594
ConjugatedThermo Fisher ScientificCat# A-21207
RRID: AB_141637Donkey anti-Rabbit IgG (H + L) Antibody, Alexa Fluor 647
ConjugatedThermo Fisher ScientificCat# A-31573
RRID: AB_2536183Donkey anti-mouse IgG (H + L) Antibody, Alexa Fluor 488
ConjugatedThermo Fisher ScientificCat# A21202
RRID: AB_141607Donkey anti-mouse IgG (H + L) Antibody, Alexa Fluor 647
ConjugatedThermo Fisher ScientificCat# A-31571
RRID: AB_162542Donkey anti-goat IgG (H + L) Antibody, Alexa Fluor 488
ConjugatedThermo Fisher ScientificCat# A-11055
RRID: AB_2534102Donkey anti-goat IgG (H + L) Antibody, Alexa Fluor 546
ConjugatedThermo Fisher ScientificCat# A-11056
RRID: AB_142628Donkey anti-goat IgG (H + L) Antibody, Alexa Fluor 647
ConjugatedThermo Fisher ScientificCat# A-21447
RRID: AB_141844Donkey anti-rat IgG (H + L) Antibody, Alexa Fluor 594
ConjugatedThermo Fisher ScientificCat# A-21209
RRID: AB_2535795Alexa Fluor 647-AffiniPure Donkey Anti-Rat IgG (H + L)Jackson
ImmunoResearch LabsCat# 712-605-153
RRID: AB_2340694CD11b Monoclonal Antibody (M1/70), APC, eBioscienceThermo Fisher
ScientificCat# 17-0112-82
RRID: AB_469343CD45 Monoclonal Antibody (30-F11), PE-Cyanine7, eBioscienceThermo
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RRID: AB_2784648Ly-6G Monoclonal Antibody (1A8-Ly6g), FITC, eBioscienceThermo
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RRID: AB_2572531CD4 Monoclonal Antibody (RM4-5), FITC, eBioscienceThermo Fisher
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RRID: AB_464896CD8a Monoclonal Antibody (53–6.7), Alexa Fluor 700,
eBioscienceThermo Fisher ScientificCat# 56-0081-82
RRID: AB_494005CD80 (B7-1) Monoclonal Antibody (16-10A1), APC, eBioscienceThermo
Fisher ScientificCat# 17-0801-82
RRID: AB_469417CD11c Monoclonal Antibody (N418), PE, eBioscienceThermo Fisher
ScientificCat# 12-0114-83
RRID: AB_465553GM-CSF Monoclonal Antibody (MP1-22 × 109), PE, eBioscienceThermo
Fisher ScientificCat# 12-7331-82
RRID: AB_466205IFN gamma Monoclonal Antibody (XMG1.2), APC, eBioscienceThermo
Fisher ScientificCat# 17-7311-82
RRID: AB_469504IL-17A Monoclonal Antibody (eBio17B7), eFluor 450,
eBioscienceThermo Fisher ScientificCat# 48-7177-80; RRID: 11149677Bacterial and
virus strainsMycobacterium tuberculosisBecton, Dickinson, & CompanyCat#
231141Chemicals, peptides, and recombinant proteinsMOG PeptideBio-SynthesisCat#
12668-01Complete Freund’s AdjuvantSigma-AldrichCat# F5881Pertussis toxinList
Biological LaboratoriesCat# 180CuprizoneSigma-AldrichCat# 14690cOmplete Protease
Inhibitor CocktailRocheCat# 11697498001PhosSTOPRocheCat# 4906845001Ponceau S
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23-730-571T-PER Tissue Protein Extraction ReagentThermo Fisher ScientificCat#
78510SucroseSigma-AldrichCat# S0389Triton X-100Sigma-AldrichCat#
93418DAPISigma-AldrichCat# D9542ThioflavinSAAT BioquestCat# 23059ProLongGold
Antifade MountantInvitrogenCat# P36930Methoxy-X04ApexBioCat# B5769Dimethyl
Sulfoxide Anhydrous (DMSO)Sigma-AldrichCat# 276855Hanks Buffer Saline Solution
(HBSS)Thermo Fisher ScientificCat# 14025092DNase I, Grade II, from bovine
pancreasSigma-AldrichCat# 10104159001Papain, SuspensionWorthington Biochemical
CorporationCat# LS003124DMEM/F-12, no glutamineThermo Fisher ScientificCat#
21331020Fetal Bovine Serum (FBS)Thermo Fisher ScientificCat#
10082147Antibiotic-AntimycoticThermo Fisher ScientificCat#
15240096GlutamaxThermo Fisher ScientificCat# 35050061PercollCytviaCat#
17-0891-02MACS BSA Stock SolutionMiltenyi BiotecCat# 130-0910376Beta Amyloid
(1–42), humanCalifornia peptideCat# 641-15Hexafluoroisopropanol
(HFIP)Sigma-AldrichCat# 52517CypHer5E-NHS esterCytviaCat# PA15401IMDMThermo
Fisher ScientificCat# 12440053Penicillin/StreptomycinThermo Fisher
ScientificCat# 15140163Recombinant Murine M-CSFPeproTechCat#
315-02TideglusibSelleck ChemicalsCat# S2823DMEMThermo Fisher ScientificCat#
11885-084CD11b microbeads (microglia)Miltenyi BiotecCat# 130-093-634CD90.2
microbeadsMiltenyi BiotecCat# 130-121-278CD11b beads (monocytes)Miltenyi
BiotecCat# 130-049-601Fixable Viability DyeeBioscienceCat# 65-0866-14BODIPY
581/591 C11 (Lipid Peroxidation Sensor)Fisher ScientificCat# D3861Fc
BlockeBioscieneCat# 14-0161-86L-glutamineThermo Fisher ScientificCat#
25030-081Beta-mercaptoethanolThermo Fisher ScientificCat#
21985-023PMASigma-AldrichCat# P1585IonomycinSigma-AldrichCat#
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ScientificCat# NBFLuxol Fast BlueThermo Fisher ScientificCat# 212170250Lithium
CarbonateThermo Fisher ScientificCat# 446322500HematoxylinSigma-AldrichCat#
HHS128ACK Lysis BufferQuality BiologicalCat# 118-156-101TRIzolLife
TechnologiesCat# 15596018ChloroformFisher ScientificCat#
BP1145-1IsopropanolSigma-AldrichCat# I9516Critical commercial assaysPierce 660nm
Protein Assay KitThermo Fisher ScientificCat# 22660Human/Mouse AKT Pathway
Phosphorylation Array C1RayBiotechCat# AAH-AKT-1-8In Situ Cell Death Detection
Kit, FluoresceinRocheCat# 11684795910Amyloid beta 40 Mouse ELISA KitThermo
Fisher ScientificCat# KMB3481Amyloid beta 42 Mouse ELISA KitThermo Fisher
ScientificCat# KMB3441CellROX Deep Red Flow Cytometry Assay KitThermo Fisher
ScientificCat# C10491Bio-Rad Bio-Plex Pro Reagent KitBio-RadCat #
171-304070MRNeasy Micro KitQiagenCat# 74004Sensifast cDNA Synthesis
KitBiolineCat# BIO-65054Sensifast Probe No-ROX KitBiolineCat# BIO-86005Deposited
dataGene expression data for replication analysisThis paperGEO: GSE212310
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE212310Original codeThis
paperZenodo: https://zenodo.org/record/7026051#.YwkJKi-B3_QExperimental models:
Organisms/strainsMouse: 5xFAD; B6SJL-Tg(APPSwFlLon,
PSEN1∗M146L∗L286V)6799Vas/MmjaxThe Jackson LaboratoryJax Stock no.
034840-JAXMouse: B6.129P2-Syktm1.2Tara/JThe Jackson LaboratoryJax Stock no.
017309-JAXMouse: B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/JThe Jackson
LaboratoryJax stock no.
020940-JAXMouse: B6.Cg-Gt(ROSA)26Sortm6(CAG-Zsgreen1)Hze/JThe Jackson
LaboratoryJax stock no.
007906-JAXOligonucleotidesqPCR primer: SykbThermo Fisher
ScientificMm01333035_m1qPCR primer: GapdhThermo Fisher
ScientificMm99999915_g1Software and algorithmsGraphPad Prism 9GraphPadRRID:
SCR_002798Imaris 9.5.1ImarisRRID: SCR_007370Adobe
PhotoshopAdobehttps://www.adobe.com/products/photoshop/BioRenderBioRenderRRID:
SCR_018361LAS AF
SoftwareLeicahttps://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/Fiji/ImageJ
SoftwareFijiRRID: SCR_002285Noldus Ethovision XT
SoftwareNoldushttps://www.noldus.com/ethovision-xtKaluza Acquisition
SoftwareBeckman
Coulterhttps://www.beckman.com/flow-cytometry/software/kaluzaFlowJo
SoftwareFloJoRRID:SCR_008520Bio-Plex Manager
softwareBio-Radhttps://www.bio-rad.com/en-us/product/bio-plex-200-systemsR
Studio
(V4.0.5)https://rstudio.com/RRID:SCR_001905Rhttps://www.r-project.orgRRID:SCR_001905DESeq2
(v1.32.0)N/Ahttps://bioconductor.org/packages/release/bioc/html/DESeq2.htmlCell
Ranger
(V1.3.1)N/Ahttps://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installationSeurat
(v4.0.2)https://www.r-project.org/RRID:SCR_016341ToppClusterCincinnati
Children’shttps://toppcluster.cchmc.orgMonocle (v0.2.3.0)N/AN/AOtherLeica TCS
SP8 Confocal MicroscopeLeica MicrosystemsN/AMini-PROTEAN TGX Stain-Free Protein
GelBio-RadCat# 4568093Mini-PROTEAN Tetra CellBio-RadCat# 1620264Trans-Blot Turbo
Transfer SystemBio-RadCat# 1704150Bio-Spin P-6 gel Columns, Tris
BufferBio-RadCat# 7326227Mouse stereotaxic FrameStoeltingCat# 51730UNanoliter
InjectorWorld Precision InstrumentsCat# NL2010MC2TLS columnsMiltenyi BiotecCat#
130-042-401QuadroMACS magnetMiltenyi BiotecCat# 130-091-051Gallios Flow
Cytometer - Navios SystemBeckman CoulterCat# B83535Bio-Plex 200
SystemBio-Radhttps://www.bio-rad.com/en-us/product/bio-plex-200-systemsKeyence
BZ-X810
MicroscopeKeyencehttps://www.keyence.com/landing/lpc/all-in-one-fluorescence-microscope.jspCellometer
Auto 2000Nexelcom
Biosciencehttps://www.nexcelom.com/nexcelom-products/cellometer-fluorescent-viability-cell-counters/cellometer-auto-2000/NanoDrop
2000 SpectrophotometerThermo Fisher
Scientifichttps://www.thermofisher.com/order/catalog/product/ND-2000CFX384
Real-Time PCR SystemBio-RadCat# 1855484Influx Cell
SorterBDhttps://med.virginia.edu/flow-cytometry-facility/equipment/influx-cell-sorter/GENEWIZ
Next Generation
SequencingAzentahttps://www.genewiz.com/en/Public/Services/Sanger-SequencingChemiDoc
MP Imaging SystemBio-RadCat# 12003154

