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Dinkel et al., Sci. Transl. Med. 16, eadl4616 (2024) 4 December 2024
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
1 of 14
NEURODEGENERATION
Myeloid cell–specific loss of NPC1 in mice recapitulates
microgliosis and neurodegeneration in patients with
Niemann- Pick type C disease
Lina Dinkel1†, Selina Hummel2†, Valerio Zenatti1, Mariagiovanna Malara1, Yannik Tillmann1,
Alessio Colombo1, Laura Sebastian Monasor1, Jung H. Suh3, Todd Logan3, Stefan Roth4,
Lars Paeger1, Patricia Hoelner5,6, Oliver Bludau5, Andree Schmidt1,6,7, Stephan A. Müller1,7,
Martina Schierer1,8, Brigitte Nuscher9, Jasenka Rudan Njavro1, Matthias Prestel1,
Laura M. Bartos2, Karin Wind- Mark1,2, Luna Slemann2, Leonie Hoermann2, Sebastian T. Kunte2,
Johannes Gnörich2, Simon Lindner2, Mikael Simons1,4,8,10, Jochen Herms1,8,11, Dominik Paquet4,8,
Stefan F. Lichtenthaler1,7,8, Peter Bartenstein2, Nicolai Franzmeier4,8,12, Arthur Liesz4,8,
Antje Grosche5, Tatiana Bremova- Ertl13,14, Claudia Catarino15, Skadi Beblo16, Caroline Bergner17,
Susanne A. Schneider13, Michael Strupp13, Gilbert Di Paolo3,
Matthias Brendel1,2,8*, Sabina Tahirovic1*
Niemann- Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1
gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration.
Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1
in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we re-
port severe alterations in microglial lipidomic proles, including the enrichment of bis(monoacylglycero)phosphate,
increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hy-
peractivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a path-
ological cascade resembling NPC- like phenotypes, including a shortened life span, motor impairments, astrogliosis,
neuroaxonal pathology, and increased neurolament light chain (NF- L), a neuronal injury biomarker. As observed in
the mouse model, patients with NPC showed increased NF- L in the blood and microglial hyperactivity, as visualized
by TSPO- PET imaging. Reduced TSPO expression in blood- derived macrophages of patients with NPC was measured
after N- acetyl- l- leucine treatment, which has been recently shown to have benecial eects in patients with NPC,
suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results
demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further
investigated for therapeutic targeting and disease monitoring.
INTRODUCTION
Niemann- Pick type C (NPC) disease is an inherited metabolic lyso-
somal storage disorder that manifests with progressive neurodegen-
eration. NPC is caused by autosomal recessive mutations in the NPC1
(95%) or NPC2 (5%) gene. e encoded proteins reside in late endo-
somal (LE)/lysosomal compartments and jointly regulate cholesterol
egress (1). It has been postulated that the small luminal protein NPC2
transfers cholesterol to the transmembrane protein NPC1, which expor ts
cholesterol out of LE/lysosomes (2–4). Disease- causing mutations
in NPC1 lead to disturbances in cellular lipid metabolism, result-
ing in storage of adverse lipids, including unesteried cholesterol or
glycosphingolipids (5–8).
NPC manifests mostly in early childhood with a complex, heterog-
enous, and oen severe clinical phenotype. Neurological symptoms of
NPC include ataxia, dystonia, supranuclear saccade and gaze palsy,
epileptic seizures, and cognitive impairment, leading to premature
death (1,9,10). Pathological hallmarks include neurobrillary tangles,
axonal swellings, neuroinammation, and hypomyelination (1, 11–14).
Molecular mechanisms such as aberrant endolysosomal tracking
and autophagy seem to contribute to NPC pathology (15–19). e
neurodegenerative aberrations are region specic, encompassing vul-
nerable Purkinje cells in the cerebellum and retinal pathology. Retinal
1German Center for Neurodegenerative Diseases (DZNE) Munich, 81377 Munich,
Germany. 2Department of Nuclear Medicine, LMU University Hospital, LMU Munich,
81377 Munich, Germany. 3Denali Therapeutics Inc., South San Francisco, CA 94080, USA.
4Institute for Stroke and Dementia Research (ISD), LMU University Hospital, LMU
Munich, 81377 Munich, Germany. 5Department of Physiological Genomics, Biomed-
ical Center (BMC), Faculty of Medicine, Ludwig- Maximilians- Universität München,
82152 Planegg- Martinsried, Germany. 6Graduate School of Systemic Neurosciences,
Ludwig Maximilian University, 82152 Planegg- Martinsried, Germany. 7Neuropro-
teomics School of Medicine and Health, Klinikum rechts der Isar, Technical Univer-
sity of Munich, 81675 Munich, Germany. 8Munich Cluster for Systems Neurology
(SyNergy), 81377 Munich, Germany. 9Metabolic Biochemistry, Biomedical Center
(BMC), Faculty of Medicine, LMU Munich, 81377 Munich, Germany. 10Institute of
Neuronal Cell Biology (TUM- NZB), Technical University of Munich, 80802 Munich,
Germany. 11Center for Neuropathology and Prion Research, Ludwig- Maximilians-
University München, 81377 Munich, Germany. 12Department of Psychiatry and
Neurochemistry, University of Gothenburg, Sahlgrenska Academy, Institute of
Neuroscience and Physiology, SE- 413 90 Mölndal and Gothenburg, Sweden. 13De-
partment of Neurology, LMU University Hospital, LMU Munich, 81377 Munich,
Germany. 14Department of Neurology, University Hospital Bern, 3010 Bern, Switzer-
land. 15Friedrich Baur Institute, Department of Neurology, LMU University Hospital,
LMU Munich, 80336 Munich, Germany. 16Center for Pediatric Research Leipzig, De-
partment of Women and Child Health, Hospital for Children and Adolescents, Uni-
versity Hospital Leipzig; Leipzig University Center for Rare Diseases, 04103 Leipzig,
Germany. 17Department of Neurology, University Hospital Leipzig, 04103 Leipzig,
Germany.
*Corresponding author. Email: matthias. brendel@ med. uni- muenchen. de (M.B.); sa-
bina. tahirovic@ dzne. de (S.T.)
†These authors contributed equally to this work.
Copyright © 2024 The
Authors, some rights
reserved; exclusive
licensee American
Association for the
Advancement of
Science. No claim to
original U.S.
Government Works
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Dinkel et al., Sci. Transl. Med. 16, eadl4616 (2024) 4 December 2024
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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axonal degeneration has been shown to correlate with motor symp-
toms and disease progression (20,21).
Currently, there is no cure for NPC. In many countries, the gan-
glioside synthesis inhibitor miglustat is the rst approved treatment
with potential to ameliorate disease progression (22). In recent clinical
trials, it was shown that the modied amino acid N- acetyl- - leucine
(NALL) improved clinical signs and symptoms in patients with NPC
(23,24). Besides therapeutics, there is also an unmet need to accel-
erate the diagnosis of NPC and enable the precise monitoring of the
therapeutic benets (25).