RESOURCE AVAILABILITY

LEAD CONTACT

Further information and requests for resources and reagents should be directed
to and will be fulfilled by the lead contact, John R. Lukens
(Jrl7n@virginia.edu).

MATERIALS AVAILABILITY

This study did not generate new unique reagents.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

MICE

All mouse experiments were performed in accordance with the relevant guidelines
and regulations of the University of Virginia and approved by the University of
Virginia Animal Care and Use Committee. 5xFAD mice (Stock #34848-JAX), Sykfl/fl
mice (Stock #017309) and Cx3cr1ERT2cre mice (Stock #020940) were obtained from
The Jackson Laboratory and were crossed to generate Sykfl/fl (denoted as
Sykcon), Sykfl/fl Cx3cr1ERT2cre (denoted as SykΔMG), 5xFAD Sykfl/fl (denoted as
5xFAD), and 5xFAD Sykfl/fl Cx3cr1ERT2cre (denoted as 5xFAD SykΔMG) experimental
mice. Upon weaning, female Sykcon and SykΔMG, 5xFAD, and 5xFAD SykΔMG
littermates were fed tamoxifen diet (Envigo Teklad #TD.130858) ad libitum for
two weeks and then returned to normal chow. Ai6-ZsGreen (Stock #007906-JAX)
reporter mice were generously provided by Dr. Tajie Harris and crossed with
Syk+/+ Cx3cr1ERT2Cre and Sykfl/fl Cx3cr1ERT2Cre mice. In EAE experiments,
Sykcon, SykΔMG, Sykcon−Ai6, and SykΔMG−Ai6 mice were fed tamoxifen diet for two
weeks upon weaning and then returned to normal chow. In cuprizone experiments,
Sykcon and SykΔMG were fed tamoxifen diet for two weeks upon weaning and then
returned to normal chow. Mice were housed for AD and demyelinating experiments
in specific pathogen-free conditions under standard 12-h light/dark cycle
conditions in rooms equipped with control for temperature (21 ± 1.5°C) and
humidity (50 ± 10%).

EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) INDUCTION AND SCORING

On the day of immunization (Day 0), mice were injected subcutaneously over each
shoulder with 100 μL of an emulsion containing 0.5 mg/mL MOG35-55 peptide
(Bio-Synthesis, 12-668-01) and 1.25 mg/mL heat-killed Mycobacterium tuberculosis
(Becton, Dickinson, & Company, 231-141) in Complete Freund’s Adjuvant (Sigma
Aldrich, F5881). Mice were intraperitoneally injected with 200 ng of Pertussis
toxin (List Biological Laboratories, 180) on days 0 and 2 post-immunization.

Beginning at approximately day 7 post-immunization, mice were monitored daily
for onset of hindlimb paralysis and scored for EAE severity using the following
5-point clinical scoring system: 0 = normal tail; 0.5 = limp at tip of tail; 1 =
completely limp tail; 1.5 = partial hindlimb weakness/mouse can be flipped onto
its back; 2 = complete hindlimb weakness with abnormal gait; 3 = partial
hindlimb paralysis; 3.5 = complete hindlimb paralysis in both legs; 4 = hind-
and fore-limb paralysis; 5 = moribund/dead.

CUPRIZONE MODEL

For the cuprizone model, adult mice were fed regular chow mixed with 0.3%
cuprizone (Sigma, 14,690) ad libitum for 5 weeks to induce demyelination.

METHOD DETAILS

WESTERN BLOTTING

MACS-sorted microglia, monocytes, and T cells were resuspended in Western blot
lysis buffer [dH2O, RIPA, cOmplete Protease Inhibitor Cocktail (Roche), and
PhosSTOP (Roche)]. Protein concentration was measured using Pierce 660 nm
Protein Assay Reagent (Thermo Scientific, 22-660). 4X SDS loading dye was added
to protein lysates and incubated at 95°C for 3 min. For each sample, 25 ug of
protein was loaded per lane of a 4–20% Mini-PROTEAN TGX Stain-Free Protein Gel
(BioRad, 4-568-093) and run at 120 V for 1.5 h using Mini-PROTEAN Tetra Cell
(BioRad, 1-658-004). Proteins were transferred to an Immun-Blot Low Fluorescence
PVDF membrane (BioRad, 1-620-264) using Trans-Blot Turbo transfer system
(BioRad, 1-704-150) set to 2 mini gels and mixed MW for 21 min. SYK protein was
probed using anti-SYK (D3Z1E) XP Rabbit mAb (Cell Signaling Technologies,
13,198, 1:1000 overnight at 4°C) and goat anti-rabbit IgG StarBright Blue 700
secondary antibody (BioRad, 12-004-161, 1:1000 for 2 h at room temperature). The
stain-free gel and blotted membrane were imaged using ChemiDoc MP Imaging System
(BioRad, 12-003-154). Total protein loaded was quantified using Image lab touch
software (BioRad), Beta-Actin (Cell Signaling, 12-620), Ponceau S stain
(Sigma-Aldrich, P7170) and SYK protein were quantified using Fiji. Samples
underwent the same preparation for the AKT pathway phosphorylation kit
(RayBiotech, AAH-AKT-1-8). The AKT assay was performed in accordance to manual
instructions and pixel density was analyzed using Adobe Photoshop.

BRAIN SAMPLE PREPARATION

Mice were euthanized using CO2 asphyxiation and transcardially perfused with
20 mL of 1xPBS. For AD experiments, brains were dissected out with the left
hemisphere drop-fixed in 4% paraformaldehyde over night at 4°C and the right
hemisphere flash-frozen and stored at −80°C. Drop-fixed samples were transferred
to 30% sucrose for 48 h and then mounted and frozen in Tissue-Plus OCT compound
(Fisher Scientific). These brains were then sectioned at 50 μm in thickness
using a cryostat (Leica) and stored in PBS +0.05% sodium azide at 4°C for
downstream staining and imaging. The flash-frozen brains were thawed for RNA and
protein extraction and mechanically homogenized in 500 μL of Tissue Protein
Extraction Reagent T-PER (Thermo Fisher, 78-510) containing phosphatase
inhibitor cocktail PhosSTOP (Roche, 04-906-845-001) and protease inhibitor
cocktail cOmplete (Roche, 11-873-580-001). Following homogenization, 50 μL of
the brain sample was diluted in 500 μL Trizol for future RNA extraction and
stored at −80°C. The stock brain samples were then spun down at 16,000 rpm for
10 min and the supernatant and pellet were isolated for soluble and insoluble
amyloid beta analyses, respectively. For EAE experiments, brain tissue was
dissected and immersion fixed in 4% paraformaldehyde for 48 h, followed by
dehydration in 30% sucrose and freezing in OCT. Free-floating cryosections were
cut (40 μm) and collected in PBS containing 0.02% sodium azide and stored at 4°C
until further analysis.

ELISA

Brain samples underwent guanidine-extraction in which pelleted brain samples
were incubated 1:6 in 5 M guanidine HCL/50 mM tris, pH = 8.0 at room temperature
for 3 h, then diluted 1:5 in PBS containing protease inhibitor cocktail cOmplete
(Roche, 11-873-580-001), centrifuged at 16,000 g for 20 min at 4°C, and the
supernatant was collected and stored at −80°C pending ELISA. Triton X-
extraction was performed by diluting the pelleted brain samples 1:5 in 1% Triton
X-100 in T-PER buffer and sonicating the samples for 30 min at room temperature,
then spun down at 16,000 g for 20 min at 4°C and the supernatant stored at −80°C
until used for Aβ measurement by ELISA. Amyloid beta 40 or 42 Mouse ELISA kits
were utilized (Thermo Fisher, KMB3481, KMB3441) on samples obtained with the
soluble fraction (T-PER extracted supernatant) diluted 1:10, Triton X- fraction
diluted 1:40, and the guanidine fraction diluted 1:200 following manufacturer’s
instructions.