Early- onset human NPC disease is well recapitulated in the whole-
body Npc1 knockout mouse model (here abbreviated as Npc1−/−) that
provides an excellent tool for preclinical studies (26,27). is model
displays prominent neuronal pathology, such as axonal spheroids
and region- specic neurodegeneration, particularly evident in the
cerebellum (Purkinje cell loss), thalamus, and retina (21,28–30).
Further pathological hallmarks of NPC are recapitulated in mice,
such as demyelination, and severe microgliosis and astrogliosis, which
are emerging as active players in disease pathology (16,31–34). Ben-
ecial eects of NALL treatment have also been reported in Npc1−/−
mice, including reduced lipid storage and improvements in energy
metabolism and behavioral skills associated with reduced microglial
activation (35).
Cell type–specic depletion of NPC1 and rescue experiments
identied a cell- autonomous role of NPC1 in neurons and oligo-
dendrocytes, whereas its cell- autonomous role in astrocytes is still
debated (36). Our recent work began to address the role of NPC1
in microglia and demonstrated the requirement of NPC1 for en-
dolysosomal lipid tracking. We showed an accumulation of lipid-
enriched substrates such as myelin debris within LE/multivesicular
bodies (MVBs) of Npc1−/− microglia and demonstrated that mi-
croglial molecular and functional alterations occur before neuro-
degeneration (16).
It is now unknown how neuroinammation and microglial malfu nc-
tion contribute to neurodegeneration in NPC. To monitor neuro-
inammation in vivo, translocator protein 18 kDa (TSPO) is commonly
used as a positron emission tomography (PET) ligand for visual-
izing microglial activation (37). Although the mitochondrial pro-
tein TSPO is also expressed by endothelial cells and astrocytes,
upon inammatory and neurodegenerative conditions, it oen be-
comes enriched in microglia, suggesting its suitability for monitor-
ing microglial activation (38,39). Increased neuroinammation in
the white matter of patients with NPC has been detected by TSPO-
PET imaging, but no association with disease severity was ob-
served (13), questioning whether microglial activation contributes
to NPC disease progression.
Here, by exploring a mouse model with a depletion of NPC1
in myeloid cells, we showed that NPC1 regulates microglial lipid
homeostasis and that its loss is sucient to trigger microglial acti-
vation, followed by astrogliosis and neuroaxonal pathology. e
neuroaxonal pathology is also supported by the increase of its
blood surrogate marker neurolament light chain (NF- L) in the
mouse model and in patients with NPC. Our work shows a patho-
logical cross- talk between microglia, astrocytes, and neurons, sup-
porting the contribution of these various brain cells to NPC
disease. We further reveal that microglial dysfunction, recapit-
ulated in blood- derived macrophages of patients with NPC,
was ameliorated after a 6- week treatment with NALL, as deter-
mined by reduced TSPO expression and a trend toward reduced
phagocytosis. ese ndings suggest that microglia actively con-
tribute to NPC disease and that blood- derived macrophages show
similar pathological alterations, oering an ideal tool to monitor
therapeutic interventions.
RESULTS
Loss of NPC1 in myeloid cells compromises endolysosomal
tracking and microglial lipid metabolism
To analyze a cell- autonomous pathomolecular function of NPC1
in microglia, we used our recently generated conditional mouse
model Npc1ox/ox; Cx3cr1Cre (Cre+), which depletes NPC1 in mi-
croglia, and other Cx3cr1- expressing myeloid cells (16,40,41). In
contrast with the whole- body Npc1 knockout (Npc1−/−) that reaches
humane end points at the age of approximately 3 months (42), the
myeloid- specic model reaches humane end points at the age of 11 to
12 months (g. S1, A and B). To characterize a key cellular pheno-
type in NPC, namely, enhanced phagocytosis and impairment in
lipid tracking, we cultured Cre− and Cre+ microglia in the pres-
ence of pHrodo- labeled myelin. Cre+ microglia displayed signi-
cantly (P < 0.001) enhanced phagocytic uptake of myelin (Fig. 1, A
and B). Cre− microglia were able to process myelin into lipid drop-
lets, as judged by positive staining for neutral lipids (Nile Red) at
the cell periphery (Fig. 1, C and D). In contrast, lipid droplets were
almost completely absent at the cell periphery of Cre+ microglia.
Electron microscopy conrmed impairments in myelin turnover
into lipid droplets and its accumulation in LE/MVBs in Cre+ mi-
croglia (Fig. 1E). e lipid accumulation within LE/MVBs was
further corroborated by the nding that the number of lysosomes
was decreased in Cre+ microglia under this myelin- fed condition
(Fig. 1, E and F).
Previous work has demonstrated altered lipidomic proles in
NPC at the level of the whole brain (43), but lipid proles were not
analyzed in specic brain cell types. To characterize the lipid pro-
le of microglia from Cre− and Cre+ mice at early stages (2 months)
of disease pathology, we performed a lipidomic analysis using
liquid chromatography–mass spectrometry (LC- MS) (table S1).
We detected increased cholesterol and decreased cholesteryl esters
(CEs) in Cre+ microglia (Fig. 1, G to I), in accordance with the
absence of Nile Red–positive lipid droplets observed in vitro (Fig.
1, C and D). Multiple bis(monoacylglycero)phosphate (BMP) spe-
cies, which are lipids enriched in MVBs and linked to cholesterol
dyshomeostasis in NPC (44–46), were prominently increased in
Cre+ microglia (Fig. 1, G and H). Further, we detected ceramide
derivates such as lactosylceramide, myelin- enriched lipids like sul-
fatides, and gangliosides among the most up- regulated lipids in
Cre+ microglia (Fig. 1, G and H). In addition to CEs, we detected
reduced coenzyme Q10, phospholipids, cholesterol sulphate, and
di- and triglycerides (Fig. 1, G and I). Signicantly increased cho-
lesterol (P < 0.05) and a trend toward an increase in GM2 were
also quantied by immunocytochemistry of cultured Cre+ microg-
lia (Fig. 1, J and K).
To estimate the potential contribution of the compromised brain
environment in NPC to the lipidomic prole of microglia, we per-
formed a lipidomic analysis in Npc1−/− mice at 2 months of age,
corresponding to a late pathological stage with severe neurode-
generation (fig. S2A and table S2). The lipidomic profile of
Npc1−/− microglia and Cre+ microglia showed a high overlap, in-
cluding an increase in cholesterol and BMP species (g. S2, A to C).
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Dinkel et al., Sci. Transl. Med. 16, eadl4616 (2024) 4 December 2024
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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We further investigated the lipid prole of microglia from early
pathological stages (1 week) of Npc1−/− mice, which resembled
the lipidomic prole of Npc1−/− microglia at late pathological stages,
underscoring increased cholesterol and BMP species and decreased
CE (g. S2D and table S3). is is in line with an increased LE/
MVB area, whereas the number of lysosomes was not changed (g.
S2, E to G). Several CE species showed the highest reduction
at the early pathological stage, suggesting that CEs were more
abundant during development. Our results demonstrate that lipid
changes, as well as enrichment of LE/MVBs in microglia, occur
very early in NPC disease and are driven by the cell- autonomous
function of NPC1.
TSPO- PET imaging reveals strong microglial activation in
mice lacking NPC1 in myeloid cells
Next, we investigated whether lipid tracking defects are associated
with microglial activation and neuroinammation. To visualize
microglial activation, we performed TSPO- PET imaging at early
(2 months), intermediate (7 months), and late (11 months) stages of
disease pathology in Cre− and Cre+ mice. We detected an increase in
Cre
-
Cre
+
0
20
40
60
80
100
Lipid droplet
-
positive cells (%)
GM2 Filipin
ABC
D
CD68 GM2 filipin
Cre+
Cre
-
1200× 5000×
G
J
CD68
Cre+
Cre
-
H
Coenzyme Q10CE(18:1)
CholesterolBMP(18:1/18:1
)
LacCer(d18:1/18:0) GM2(d38:1)
PS(16:0_22:6) TG(18:0_36:2)
I
E
Cre
-
Cre+
Myelin
F
Cre
-
+ CD
Cre+ + CD
K
Cre
-
Cre+
CD
--
++
Myelin
Nile Red
CD68
my.Nile CD68 Red
Cre+
Cre
-
H
**
*
**
*
Cre
-
Cre
+
0.00
0.02
0.04
0.06
0.08
0.10
Lysosomes perµm²
0
100
200
300
Mean intensityper cell (%)
Cre
-
Cre+
0
1
2
3
4
5
Cre
-
Cre+
0.0
0.5
1.0
1.5
2.0
2.5
Cre
-
Cre+
0
1
2
3
4
5
Cre
-
Cre+
-
10
-
8
-
6
-
4
-
2
0
Cre
-
Cre
+
-
6
-
4
-
2
0
2
Cre
-
Cre
+
0
1
2
3
4
Cre
-
Cre
+
-
5
-
4
-
3
-
2
-
1
0
Cre
-
Cre
+
-
4
-
2
0
2
4
RUV-adjusted area ratios (log2)
RUV-adjusted area ratios (log2)
FilipinGM2
0
2000
4000
6000
Mean intensitypercell(a.u.)
Cre
-
Cre
+
ns
Down
Up
Stable
Fig. 1. Loss of NPC1 impairs myelin tracking
and lipid homeostasis in microglia. (A) Analysis
of phagocytic uptake of pHrodo- labeled myelin
in cultured Cre− (n = 3) and Cre+ (n = 3) microglia.
Cytochalasin D (CD) was used as a negative control.
Scale bars, 200 μm. (B) Quantication of the uo-
rescent mean intensity of pHrodo 6 hours after feed-
ing. Values were normalized to Cre− controls and
are depicted as means ± SEM, and a one- way analysi s
of variance (ANOVA) with Tukey’s multiple compari-
son test was performed (***P < 0.001). (C) Immu-
nocytochemical analysis of myelin process ing in
cultured Cre− and Cre+ microglia. Microglia were
fed with uorescently labeled myelin (my., green), and
the presence of lipid droplets (Nile Red, magenta)
was analyzed after 48 hours. Cells were immunos-
tained using CD68 (white). A representative microg-
lial cell is depicted, and one lipid droplet is marked
with an arrow. Hoechst (H) was used as the nuclear
stain (cyan). Scale bars, 10 μm. (D) Quantication of
lipid droplet–positive cells in myelin- fed Cre− (n =
3) and Cre+ (n = 3) microglia. Data are presented as
means ± SEM. A two- tailed unpaired Student’s t test
was performed (***P < 0.001). (E) Representative
electron microscopy images of myelin- fed Cre−
and Cre+ microglia 48 hours after feeding. Myelin
accumulation is shown by yellow asterisks, and
lysosomes are indicated by red arrows. Dierent
identities of lipid droplets are visualized by blue
and green arrows. Scale bars, 2 μm (1200×) and 1 μm
(5000×). (F) Quantication of lysosomal number
in myelin- fed Cre+ (n = 3) and Cre− microglia (n =
3). Data are presented as means ± SEM, and a two-
tailed unpaired Student’s t test was performed (*P <
0.05). (G) Lipidomic analysis of acutely isolated
microglia from 2- month- old Cre− (n = 5) and Cre+
mice (n = 5). Signicantly (adjusted P value < 0.05)
increased (up) or decreased (down) lipids are de-
picted in red and blue, respectively. Lipids that are
not signicantly changed (adjusted P value > 0.05)
are depicted in gray (stable). The dotted line repre-
sents a P value of 0.05. (H and I) Selected up-
regulated (H) and down- regulated (I) lipids from
the lipidomic analysis of Cre− and Cre+ microglia.
The log2- transformed RUV (removal of unwanted
variables)–adjusted area ratios are depicted as
means ± SEM (the nonadjusted P value is depicted,
*P < 0.05, **P < 0.01, ***P < 0.001). (J) Cultured
Cre− (n = 3) and Cre+ (n = 3) microglia isolated from 2- month- old mice were immunostained against CD68 (magenta) and GM2 lipid (green). Cholesterol was stained using
lipin (cyan). Scale bars, 10 μm. (K) Quantication of the uorescent mean intensity [arbitrary unit (a.u.)] of lipin and GM2 was measured using ImageJ and is represented
as means ± SEM. A two- tailed unpaired Student’s t test was performed for each lipid (ns, P > 0.05; *P < 0.05).
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TSPO- PET signals in the midbrains and brainstems of Cre+ mice at
intermediate disease stages (7 months) and across all analyzed brain
regions at late stages of disease pathology (Fig. 2, A and B). A com-
parison of TSPO- PET signals in Cre+ and Cre− mice revealed the
midbrain and brainstem as hotspot regions of microglial activation
upon loss of NPC1 (Fig. 2C). To corroborate the microglial contribu-
tion to increased TSPO- PET signals, we used our recently established
single- cell radiotracing technology (scRadiotracing) (47). Cre− mice
showed enriched TSPO tracer uptake in microglia compared with
astrocytes (Fig. 2D), arguing for microglia- specic uptake of the
TSPO- PET tracer. Moreover, microglia isolated from the Cre+ mice
showed a 1.7- fold increased TSPO tracer uptake per cell compared
with the Cre− mice (Fig. 2D), implying that the increased TSPO- PET
imaging signals not only were a consequence of microgliosis but also
indicated higher TSPO per cell. ese results were further supported
by immunohistochemical analysis, revealing a signicantly (P <
0.001) increased expression of TSPO in Iba1- positive microglia of
Cre+ mice at the age of 12 months that was not observed at 2 months
(Fig. 2, E to H). In contrast to microglia, we did not detect changes in
TSPO expression in astrocytes (g. S3, A and B).
Next, we asked whether microglial activation occurs also at ear-
lier time points by performing time- dependent immunohistochem-
ical analysis using microglial LE/lysosomal marker CD68. ese
experiments determined a signicantly (P < 0.001) increased CD68
coverage in Cre+ mice already at an early stage (Fig. 2I and g. S4, A
and B), which was further enhanced at intermediate and late patho-
logical stages in a region- specic manner, with the midbrain and
brainstem being most aected (Fig. 2J and g. S4, C to F). Similar
time- and region- dependent phenotypic changes in microglia were
also evident by increased Iba1 coverage in Cre+ mice (g. S4, G and
H). Along with enhanced microglial activity, phagocytic cups were
prominent in Cre+ mice at late stages, providing in vivo evidence of
increased phagocytosis (g. S4, I and J). us, microglial activation
and alterations in the endolysosomal pathway can be captured in
Cre+ mice already at early pathological stages, whereas alterations of
TSPO become evident at intermediate and late stages of pathology.