IMMUNOFLUORESCENCE MICROSCOPY

Floating brains sectioned stored in PBS +0.05% sodium azide were blocked with 2%
donkey serum, 1% BSA, 0.1% Triton-X, 0.05% Tween20 in PBS for 1 h at room
temperature before applying the primary antibody master mix diluted in this
block overnight at 4°C. Samples were stained with anti-Aβ (D54D2, Cell
Signaling, 1:300; or 6e10, BioLegend, 1:1000) to label plaques. To study
microglial morphology and numbers, sections were stained with Iba1 (ab5076,
Abcam, 1:300). To further characterize microglia, we labeled with Ki67-EF660
(SoIA15, Thermo Fisher, 1:100), anti-Clec7a (R1-8g7, Invivogen, 1:30), Tmem119
(ab209064, Abcam, 1:300), and anti-CD68 (FA-11, BioRad, 1:1000). Neuronal health
was probed by staining for anti-APP (Y188, ab32136, Abcam, 1:750),
anti-phospho-tau (AT8, Thermo Fisher, 1:500), and anti-NeuN (MAB377, Millipore
Sigma, 1:500). For cuprizone experiments, sections were stained with PDGFRα
(AF1062, R&D Systems, 1:200), CC1 (OP80, 1:200, Millipore), Olig2 (AB9610,
1:500, Millipore), dMBP (AB5864, Millipore Sigma, 1:2000), and MBP (ab7349,
Abcam, 1:1000). Sections were then washed 3 times for 10 min at room temperature
in PBS and 0.05% tween 20, then incubated in matched donkey Alexa Fluor 488,
594, 647 anti-rabbit, -goat, -rat, -streptavidin, and -mouse (Thermo Fisher,
1:1000 dilution) at room temperature for 2 h. Samples were washed again 3 times
for 10 min at room temperature and incubated with DAPI (1:1000) for 10 min at
room temperature, or stained for plaques with ThioflavinS (Sigma-Aldrich,
2 mg/10mL) for 8 min followed by three 2-min washes with 50% ethanol at room
temperature. To investigate cell death, sections were stained by TUNEL
(Millipore Sigma, 11-684-795-910) following the manufacturer’s protocol. All
tissue sections were then transferred to wells containing PBS before being
mounted to glass slides with ProLongGold antifade reagent (Invitrogen, P36930)
and coverslips. Mounted slides were stored at 4°C until imaged using LAS AF
software (Leica Microsystems) on a Leica TCS SP8 confocal microscope.
Quantification of images were performed using Fiji software or Imaris software
(9.5.1).

BEHAVIOR

All behavior experiments were performed between 8 am and 12 pm in a blinded
fashion. All mice were 4 months old at the time of the assay. Mice were
transported from their home vivarium room to the behavior core and allowed
30 min to habituate before beginning each test.

MORRIS WATER MAZE

The MWM was done as described previously (Da Mesquita et al., 2018). In brief,
the test involved four days of training consisting of four trials and one day of
probe consisting of one trial. Mice were alternately placed facing different
visual cues for each trial in a 23°C pool made opaque with white paint. The
hidden platform was placed 1 cm below the water surface. Each trail lasted 60 s,
and the mouse was placed on the hidden platform for 5 s if unable to locate it
within the 60 s trial. All trials were tracked and scored using video tracking
software (Noldus Ethovision XT).

ELEVATED PLUS MAZE

EPM was used to investigate anxiety in mice and was performed as described
previously (Lammert et al., 2020). The maze has two open arms (35 × 6 cm2) and
two closed arms (35 × 6 cm2) with 20 cm-tall black plexiglass walls elevated
121 cm from the floor. The mice were placed in the center square connecting the
open and closed arms and allowed to explore during a 5 min trial. Activity was
monitored and scored using video tracking software (Noldus Ethovision XT).

IN VIVO AΒ PHAGOCYTOSIS ASSAY

5xFAD, 5xFAD SykΔMG, and littermate controls were intraperitoneally injected
with 10 mg/kg methoxy-X04 (ApexBio, B5769) in a 1:1 ratio of PBS and DMSO. A
brain harvest was completed 3 h after injection in which mice were euthanized
using CO2 asphyxiation before being intracardially perfused with 20 mL of PBS +
Na heparin (5 units/mL). The brains were placed in a Hanks buffer saline
solution (HBSS) (Thermo Fisher, 14-025-092) with DNAse I (50 U/mL)
(Sigma-Aldrich, 10-104-159-001) and papain (2 mg/mL) (Worthington, LS003124) and
homogenized using a 10 mL serological pipette. The brain homogenates were then
incubated at 37°C for 15 min. This process was repeated for a total of 3 times
with the final two homogenizations accomplished using a 5 mL serological
pipette. The brain homogenates were then passed through a 70 μm strainer to
create a single-cell suspension. The cell suspension was then placed in 20 mL of
DMEM/F12 buffer (21-331-020, Thermo Fisher) with 10% fetal bovine serum (FBS)
(Thermo Fisher, 10-082-147), 1% Anti-anti (Thermo Fisher, 15-240-096), and 1%
Glutamax (Thermo Fisher, 35-050-061) and spun down with a slow brake at 300 g
for 10 min. The cell pellet was then resuspended in 13 mL of 37% Percoll
(Cytvia, 17-0891-02) and spun down with no brake at 2000 rpm for 12 min. Myelin
was removed and cells were resuspended in MACS buffer (Miltenyi Biotec,
130–0910376) to wash. Flow cytometry for microglia was then performed (as
described below) with additional gating for methoxy-X04.

CYPHER5E-AΒ PREPARATION

Monomerization of Aβ (1–42) (641-15, California peptide) was achieved using a
previously published protocol (Stine et al., 2011), using hexafluoroisopropanol
(HFIP) (52-517, Sigma-Aldrich). 5 mM monomeric Aβ samples were incubated for
24 h at 4°C in F12 media to make a 200 μM stock of oligomeric Aβ. Samples were
then incubated with CypHer5E-NHS ester (PA15401, Cytvia) diluted in 0.1 M sodium
bicarbonate for 30 min covered and at room temperature. Following incubation,
Biospin columns (7-326-227, Bio-Rad) were used to quench unbound dye.
CypHer5E-tagged Aβ oligomers were stored at 4°C prior to BMDM treatment.

IN VITRO AΒ PHAGOCYTOSIS ASSAY

Bone marrow-derived macrophages (BMDMs) were isolated from the hindlimbs of WT
and Sykfl/fl LysMCre mice. Marrow-containing bones were sprayed with 70% ethanol
before being placed in IMDM (Thermo Fisher, 12-440-053) containing
penicillin/streptomycin (P/S) (Thermo Fisher, 15-140-163). A 25-gauge needle was
used to flush marrow from the bones using 20 mL of IMDM containing P/S. An
18-gauge needle was then used to triturate flushed bone marrow 5 times to make a
single-cell suspension. Samples were spun down at 1500 rpm for 5 min at 4°C.
Cell pellets were resuspended in BMDM media containing IMDM, 10% FBS, 1%
non-essential amino acids, 1% P/S, and 50 ng/mL M-CSF (PeproTech, 315-02). Cell
plating was performed using 150 × 25 mm culture dishes (430-597, Thomas
Scientific). After three days, 5 mL of BMDM media was added to each dish. Six
days post initial cell plating, media was aspirated from dishes and 10 mL of PBS
was added to each plate and incubated for 10 min at 4°C. BMDMs were removed from
the dish using a scraper and transferred to a conical tube, spun down, and
resuspended in BMDM media. 100,000 cells/well were pipetted in into a flat
bottom 96 well plate. The next day, BMDMs were treated with vehicle or 10 μM of
Tideglusib (Selleck Chemicals, S2823) 1 h prior to treatment with 10 μM of
oligomeric Aβ tagged with CypHer5E. 24 h post Aβ treatment, BMDMs were then
collected and flow cytometry was used to assess CypHer5E fluorescence.