Observed regional vulnerabilities in the Cre+ mice dier from the
Npc1−/− mice, which showed the highest CD68 immunoreactivity in
the cerebellum (g. S5), with a well- described neurodegeneration of
Purkinje cells (29).
To further study the dierential vulnerability, we compared the
cerebellar with the cerebral (brain without cerebellum) proteome
in Cre− and Cre+ mice at late pathological stages. e number of
false discovery rate (FDR)–adjusted signicantly altered proteins
was higher in the cerebrum (273 proteins) compared with the
cerebellum (36 proteins) (g. S6A and table S4). Both proteomes
included an enrichment of complement factors (C1QA, C1QB,
C1QC, and C4B), lysosomal proteins (HEXA, HEXB, MAN2B1,
CTSZ, CTSH, and NAGA), disease- associated microglia (DAM)
proteins (LGALS3, APOE, and GRN), and astrocytic glial brillary
acidic protein (GFAP) in Cre+ mice. e microglial homeostatic
protein P2RY12, together with proteins associated with neuro-
nal function (SLC6A5, SYT2, and KCNC3), were significantly
decreased (FDR- adjusted P < 0.05) in the Cre+ cerebral proteome
(g. S6A). e underlying altered molecular pathways are involved
in regulation of immune responses and development and prolif-
eration of glial cells (g. S6B). ese results suggest that microglial
loss of NPC1 has profound eects on brain cell homeostasis espe-
cially in the cerebrum.
Microglia- to- astrocyte propagation of pathological insults
in mice lacking NPC1 in myeloid cells
Prominent microglial pathology upon loss of NPC1 may have direct
consequences on the homeostasis of other brain cells. Microglia- to-
astrocyte propagation of pathological insults is a well- supported
phenomenon (48), and the brain proteome of Cre+ mice revealed
phenotypic changes in astrocytes (g. S6, A and B). To track poten-
tial changes in astrocytes in vivo, we explored monoamine oxidase B
(MAOB)–PET as an astrocyte imaging tool using the tracer [18F]
uorodeprenyl- D2 (49). We observed an increase in the MAOB-
PET signal in all analyzed brain regions at intermediate and late
pathological stages in Cre+ mice (Fig. 3, A and B), with the midbrain
and brainstem being the hotspot regions of MAOB- positive astro-
cytic reactivity (Fig. 3C).
e regional overlap of the highest TSPO- PET (Fig. 2C) and
MAOB- PET signal (Fig. 3C) suggests that astrocyte reactivity
may be a response to pathological alterations in microglia. Using
scRadiotracing technology, we conrmed that astrocytes are a ma-
jor cellular source of the increased MAOB- PET signal (Fig. 3D). In
contrast to microglia, cellular uptake of the MAOB radiotracer was
comparable in the astrocyte- enriched fraction from Cre− and Cre+
mice (Fig. 3D), suggesting that the increased MAOB- PET signal is
a consequence of astrogliosis. e astrocytic response was further
veried by Western blot and immunohistochemical analyses of GFAP,
a common astrocytic marker (50). Western blot analysis revealed a
trend toward increased GFAP immunoreactivity in Cre+ mice al-
ready at early pathological stages, and signicantly increased GFAP
was detected at intermediate (P < 0.05) and late (P < 0.01) patho-
logical stages (Fig. 3, E and F). Similar results were also obtained
by immunohistochemical analysis (Fig. 3, G and H). Despite the
prominent alterations in microglia and astrocytes, we did not de-
tect any obvious dierence in myelination at late pathological stages
in Cre+ mice by looking at the oligodendrocyte marker 2ʹ, 3ʹ -cyclic
nucleotide phosphodiesterase (CNPase) (g. S7), suggesting that
microglial NPC1 depletion is not a major driver ʹof myelination de-
fects observed in NPC (16).
Region- specic neurodegeneration in mice lacking NPC1 in
myeloid cells
Neurodegeneration in NPC is best illustrated by the loss of Purkinje cells
in the cerebellum as well as retinal degeneration (21,29). To determine
the impact of myeloid cell–specic loss of NPC1 on neuronal function,
we rst analyzed the morphology of the Purkinje cell layer by immuno-
histochemistry using the marker calbindin. We revealed an intact
Purkinje cell layer even at late pathological stages in Cre+ mice (Fig. 4A).
Total dendrite length of Purkinje cells and branching pattern quantied
by Sholl analysis showed no dierences between Cre− and Cre+ mice (Fig.
4, B to E). is result supports the hypothesis that neurodegeneration of
Purkinje cells observed in Npc1−/− mice is likely driven by the loss of the
neuronal (40) rather than microglial NPC1 function. Next, we examined
neurodegeneration in the well- stratied retinal tissue. Although reti-
nal layer thickness was not signicantly altered at late pathological
stages in Cre+ mice (Fig. 4, F and G), we observed a trend toward a
reduction in calretinin- positive neurons and a signicantly (P < 0.01)
reduced number of protein kinase Cα–positive neuronal popula tions
in the inner nuclear layer (Fig. 4H). Similar to other analyzed brain
regions, the retina had increased CD68 immunoreactivity (g. S8A).
To characterize neuronal function across dierent pathological
stages and brain regions, we determined glucose uptake by applying
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Cre
-
Cre+
0
20
40
60
80
TSPO perIba1-positive cell (µm³)
ns
Cre
-
Cre
+
0
100
200
300
400
500
TSPO perIba1-positive cell(µm³)
0
1×10
-
6
2×10
-
6
3×10
-
6
Cellular TSPO tracer uptake
(%ID * weight/cell)
Cre+
Cre
-
Microglia-
enriched
fraction
Astrocyte-
enriched
fraction
ns
F
CD68
Cre
-
12
1
2
Cre
+
34
43
Ctx Ctx
bs bs
123
4
Cre
+
CD68
Cre
-
Ctx Ctx
bs bs
12 43
A
Cre+
Cre
-
)VUS( TEP-OPST
1.0
shtnom 2
7 months11 months
0.1
B
C
11 months
DE
TSPO-PET (SUV)
2 months
Midbrain
GH
12 months
Midbrain
Iba1TSPO
Cre
-
Cre+
IJ
2 months 12 months
Iba1TSPO
Cre
-
Cre+
2 months 12 months
TSPO Iba1
TSPO Iba1
0% 80%
Cre+ vs. Cre
-
TSPO-PET (SUV)
Cortex Midbrain Brainstem Cerebellum
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Age (months)
G*A,T = 1.962
ns
2711
ns
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Age (months)
G*A,T = 2.001
ns
2711
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Age (months)
G*A,T = 1.894
ns
2711
ns
ns Cre+
Cre
-
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Age (months)
G*A,T = 2.574
P = 0.0134
2711
ns
Fig. 2. TSPO- PET reveals region- specic microglial hyperactivity. (A) TSPO- PET images of Cre− and Cre+ mouse brains imaged at 2, 7, and 11 months of age. The shown sagit-
tal brain sections represent group average images of the standardized uptake value (SUV) projected on a standard MRI T1w atlas. (B) Quantication of individual TSPO- PET SUVs
derived from the cortex, midbrain, brainstem, and cerebellum. Dotted lines represent linear associations between age and TSPO- PET quantication with their 95% condence
interval (lled area). The genotype × age (G * A) interaction was calculated. A multiple linear regression was performed for statistical analysis with the following sample sizes:
Cre− n = 8 and Cre+ n = 5 for 2 months, Cre− n = 10 and Cre+ n = 8 for 7 months, and Cre− n = 12 and Cre+ n = 6 for 11 months (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P
< 0.001). (C) Percentage dierence in TSPO- PET (SUV) between Cre+ and Cre− mice at 11 months of age. (D) Quantication of cellular TSPO tracer uptake in microglia- and
astrocyte- enriched fractions of 12- month- old mice by scRadiotracing. Data are presented as means ± SEM, and a one- way ANOVA with Tukey’s multiple comparison test was
performed (ns, P > 0.05; **P < 0.01; ***P < 0.001) with the following sample sizes: Cre− n = 8 and Cre+ n = 6 (microglia- enriched fraction), and Cre− n = 8 and Cre+ n = 5 (astrocyte-
enriched fraction). (E) Immunohistochemical analysis of TSPO (green) in Iba1- positive cells (magenta) in the midbrains of Cre− and Cre+ mice at 2 months of age. Scale bars,
50 μm. (F) Quantication of TSPO volume per Iba1- positive cell in Cre− (n = 3) and Cre+ (n = 3) mice at 2 months of age. Data are presented as means ± SEM, and a two- tailed
unpaired Student’s t test was performed (ns, P > 0.05). (G) Immunohistochemical analysis of TSPO (green) and Iba1 (magenta) in the midbrains of Cre− and Cre+ mice at 12 months
of age. Scale bars, 50 μm. (H) Quantication of TSPO volume per Iba1- positive cell of Cre− (n = 3) and Cre+ (n = 3) mice at 12 months of age. Data are presented as means ± SEM,
and a two- tailed unpaired Student’s t test was performed (***P < 0.001). (I and J) Sagittal brain sections of 2- (I) and 12 (J)- month- old Cre− and Cre+ mice were immunostained
against CD68 (white). Numbers 1 to 4 represent zoom- ins from the respective tile scans. Scale bars, 2000 μm (tile scans) and 500 μm (zoom- ins). Ctx, cortex; bs, brainstem.
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6 of 14
2- deoxy- 2- [18F]uoro- D- glucose (FDG)–PET imaging. Reduced glu-
cose metabolism is used as a surrogate marker of neuronal dysfunction
and neurodegeneration (51). e FDG- PET signal was signicantly
(P < 0.05) reduced at intermediate (cortex and midbrain) or late (brain-
stem) pathological stages in Cre+ mice (Fig. 5, A and B), illustrating a
widespread neuronal dysfunction upon loss of NPC1 in myeloid cells.
Next, we assessed NF- L in the serum, a well- accepted biomarker for
neuroaxonal injury (52,53). In line with the reduced FDG- PET, we
detected signicantly increased NF- L in sera at intermediate (P < 0.01)
and late (P < 0.001) pathological stages in Cre+ mice (Fig. 5C). Im-
munohistochemical analysis of neurolament heavy chain (NF200)
revealed a signicant (P < 0.001) accumulation at intermediate patho-
logical stages in the midbrains and brainstems of Cre+ mice (Fig. 5, D
and E), which indicates axonal swelling, a well- described neuronal pa-
thology in NPC disease (31,34). Neuronal defects were further sup-
ported by accumulation of the autophagy marker p62 in NeuN- positive
cells in the cortex, midbrain, and brainstem (Fig. 5, F and G), which
was not detected in the cerebellum (g. S8B). To test whether defects
caused by activated microglia lacking NPC1 have consequences on be-
havior, we assessed motor performance using a rotarod test. Cre+ mice
showed reduced latency to fall at intermediate pathological stages
compared with Cre− mice (Fig. 5H).
0
2×10
-
10
4×10
-
10
6×10
-
10
8×10
-
10
Cellular MAOB tracer uptake
(%ID * weight/cell)
Cre+
Cre
-
ns
ns
ns
Astrocyte-
enriched
fraction
Astrocyte-
depleted
fraction
0
2
4
6
8
10
GFAP fold change
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Age (months)
MAOB-PET (SUV)
G * A
T = 1.897
ns
2711
ns
A
D
MAOB-PET (SUV)
1.0
0.1
Cre+
Cre
-
shtnom 2
7 months
11 months
Cortex Midbrain Brainstem
B
C
0% 80%MAOB-PET (SUV)
Cre+ vs. Cre
-
11 months
E
F
GFAP
50 kDa -
37 kDa -
Cre
-
Cre+
37 kDa - GAPDH
GFAP
GAPDH
GFAP
GAPDH
50 kDa -
37 kDa -
37 kDa -
shtnom 2
shtnom 7
shtnom 21
2 months
50 kDa -
37 kDa -
37 kDa -
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Age (months)
G * A
T = 3.038
P = 0.005
2711
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Age (months)
G * A
T = 2.227
P = 0.03
2711
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Age (months)
G * A
T = 1.628
ns
2711
ns
Cerebellum
Cre+
Cre
-
G
Cre
-
Cre+
GFAP
H
CortexMidbrainBrainstem
7 months
Cre
-
Cre+
12 months
Cre
-
Cre+
2 months 7 months 12 months
Cortex Midbrain Brainstem
0.0
0.2
0.4
0.6
0.8
1.0
1.2
GFAP coverage (%)
ns
ns
Cortex Midbrain Brainstem
0
2
4
6
8
10
12
GFAP coverage (%)
Cortex Midbrain Brainstem
0
5
10
15
20
GFAP coverage (%)
Fig. 3. NPC1- decient microglia cause astro-
gliosis. (A) MAOB- PET images of Cre− and Cre+
mouse brains at 2, 7, and 11 months of age. The
shown sagittal brain sections represent group
average images of SUVs projected on a stan-
dard MRI T1w atlas. (B) Quantication of indi-
vidual MAOB- PE T SUVs derived from the cortex,
midbrain, brainstem, and cerebellum. Dotted
lines represent linear associations between age
and MAOB- PET quantication with their 95%
condence interval (lled area). The genotype ×
age (G * A) interaction was calculated. A multi-
ple linear regression was performed for statisti-
cal analysis with the following sample sizes for
the cortex, midbrain, and brainstem: Cre− n = 5
and Cre+ n = 4 for 2 months, Cre− n = 5 and Cre+
n = 6 for 7 months, and Cre− n = 6 and Cre+ n =
5 for 11 months. For the cerebellum, the sample
sizes were Cre− n = 5 and Cre+ n = 4 for 2 months,
Cre− n = 6 and Cre+ n = 6 for 7 months, and Cre− n =
5 and Cre+ n = 5 for 11 months (ns, P > 0.05; *P <
0.05; **P < 0.01; ***P < 0.001). (C) Percentage
dierence in MAOB- PET (SUVs) between Cre−
and Cre+ mice at 11 months of age. (D) Quanti-
cation of cellular MAOB- PET tracer uptake in
astrocyte- enriched and astrocyte- depleted frac-
tions of 10- month- old mice by scRadiotracing.