INTRAHIPPOCAMPAL INJECTION

5xFAD and 5xFAD SykΔMG mice were anesthetized before receiving a bilateral
hippocampal injection of 2 μL of vehicle or 2 μg pustulan into the right and
left hemisphere of the hippocampus (at ±2 mm lateral, −2 mm posterior, and −2 mm
ventral relative to the intersection of the coronal and sagittal suture (bregma)
at a rate of 200 nL/min) using a stereotaxic frame (51730U, Stoelting) and
nanoliter injector (NL2010MC2T, World Precision Instruments). Seven days post
injection, mice were euthanized using CO2 and transcardially perfused before
preparing brains for immunofluorescent staining to evaluate Aβ clearance in the
hippocampus. Images were analyzed using Fiji software and Imaris software
(9.5.1).

MACS ISOLATION OF MICROGLIA, T CELLS, AND MONOCYTES

Mice were euthanized with CO2 and immediately transcardially perfused with 20 mL
1X PBS. Brains were collected and their meninges and choroid plexus were removed
before using a previously described MACS-sorting protocol (Norris et al., 2018)
to isolate microglia. Spleens from these mice were collected and kept on ice in
DMEM (Thermo Fisher, 11-885-084) with penicillin/streptomycin (Thermo Fisher,
15-140-163) and homogenized by gently mashing through a 70 μm cell strainer.
Spleen homogenates were then centrifuged at 1500 rpm for 5 min then resuspended
in 2 mL ACK lysis buffer (Quality Biological,118-156-101) and incubated at room
temperature for 3 min to lyse red blood cells. Finally, the MACS-sorting
protocol was used to isolate T cells and monocytes. Spinal cords were dissected
and kept on ice in DMEM (Thermo Fisher, 11-885-084) with penicillin/streptomycin
(Thermo Fisher, 15-140-163). Tissues were homogenized by gently mashing through
a 70 μm cell strainer. Homogenates were centrifuged at 1500 rpm for 5 min then
resuspended in 13 mL 37% isotonic Percoll (GE Healthcare, 17-0891-01) in 1X PBS.
Samples were centrifuged at 2000 rpm for 12 min at room temperature with no
brake. After centrifugation, the top myelin layer and supernatant were aspirated
and the cell pellet was resuspended in MACS buffer before using the MACS-sorting
protocol to isolate microglia (Miltenyi Biotec, 130–0910376). We proceeded with
column purification of microglia using CD11b microbeads (Miltenyi, 130-093-634),
purification of T cells using CD90.2 microbeads (Miltenyi, 130-121-278), and
purification of monocytes using CD11b microbeads (Miltenyi, 130-049-601). We
performed sorting utilizing LS columns and a QuadroMACS magnet (Miltenyi,
130-042-401 and 130-091-051) according to manufacturer’s instructions.
Column-bound cells were analyzed for purity by flow cytometry, probed for SYK
expression by qPCR and Western blotting, or submitted for RNA-seq.

FLOW CYTOMETRY

For MACS-sort validation, an aliquot of the microglia-positive and -negative
fractions were transferred to a 96-well round bottom plate, then washed with 1X
PBS and spun down at 1600 rpm for 5 min. The cells were then stained with
fixable viability dye (eBioscience, 65-0866-14) at 1:1000 for 30 min at 4°C.
Following incubation, cells were then washed with FACS buffer (pH 7.4; 0.1 M
PBS; 1 mM EDTA, and 1% BSA). Cells were then stained 1:200 with CD11b (APC),
CD45 (PE-Cy7), and TCR β chain (Brilliant Violet 510) flow antibodies (all from
eBioscience) in FACS buffer (pH 7.4; 0.1 M PBS; 1 mM EDTA, and 1% BSA) for
15 min at 4°C. The cell pellets were then washed with FACS buffer then
resuspended in 100 μL of FACS buffer. Microglia were identified as the CD45int
and CD11bhi after gating for single and live cells.

For lipid-droplet-accumulation and reactive oxygen species (ROS) assessment in
microglia, mice were euthanized with CO2 at 8 months of age and immediately
transcardially perfused with 20 mL 1X PBS. Brains were collected and their
meninges and choroid plexus removed and prepped as a single cell suspension as
described in (Norris et al., 2018). Brain homogenates were centrifuged at
1500 rpm for 5 min then resuspended in 13 mL 37% isotonic Percoll (GE
Healthcare, 17-0891-01) in 1X PBS. Samples were centrifuged at 2000 rpm for
12 min at room temperature with no brake. After centrifugation, the top myelin
layer and supernatant were aspirated and the cell pellet was washed with PBS.
Cells were then stained either 1:500 with CellROX (Thermo Fisher, C10491) for
30 min or 1:2000 with BODIPY (Invitrogen, D3861) for 10 min diluted in PBS at
37°C. Cells were spun down and washed with FACS buffer. Cells were then stained
1:200 with CD11b (APC), CD45 (PE-Cy7), and TCR β chain (Brilliant Violet 510)
flow antibodies (all from eBioscience) in FACS buffer for 15 min at 4°C. The
cell pellets were then washed with FACS buffer then resuspended 1:5000 with DAPI
in 100 μL of FACS buffer. Microglia were identified as the CD45int and CD11bhi
after gating for single and live cells.

For flow cytometry staining of BMDMs, cells were washed with FACS buffer and
centrifuged at 1500 rpm for 5 min, and resuspended in 100 μL of FACS buffer with
fluorescently labeled antibodies (all from eBioscience) specific for CD11b
(clone M1/70) and F4/80 (clone BM8) diluted 1:200. Cells were incubated in the
dark for 20 min at room temperature, washed with FACS buffer, and resuspended
1:5000 with DAPI in 100 μL of FACS buffer. BMDMs were identified as the CD11bhi
and F4/80hi after gating for single and live cells.

For flow cytometry staining in EAE experiments, cells were plated (100 μL of
resuspended spinal cord or 1x106 splenocytes) in a 96-well plate and washed with
FACS buffer. After centrifugation at 1500 rpm for 5 min and removal of
supernatants, cells were resuspended in 100 μL 1X PBS with 1:1000 fixable
viability dye (eBioscience, 65-0866-14) and 1:1000 Fc Block (eBioscience,
14-0161-86). Cells were incubated at 4°C for 30 min. Cells were then washed with
FACS buffer, centrifuged at 1500 rpm for 5 min, and resuspended in 100 μL of
FACS buffer with fluorescently labeled antibodies (all from eBioscience)
specific for CD45 (clone 30-F11), CD11b (clone M1/70), Gr-1 (clone RB6-8C5),
MHC-II (clone M5/114.15.2), TCRβ (clone H57-597), CD4 (clone RM4-5), CD8 (clone
53–6.7), CD11c (clone N418), and CD80 (clone 16-10A1) diluted 1:200. Cells were
incubated in the dark for 20 min at room temperature, washed twice with FACS
buffer, and fixed with 1% paraformaldehyde in FACS buffer.