A one- way ANOVA with Tukey’s multiple com-
parison test was performed with n = 4 for Cre−
and Cre+ samples (ns, P > 0.05; **P < 0.01).
(E) Western blot showing the expression of GFAP
in Cre− (n = 3) and Cre+ (n = 3) mouse brain
tissue harvested at 2, 7, and 11 months of
age. GAPDH (glyceraldehyde- 3- phosphate de-
hydrogenase) was used as a loading control.
(F) Fold change of GFAP quantied by densitom-
etry (MultiGauge). Values were normalized to
Cre− and are depicted as means ± SEM. A two-
tailed unpaired Student’s t test was performed
for each age (ns, P > 0.05; *P < 0.05; **P < 0.01).
(G) Immunohistochemical analysis of GFAP in
the cortices, midbrains, and brainstems of 2- ,
7- , and 12- month- old Cre− and Cre+ mice. Scale
bars, 100 μm. (H) Quantication of GFAP cover-
age in the cortices, midbrains, and brainstems
of Cre− (n = 3) and Cre+ (n = 3) mice at 2, 7, and
12 months of age. Data are presented as means
± SEM, and a two- way ANOVA with Šidák’s mul-
tiple comparisons test was performed (ns, P >
0.05; **P < 0.01; ***P < 0.001).
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7 of 14
Human iPSC- derived NPC1- decient microglia display
increased phagocytic function but are not neurotoxic
We have shown that loss of NPC1 in myeloid cells is sucient to
cause neuronal pathology in the brain, but whether microglia can
directly trigger neurodegeneration remains unclear. erefore, us-
ing CRISPR- Cas9, we generated a human- induced pluripotent stem
cell (hiPSC) line carrying the most common patient NPC1 mutation
I1061T (g. S9A) and established cocultures of microglia carrying
the NPC1 mutation (iMG NPC) and hiPSC- derived wild- type neu-
rons (iN WT). Successful generation of iN [70 days in vitro (DIV)]
and iMG (28 DIV) was monitored by the reduction in pluripotency
(g. S9B) and induction of neuronal (g. S9C) and microglial markers
(g. S9D), respectively. iMG NPC (28 DIV) showed no dierence in
NPC1 mRNA compared to iMG WT (g. S9E), but substantial re-
duction in NPC1 protein (g. S9, F and G) was reported for this
mutation (54). As shown for Cre+ microglia, iMG NPC (16 DIV)
displayed increased phagocytic uptake of myelin (g. S9, H and I).
To test whether iMG NPC are neurotoxic, we rst dierentiated
iMG and iN in monoculture for 16 and 60 DIV, respectively, and
then cocultured either iMG NPC or iMG WT with iN WT for an
additional 8 days and monitored their survival by quantifying the
neuronal marker MAP2. No obvious dierences were observed in
MAP2 coverage, number of MAP2- positive cells, or MAP2 protein
in cocultures of iN WT and iMG NPC compared to iMG WT (g. S9,
J to M). is result implies that more complex neuronal- glial inter-
actions and long- term microglial deficiency in vivo may be re-
sponsible for neuronal defects rather than a direct microglia- driven
neurotoxicity.
Emerging clinical tools for NPC disease monitoring
As a proof of concept that microglial activation is of relevance for
human NPC pathology, we performed TSPO- PET imaging (37) in
0
40
80
120
160
200
240
280
0
10
20
30
40
50
60
Radius (µm)
#Intersections
Shollintersections
Cre
-
Cre+
Cre
-
Cre
+
0
50
100
150
200
250
Retinal thickness (µm)
ns
Cre
-
Cre+
0
2000
4000
6000
8000
10,000
Area under the curve
ns
GCL INLINL
Calretinin
+
PKC
+
Cre
-
Hoechst calbindinCalbindin
AC
12 months
Cre+
BDE
FGH
Retinal section (12 months)
Calretinin PKC
GCL
INL
ONL
IPL
OPL
Calretinin
Cre
-
PKCDAPI Calretinin PKC
GCL
INL
ONL
IPL
OPL
Cre+
Calretinin
PKC
DAPI
ILM
OLM
ILM
OLM
0
10
20
30
Cellcountper scanfield
ns ns
Cre
-
Cre+
0
2000
4000
6000
8000
10,000
Total dendritelength(µm)
ns
Cre
-
Cre+
Fig. 4. NPC1- decient microglia drive region- specic neurodegeneration. (A) Immunohistochemical analysis of Purkinje cells (calbindin, green) in 12- month- old Cre−
and Cre+ mice. Hoechst was used as the nuclear stain (cyan). Scale bars, 500 μm. (B to E) Morphological characterization of Purkinje cells in 7- month- old Cre− and Cre+
mice. Three- dimensional reconstructions of single Purkinje cells from Cre− and Cre+ mice and after biocytin- backll and subsequent labeling with streptavidin–Alexa
Fluor 647 (red insets). Scale bars, 30 μm (B). Analysis of the total length of the dendritic tree of Purkinje cells from Cre− (n = 4 cells from two mice) and Cre+ (n = 4 cells from
one mouse) animals. Data are presented as means ± SEM, and a two- tailed unpaired Student’s t test was performed (ns, P > 0.05) (C). Sholl analysis depicting the mean
number of intersections along the distance to the soma (D). Analysis of the area under the curve obtained from (D). Data are presented as means ± SEM, and a two- tailed
unpaired Student’s t test was performed (ns, P > 0.05) (E). (F to H) Analysis of retinal morphology in 12- month- old Cre− and Cre+ mice. Retinal sections were stained for
calretinin (magenta) and PKCα (green). DAPI was used as the nuclear stain (cyan). Grayscale images represent single channels for calretinin and PKCα. The dashed line
represents the borders of the ILM and OLM. Scale bars, 20 μm (F). The retinal thickness from ILM to OLM (G) or the number of calretinin- or PKCα- positive cells (H) was
quantied. Data are presented as means ± SEM, and simple nonparametric, unpaired Mann- Whitney t test was performed with the following sample sizes: n = 5 for Cre−
samples, and n = 7 for Cre+ samples (ns, P > 0.05; **P < 0.01). ILM, internal limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer;
OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane.