For intracellular cytokine staining, cells were plated (100 μL of resuspended
spinal cord or 1x106 splenocytes) in a 96-well plate in IMDM stimulation media
(Iscove’s Modified Dulbecco’s Media) (Thermo Fisher, 12-440-053),
penicillin/streptomycin (Thermo Fisher, 15-140-163), 10% heat-inactivated fetal
bovine serum (Thermo Fisher, 10-082-147), 1% L-glutamine (Thermo Fisher,
25-030-081), and 50 μM beta-mercaptoethanol (Thermo Fisher, 21-985-023)] with
20 ng/mL PMA (Sigma-Aldrich, P1585), 1 μg/mL ionomycin (Sigma-Aldrich, I9657),
and 1:1000 monensin (eBioscience, 00-4505-51). Cells were incubated for 5 h at
37°C with 5% CO2, then washed with 1X PBS prior to proceeding with surface
staining for flow cytometry as described above. Cells were fixed and
permeabilized using IC fixation buffer (eBioscience, 00-8222-49) and
permeabilization buffer (eBioscience, 00-8333-56) following manufacturer’s
instructions. Cells were then stained with 100 μL fluorescently labeled
antibodies (all from Thermo Fisher) for GM-CSF (clone MP1-22 × 109), IFN-γ
(clone XMG1.2), and IL-17A (clone eBio17B7) diluted 1:200 in 1X permeabilization
buffer for 20 min at room temperature. Cells were washed twice with 1X
permeabilization buffer, then twice with FACS buffer.

Sample data were acquired within a few days of fixation using a Gallios flow
cytometer (10 colors, 3 lasers, B5-R1-V2 Configuration with Kaluza Acquisition;
Beckman Coulter) and analyzed using FlowJo software (Becton, Dickinson, &
Company).

MULTIPLEX CYTOKINE ASSAY

Immune cells isolated from spleens were plated in a 96-well plate at up to 2x105
cells/well and stimulated with 30 μg/mL MOG35-55 peptide in IMDM stimulation
media for 48 h at 37°C with 5% CO2. After incubation, cells were centrifuged at
1600 rpm for 5 min and supernatants were collected for storage at −80°C.

Supernatants were assayed for concentrations of various cytokines using Bio-Rad
Bio-Plex Pro reagent kit (Bio-Rad, 171-304070M) and Bio-Plex Pro Mouse Cytokine
23-Plex Group I magnetic beads and detection antibodies for IL-1α, IL-1β, IL-2,
IL-4, IL-6, IL-10, IL-17, G-CSF, GM-CSF, IFN-γ, KC, and TNF-α according to
manufacturers’ instructions (Bio-Rad). Sample data for Bio-Plex Pro assays were
acquired with a Bio-Plex 200 System (Bio-Rad) and analyzed using Bio-Plex
Manager software (Bio-Rad).

HISTOPATHOLOGICAL ANALYSIS OF EAE SPINAL CORDS

Mice were euthanized with CO2 and immediately transcardially perfused with 20 mL
1X PBS followed by 20 mL 10% neutral buffered formalin (NBF; Azer Scientific).
Spinal columns were dissected and immersion fixed in 10% NBF for at least 48 h.
The thoracic region of the spinal cord was carefully dissected from the column,
embedded in paraffin, sectioned coronally from the rostral end of each piece,
and mounted on slides. Sections were stained with Luxol Fast Blue (LFB; Thermo
Fisher, 212-170-250) to label myelin. In brief, after deparaffinization,
sections were incubated at 60°C for 16–24 h in 0.1% Luxol Fast Blue in 95%
ethanol +0.05% acetic acid. Excess stain was removed with 95% ethanol and slides
were dipped in 0.05% lithium carbonate (Thermo Fisher, 446-322-500) in dH2O for
10–20 s then in 70% ethanol to remove LFB staining from gray matter but not from
myelinated white matter. Slides were washed with dH2O then counterstained with
hematoxylin (Sigma Aldrich, HHS128) to label nuclei. Brightfield images were
acquired on a Keyence BZ-X810 microscope at 4X and 40X magnification.

ISOLATION OF IMMUNE CELLS

Mice were euthanized with CO2 and immediately transcardially perfused with 20 mL
1X PBS. Spinal cords and spleens were dissected and kept on ice in DMEM (Thermo
Fisher, 11-885-084) with penicillin/streptomycin (Thermo Fisher, 15-140-163).
Tissues were homogenized by gently mashing through a 70 μm cell strainer. For
spinal cords, homogenates were centrifuged at 1500 rpm for 5 min then
resuspended in 13 mL 37% isotonic Percoll (GE Healthcare, 17-0891-01) in 1X PBS.
Samples were centrifuged at 2000 rpm for 12 min at room temperature with no
brake. After centrifugation, the top myelin layer and supernatant were aspirated
and the cell pellet was resuspended in 500 μL DMEM with penicillin/streptomycin.
For spleens, homogenates were centrifuged at 1500 rpm for 5 min then resuspended
in 2 mL ACK lysis buffer (Quality Biological, 118-156-101) and incubated at room
temperature for 3 min to lyse red blood cells. Cells were washed and resuspended
in DMEM with penicillin/streptomycin. Cells were counted using a Cellometer Auto
2000 (Nexelcom Bioscience).

RNA ISOLATION, CDNA SYNTHESIS, QPCR

RNA was isolated from the left hemisphere of the brain of 5xFAD and 5xFAD SykΔMG
mice. 50 μL of brain homogenate (described in the brain sample preparation
section) was added to 500 μL of TRIzol (Life Technologies, 15,596,018).
Following vortexing of these samples, 200 μL of chloroform (Fisher Scientific,
BP1145-1) was added and incubated for 5 min before being spun down at 14,000 rpm
at 4°C for 15 min. The top aqueous fraction was transferred to a new tube and an
equal volume of isopropanol (Sigma-Aldrich, I9516) was added then vortexed. The
samples were incubated at room temperature for 10 min and then spun down at
12,000 rpm at 4°C for 5 min. The pellet was then washed with 1 mL 70% ethanol 2
times, then allowed to air dry for 15 min before resuspending the RNA pellet in
100 μL of DNAse/RNAse free water. RNA was isolated from MACS-sorted microglia in
the brain and spinal cord using an RNeasy Micro kit (Qiagen, 74-004) according
to manufacturer’s instructions. Sample quality and quantity for AD and EAE
samples was evaluated using NanoDrop 2000 Spectrophotometer (Thermo Fisher). The
RNA was then converted to cDNA using a Sensifast cDNA Synthesis kit (Bioline,
BIO-65054). Levels of Sykb (Mm01333035_m1) and Gapdh (Mm99999915_g1) mRNA were
determined using Taqman Gene Expression Assay primer/probe mix (Thermo Fisher),
Sensifast Probe No-ROX kit (Bioline, BIO-86005), and a CFX384 Real-Time PCR
System (BioRad, 1-855-484). All reagents were used according to manufacturer’s
instructions.