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8 of 14
7 months
Midbrain
A
1.7
FDG-PET (SUV)
Cre
+
Cre
-
shtnom 2
7 months
11 months
0.4
B
Cortex Midbrain Brainstem
C
Cre
-
Cre+
NF200
Cortex Brainstem
F
Cerebellum
DE
GH
Cortex
p62 NeuN p62 NeuN
Cre
-
Cre+
7 months
Midbrain Brainstem
p62 NeuN p62 NeuN p62 NeuN p62 NeuN
0
500
1000
1500
2000
NF-L in serum(pg/ml)
Cre
-
Cre+
ns
ns
2months7months 12 months
Cortex Midbrain Brainstem
0
10
20
30
40
NF200aggregates perimage
Cre
-
Cre
+
ns
ns
Cre
-
Cre+
0
50
100
150
200
250
Latency tofall(s)
0.0
0.4
0.8
1.2
1.6
2.0
2.4
Age (months)
FDG-PET (SUV)
G * A
T = 2.362
P = 0.02
2711
ns
Age (months)
G * A
T = 2.522
P = 0.02
2711
ns ns
Age (months)
G * A
T = 2.250
P = 0.03
2711
ns ns
Age (months)
G * A
T = 1.674
ns
2711
ns
ns
Cortex Midbrain Brainstem
0
20
40
60
80
100
p62/NeuN-positivecells (%)
Cre
-
Cre+
ns
0.0
0.4
0.8
1.2
1.6
2.0
2.4
0.0
0.4
0.8
1.2
1.6
2.0
2.4
0.0
0.4
0.8
1.2
1.6
2.0
2.4
Fig. 5. NPC1- decient microglia drive neuronal dysfunction. (A) FDG- PET images of Cre− and Cre+ mouse brains imaged at 2, 7, and 11 months of age. The shown
sagittal brain sections display group average images of SUVs projected on a standard MRI T1w atlas. (B) Quantication of individual FDG- PET SUVs derived from the cortex,
midbrain, brainstem, and cerebellum. Dotted lines represent linear associations between age and FDG- PET quantication with their 95% condence interval (lled area).
The G * A interaction was calculated. A multiple linear regression was performed for statistical analysis with the following sample sizes: Cre− n = 13, Cre+ n = 5 for 2
months, Cre− n = 13, Cre+ n = 7 for 7 months, and Cre− n = 8, Cre+ n = 5 for 11 months (ns, P > 0.05; *P < 0.05). (C) NF- L enzyme- linked immunosorbent assay (ELISA) of
blood sera from 2- , 7- , and 12- month- old Cre− (n = 4) and Cre+ (n = 4) mice. Data are presented as means ± SEM, and a two- way ANOVA with Bonferroni’s multiple com-
parison correction was performed (ns, P > 0.05; **P < 0.01; ***P < 0.001). (D) Immunohistochemical analysis using an antibody against NF200 (white) at 7 months of age.
NF200 aggregates in Cre+ mice are marked with magenta arrows. Scale bars, 50 μm. (E) Quantication of NF200 aggregates in Cre− (n = 3) and Cre+ (n = 3) mice in the
cortices, midbrains, and brainstems at 7 months of age. Data are presented as means ± SEM, and a two- way ANOVA with Tukey’s multiple comparisons test was performed
(ns, P > 0.05; **P < 0.01; ***P < 0.001). (F) Immunohistochemical analysis using an antibody against p62 (green) and the pan–neuronal marker NeuN (magenta) in Cre−
and Cre+ mice at 7 months of age. Scale bars, 50 μm. (G) Quantication of p62/NeuN- positive cells relative to the total number of NeuN- positive cells in the cortices,
midbrains, and brainstems at 7 months of age. Data are presented as means ± SEM, and a two- way ANOVA with Tukey’s multiple comparisons test was performed (ns,
P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). (H) Motor function and coordination of 7- month- old Cre− (n = 20) and Cre+ (n = 17) mice were tested using the rotarod. Data
are presented as means ± SEM, and a two- tailed unpaired Student’s t test was performed (***P < 0.001).
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9 of 14
ve patients with NPC. We detected a profound up- regulation of the
TSPO- PET signal across dierent brain regions with pronounced
signal elevations in the insula, brainstem, and cerebellum (Fig. 6, A
and B). Moreover, these regional TSPO- PET signals were higher in
patients with NPC compared with patients with Alzheimer’s disease
(AD) or 4- repeat tauopathies, supporting its utility as a clinical tool
for NPC (Fig. 6B).
Next, we explored the translational potential of peripheral myeloid
cells for NPC disease monitoring. We showed that rodent Npc1−/−
bone marrow–derived macrophages recapitulate major hallmarks of
NPC1- decient microglia, including cholesterol accumulation, up-
regulation of intraluminal vesicle (ILV)–resident CD63, increased
phagocytic uptake, lipid tracking defects, and alterations in choles-
terol metabolism (g. S10, A to H, and table S5). is is in line with
our previous characterization of blood- derived macrophages in pa-
tients with NPC that also recapitulated functional hallmarks of NPC
(16). us, blood- derived macrophages may provide a potential clini-
cal tool for therapeutic monitoring. Preclinical studies suggested that
NALL treatment of Npc1−/− mice ameliorates disease progression and
is associated with reduced microglial activation (35). NALL treatment
showed benecial eects in an NPC clinical trial (24), which prompted
us to test whether blood- derived macrophages of patients with NPC
may be altered upon NALL treatment. To this end, we performed an
ex vivo assay for Aβ plaque clearance—a surrogate marker to measure
increased phagocytosis in NPC (16). A 6- week treatment of patients
with NALL displayed a trend toward reduced phagocytic hyperactiv-
ity in all three longitudinally analyzed patients (Fig. 6C). Subsequently,
we compared the proteomic signatures of patient macrophages before
and aer a 6- week treatment with NALL (table S6). We measured a
35% reduction in TSPO in blood macrophages aer treatment of pa-
tients with NALL (Fig. 6D), pointing to a benecial eect of the drug
on ameliorating hyperactivation of myeloid cells. Collectively, these
data underscore the importance of brain microglia and peripheral
myeloid cells in the course of NPC disease and document a potential
for TSPO as a monitoring biomarker in clinical trials.
Next, we explored the potential of blood- based markers that
show promise in other neurodegenerative diseases, namely, the neu-
ronal injury markers NF- L and ubiquitin C- terminal hydrolase L1
(UCH- L1) and the astrocytic marker GFAP (55,56). Plasma concen-
trations of NF- L, UCH- L1, and GFAP were signicantly (P < 0.05)
higher in patients with NPC compared with their age- matched controls
(Fig. 6E), favoring their potential as tools for disease monitoring.
In summary, we envisage the utility of markers capable of capturing
microglial (TSPO), astrocytic (GFAP), and neuronal (NF- L and UCH-
L1) pathology as a promising avenue to explore in NPC. It remains to
be tested whether concentrations of NF- L, UCH- L1, and GFAP may be
sensitive to treatment, as we could show for TSPO, underscoring their
potential for disease monitoring in future clinical trials.
DISCUSSION
Microglia are strongly engaged in NPC pathology, and their altera-
tions occur before neurodegeneration, suggesting that they may
play an active role during disease progression (16,31, 32). We ex-
plored a myeloid NPC1 depletion mouse model as a tool to study
the consequences of microglial hyperactivity on brain homeostasis
and delineate the contribution of microglia to NPC pathology. Loss
of NPC1 in myeloid cells was sucient to trigger astrogliosis, neu-
ronal pathology, and region- specic neurodegeneration, supporting
neuronal- glial cross- talk in NPC (36). No neurotoxicity could be de-
tected aer 8 days of coculturing iPSC- derived iN WT and iMG
NPC, suggesting that long- term microglial deciency in vivo may be
responsible for neuronal defects. Furthermore, our ndings from the
mouse model translated well to human patients with NPC as illus-
trated by the increased TSPO and accumulation of the neuroaxonal
injury biomarker NF- L and astrocytic marker GFAP in the blood.