FACS SORTING FOR RNA SEQUENCING

Ai6-ZsGreen reporter mice possess a LoxP-flanked STOP cassette that prevents the
expression of green fluorescent protein variant ZsGreen1 until the stop site is
excised after tamoxifen induction of Cre-recombinase activity. Following the
withdrawal of tamoxifen, short-lived Ai6-ZsGreen expressing cells in the
periphery are promptly replaced by newly derived cells that lack Ai6-ZsGreen
expression. In contrast, the long-lived nature of microglia allows them retain
their Ai6-ZsGreen signal for months post-tamoxifen withdrawal (Goldmann et al.,
2013). Sykcon−Ai6 and SykΔMG−Ai6 mice were euthanized on EAE Day 35 with CO2 and
immediately transcardially perfused with 20 mL ice-cold 1X PBS. Spinal cords
were dissected and placed on ice in DMEM +10% FBS. Tissues were gently
homogenized by mashing through a cell strainer then centrifuged at 1500 rpm for
5 min. Pellets were resuspended in 13 mL 37% isotonic Percoll (GE Healthcare,
17-0891-01) in 1X PBS. Samples were centrifuged at 2000 rpm for 12 min at room
temperature with no brake. After centrifugation, the top myelin layer and
supernatant were aspirated and the cell pellet was resuspended in FACS buffer.
Cell suspensions were incubated with Fc Block and antibodies for CD45, CD11b,
and Gr-1. After two washes with FACS buffer, cells were resuspended in FACS
buffer +0.2 mg/mL DAPI and sorted on DAPI− CD45+ ZsGreen+ cells using an Influx
Cell Sorter (BD) in the University of Virginia Flow Cytometry Core Facility.

RNA SEQUENCING DATA ANALYSIS

AD MICROGLIA RNA-SEQ

MACS-sorted microglia were sent to GENEWIZ Next Generation Sequencing. The raw
sequencing reads (FASTQ files) were aligned to the UCSC mm10 mouse genome build
using the splice-aware read aligner HISAT2. Samtools was used for quality
control filtering. Reads were sorted into feature counts with HTSeq. DESeq2
(v1.32.0) was used to normalize the raw counts based on read depth, perform
principal component analysis, and conduct differential expression analysis. The
p values were corrected with the Benjamini-Hochberg procedure to limit false
positives arising from multiple testing. The significantly repressed and
enhanced genes were put into GProfiler to gather the KEGG terms. The analysis
itself was performed using the Seq2Pathway, fgsea, tidyverse, and dplyr software
packages. Heatmaps were generated using the pheatmap R package
[https://github.com/raivokolde/pheatmap] while other plots were made with the
lattice (https://lattice.r-forge.r-project.org/) or ggplot2
[https://ggplot2.tidyverse.org] packages.

EAE MICROGLIA SCRNA-SEQ

FACS-sorted single cell suspensions were submitted to the University of Virginia
Genome Analysis and Technology Core for single-cell RNA sequencing library
preparation. The raw sequencing reads (FASTQ files) were aligned to the UCSC
mm10 mouse genome build using Cell Ranger (v1.3.1) which performs alignment,
filtering, barcode counting and unique molecular identifier (UMI) counting. R
studio (v4.0.5) was used for all downstream analyses and Seurat (v.4.0.2) was
used for filtering out low-quality cells, normalization of the data,
determination of cluster defining markers and graphing of the data on UMAP.
Low-quality cells were excluded in an initial quality-control (QC) step by
removing genes expressed in fewer than three cells, cells with fewer than 150
genes expressed, and cells expressing more than 5000 genes. Cells with more than
20% of mitochondrial-associated genes and cells with more than 5% hemoglobin
among their expressed genes were also removed. Genes with high variance were
selected using the FindVariableGenes function, then the dimensionality of the
data was reduced by principal component analysis (PCA) and identified by random
sampling 20 significant principal components (PCs) for each sample with the
PCElbowPlot function. Cells were clustered with Seurat’s FindClusters function.
Differential gene expression analysis was performed within clusters using the
ZinBWave function and DESeq2 (v1.32.0). ToppCluster (Cincinnati Children’s) was
used for network analyses to identify KEGG and GO terms in the DAM cluster. Data
was organized and graphs were created using patchwork, dplyr, tidyverse, and
Seurat. The frequency plot was created using Prism GraphPad. Pseudotime analysis
was conducted using Monocle (v0.2.3.0).

QUANTIFICATION AND STATISTICAL ANALYSIS

Mean values, SEM values, Student’s t test (unpaired), one-way ANOVA, and two-way
ANOVA were calculated using Prism software (GraphPad). Significance for pooled
EAE experiments was performed by a Mann-Whitney test. p values less than 0.05
were considered significant. ns = not significant, ∗p < 0.05, ∗∗p < 0.01,
∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

ACKNOWLEDGMENTS

This work was supported by funding from the NIH (1RF1AG071996–01, R01NS106383),
The Alzheimer’s Association (ADSF-21-816651), the Cure Alzheimer’s Fund, and The
Owens Family Foundation to J.R.L.. H.E. and K.E.Z. were supported by an NIH T32
(T32GM008136). H.E., K.E.Z., A.C.B., and C.R.L. were supported by Wagner
Fellowships. H.E. and C.H. were supported by a Double Hoo Award. E.L.F. was
supported by a National MS Foundation Fellowship (FG-1707-28590). D.A.S. was
supported by a Harrison Fellowship. C.H. was supported by a College Science
Scholars Award. A.C.B. was supported by funding from the NIH (5T32GM007267-38,
5T32AI007496–25, F30AG069396-01). C.R.L. was supported by an NIH T32
(3T32GM008328). J.A.K. was supported by an NIH T32 (T32DK007646). T.K.U. was
supported by the NIH (R01AG070973).

AUTHOR CONTRIBUTIONS

H.E., E.L.F., T.K.U., and J.R.L. designed the study; H.E., E.L.F., D.A.S., C.H.,
C.R.L., K.E.Z., and G.V. performed experiments; D.A.S. and A.C.B. performed
bioinformatics analysis; H.E., E.L.F., C.H., J.A.K., and D.A.S. analyzed data;
H.E. and J.R.L. wrote the manuscript; J.R.L. oversaw the project.

DECLARATION OF INTERESTS

The authors declare no competing interests.

INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

Recommended articles

DATA AND CODE AVAILABILITY

Bulk and Single-cell RNA-seq data have been deposited at GEO and are publicly
available as of the date of publication (accession number: GEO: GSE212310) and
can be found in the key resources table. All original code has been deposited at
Zenodo and is publicly available as of the date of publication (Zenodo:
https://doi.org/10.5281/zenodo.7026051) and is listed in the key resources
table. Any additional information required to reanalyze the data reported in
this paper will be shared by the lead contact upon request.

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CITED BY (38)

* MICROGLIAL REPOPULATION REVERSES COGNITIVE AND SYNAPTIC DEFICITS IN AN
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Over the past decade, compelling genetic evidence has highlighted the crucial
role of microglial dysregulation in the development of Alzheimer's disease
(AD). As resident immune cells in the brain, microglia undergo dystrophy and
senescence during the chronic progression of AD. To explore the potential
therapeutic benefits of replenishing the brain with new microglia in AD, we
utilized the CSF1R inhibitor PLX3397 to deplete existing microglia and induce
repopulation after inhibitor withdrawal in 5xFAD transgenic mice. Our
findings revealed the remarkable benefits of microglial repopulation in
ameliorating AD-associated cognitive deficits, accompanied by a notable
elevation in synaptic proteins and an enhancement of hippocampal long-term
potentiation (LTP). Additionally, we observed the profound restoration of
microglial morphology and synaptic engulfment following their self-renewal.
The impact of microglial repopulation on amyloid pathology is dependent on
the duration of repopulation. Transcriptome analysis revealed a high
resemblance between the gene expression profiles of repopulated microglia
from 5xFAD mice and those of microglia from WT mice. Importantly, the
dysregulated neurotrophic signaling pathway and hippocampal neurogenesis in
the AD brain are restored following microglial replenishment. Lastly, we
demonstrated that the repopulation restores the expression of brain-derived
neurotrophic factor (BDNF) in microglia, thereby contributing to synaptic
plasticity. In conclusion, our findings provide compelling evidence to
support the notion that microglial self-renewal confers substantial benefits
to the AD brain by restoring the BDNF neurotrophic signaling pathway. Thus,
targeted microglial repopulation emerges as a highly promising and novel
therapeutic strategy for alleviating cognitive impairment in AD.

* MECHANOSENSITIVE PIEZO1 CHANNEL IN PHYSIOLOGY AND PATHOPHYSIOLOGY OF THE
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Since the discovery of the mechanosensitive Piezo1 channel in 2010, there has
been a significant amount of research conducted to explore its regulatory
role in the physiology and pathology of various organ systems. Recently, a
growing body of compelling evidence has emerged linking the activity of the
mechanosensitive Piezo1 channel to health and disease of the central nervous
system. However, the exact mechanisms underlying these associations remain
inadequately comprehended. This review systematically summarizes the current
research on the mechanosensitive Piezo1 channel and its implications for
central nervous system mechanobiology, retrospects the results demonstrating
the regulatory role of the mechanosensitive Piezo1 channel on various cell
types within the central nervous system, including neural stem cells,
neurons, oligodendrocytes, microglia, astrocytes, and brain endothelial
cells. Furthermore, the review discusses the current understanding of the
involvement of the Piezo1 channel in central nervous system disorders, such
as Alzheimer's disease, multiple sclerosis, glaucoma, stroke, and glioma.

* INSULIN-DEGRADING ENZYME: ROLES AND PATHWAYS IN AMELIORATING COGNITIVE
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2023, Ageing Research Reviews
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Accumulation of amyloid-β in the central nervous system is a common feature
of Alzheimer’s disease (AD) and diabetes-related cognitive impairment. Since
the insulin-degrading enzyme (IDE) can break down amyloid-β plaques, there is
considerable interest in using this enzyme to treat both neurological
disorders. In this review, we have summarized the pre-clinical and clinical
research on the potential application of IDE for the improvement of cognitive
impairment. Furthermore, we have presented an overview of the main pathways
that can be targeted to mitigate the progression of AD and the cognitive
impairment caused by diabetes.

* EMERGING CONCEPTS TOWARDS A TRANSLATIONAL FRAMEWORK IN ALZHEIMER'S DISEASE

2023, Neuroscience and Biobehavioral Reviews
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Over the past decades, significant efforts have been made to understand the
precise mechanisms underlying the pathogenesis of Alzheimer’s disease (AD),
the most common cause of dementia. However, clinical trials targeting AD
pathological hallmarks have consistently failed. Refinement of AD
conceptualization, modeling, and assessment is key to developing successful
therapies. Here, we review critical findings and discuss emerging ideas to
integrate molecular mechanisms and clinical approaches in AD. We further
propose a refined workflow for animal studies incorporating multimodal
biomarkers used in clinical studies – delineating critical paths for drug
discovery and translation. Addressing unresolved questions with the proposed
conceptual and experimental framework may accelerate the development of
effective disease-modifying strategies for AD.

* A CELL THERAPY APPROACH TO RESTORE MICROGLIAL TREM2 FUNCTION IN A MOUSE MODEL
OF ALZHEIMER'S DISEASE

2023, Cell Stem Cell
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Alzheimer’s disease (AD) remains one of the grand challenges facing human
society. Much controversy exists around the complex and multifaceted
pathogenesis of this prevalent disease. Given strong human genetic evidence,
there is little doubt, however, that microglia play an important role in
preventing degeneration of neurons. For example, loss of function of the
microglial gene Trem2 renders microglia dysfunctional and causes an
early-onset neurodegenerative syndrome, and Trem2 variants are among the
strongest genetic risk factors for AD. Thus, restoring microglial function
represents a rational therapeutic approach. Here, we show that systemic
hematopoietic cell transplantation followed by enhancement of microglia
replacement restores microglial function in a Trem2 mutant mouse model of AD.

* AGE-DEPENDENT IMMUNE AND LYMPHATIC RESPONSES AFTER SPINAL CORD INJURY

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Spinal cord injury (SCI) causes lifelong debilitating conditions. Previous
works demonstrated the essential role of the immune system in recovery after
SCI. Here, we explored the temporal changes of the response after SCI in
young and aged mice in order to characterize multiple immune populations
within the mammalian spinal cord. We revealed substantial infiltration of
myeloid cells to the spinal cord in young animals, accompanied by changes in
the activation state of microglia. In contrast, both processes were blunted
in aged mice. Interestingly, we discovered the formation of meningeal
lymphatic structures above the lesion site, and their role has not been
examined after contusive injury. Our transcriptomic data predicted
lymphangiogenic signaling between myeloid cells in the spinal cord and
lymphatic endothelial cells (LECs) in the meninges after SCI. Together, our
findings delineate how aging affects the immune response following SCI and
highlight the participation of the spinal cord meninges in supporting
vascular repair.

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