Compared with Npc1−/− mice with a fast- progressing neurode-
generation and early lethality, pathological changes upon loss of
NPC1 in myeloid cells occur slowly, and mice survive for almost a
year. We show that hallmarks of the NPC pathology, including axonal
swelling, NF- L increase, microgliosis, astrogliosis, and motor de-
fects, occur upon loss of NPC1 in myeloid cells. However, loss of
NPC1 in myeloid cells was not sucient to trigger Purkinje cell de-
generation, reduced myelination, or early lethality. us, our study
is in line with described cell- autonomous roles of NPC1 in other cell
types that together contribute to the wide range of clinical NPC phe-
notypes (36).
NPC is described as a lysosomal storage disorder in which,
among others, unesteried low- density lipoprotein–derived choles-
terol accumulates in lamellar inclusions of LE/lysosomes (1,57). We
showed that loss of NPC1 causes a lipid tracking dysfunction and
that myelin accumulation predominates in LE/MVBs, suggesting that
the delivery of the endolysosomal cargo rather than the catalytic
capacity of lysosomes is impaired in NPC (16). e lipidomic and pro-
teomic data align with this hypothesis because NPC1- decient microg-
lia strongly accumulate BMPs and CD63, which are predominately
found in ILVs of MVBs (58), favoring MVBs as the organellar
hotspot of NPC pathology in microglia. Increased di- 22:6- BMP was
detected in urine from patients with NPC (59). Apart from defects
in BMP homeostasis, cholesterol does not reach the endoplasmic
reticulum (ER) in NPC disease and is therefore not processed into
CEs, which is in line with the reduction in lipid droplet formation
(16). is is fully supported by the reduced CEs in NPC1- decient
microglia and defects in cholesterol esterication reported in pa-
tients with NPC (60). Besides the role of NPC1 as a cholesterol trans-
porter (61), it is proposed that NPC1 also steers the cholesterol
transport from LE/MVBs to the ER by regulating membrane contact
sites (62), and defects in membrane contact sites may contribute to
broad lipid dyshomeostasis in NPC. We detected GM2 accumula-
tion in the same intracellular compartment as cholesterol in cultured
Cre+ microglia, supporting that tracking of other lipids may be im-
paired, leading to their accumulation in LE/MVBs and additional
pathologic burden (63,64). Furthermore, severe lipid accumulation
in NPC microglia may be facilitated by increased phagocytic activity
toward extracellular substrates such as cell debris and extracellular
lipids. We demonstrated that loss of NPC1 in microglia results in
increased phagocytosis in vivo as reected by the detection of
phagocytic cups in Cre+ mice, resembling defects reported during
development in NPC (65). Phagocytic cups are typically found dur-
ing development (66), which is a period of active microglia- mediated
synaptic pruning (67). In line with these ndings, we have reported
increased synaptic materials in microglia of Npc1−/− and Cre+ mice
as well as increased key regulators of synaptic pruning such as C3
and C1q (16). It has been proposed that excessive synaptic pruning
may be responsible for triggering schizophrenia- like symptoms (68),
which occur in patients with NPC but are more common in late- onset
disease. us, the microglial contribution may be even more promi-
nent in patients who display a late- onset NPC disease and present
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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with psychiatric symptoms (9). It has been shown that C1q is not a
major cause for neurodegeneration in NPC (69), arguing that en-
hanced synaptic pruning of NPC microglia is not directly responsible
for neurodegeneration. Because phagocytic cups were more promi-
nent in Cre+ mice at late pathological stages, microglia may be rather
engaged in removing already dysfunctional synapses or neurons. Con-
sidering the severe defects in lipid processing and phagocytic hyper-
activity of NPC1- depleted microglia, it is clear that microglia are
exposed to a pathological feed- forward loop by continuously accu-
mulating substrates from their environment.
Noninvasive approaches like PET imaging are emerging as im-
portant clinical tools in monitoring neurodegenerative processes
in vivo but oen lack information regarding cellular identity. Using
scRadiotracing (47), we showed that even under nonpathological
conditions, the TSPO tracer is mainly taken up by microglia, con-
rming its cell specicity. Furthermore, the TSPO- PET signal is
aberrantly increased in NPC1- depleted microglia, and this is not
only a result of microgliosis as recently suggested in human cells
(70). Increased TSPO- PET signals in Cre+ mice was in line with the
immunohistochemical analysis of CD68. However, in contrast with
TSPO, CD68 immunohistochemistry detected microglial hyperac-
tivation already at the age of 2 months, stressing that LE/lysosomal al-
terations are among the rst pathological changes in microglia of Cre+
mice. Of interest are also regional dierences in microglial activation,
with the strongest microglial hyperactivity detected in the mid-
brains and brainstems of Cre+ mice. We hypothesize that early and
broad microglial activation is triggered by cell- autonomous dys-
function of NPC1 that is further potentiated by pathological insults
NF-L
0
5
10
15
20
25
Concentration (pg/ml)
NPCAD4RT HC
0
1
2
3
4
TSPO-PET (SUVr)
NPCAD4RT HC
0
1
2
3
TSPO-PET (SUVr)
C
5
NPC #1
NPC #2
NPC #3
NPC #1
NPC #2
NPC #3
D
A
40 years
58 years
36 years27 years 34 years
Insula Brainstem Cerebellum
B
NPCAD4RT HC
0
1
2
3
TSPO-PET (SUVr)
E
GFAP
0
50
100
150
200 CTRL
NPC
UCH-L1
0
50
100
150
200
Ba
seline
NALL
0
20
40
60
80
100
Phagocytosed plaques (%)
ns
Basel
ine
NALL
-
1.0
-
0.8
-
0.6
-
0.4
-
0.2
0.0
Log2 LFQNALL/baseline (TSPO)
TEP-OPST
SUVr z score vs. HC
5.0
1.5
Fig. 6. Potential markers for therapeutic monitoring in NPC. (A) Axial and sagittal sections showing the z- scores of global mean scaled SUVs of TSPO- PET in patients
with NPC (n = 5) compared with age- matched (to the oldest patient with NPC) healthy controls (HC, n = 27). (B) Quantitative comparison of global mean scaled TSPO- PET
signals in the insulae, brainstems, and cerebella of patients with NPC (NPC, n = 5), Alzheimer’s disease (AD, n = 12), 4- repeat tauopathy (4RT, n = 12), and a subset of old
HC (n = 12). One- way ANOVA with Tukey post hoc test was performed (*P < 0.05; **P < 0.01; ***P < 0.001). (C) Ex vivo Aβ phagocytic assay of blood- derived macrophages
from patients with NPC (NPC #1 to 3) before (baseline) and after NALL treatment (NALL). Values are presented as means ± SEM. A two- tailed paired Student’s t test was
performed (ns, P > 0.05). (D) Analysis of TSPO in macrophages from patients with NPC treated with NALL. Macrophages from patients with NPC (NPC1 #1 to 3) were ana-
lyzed before (baseline) and after a 6- week NALL treatment via LC- MS/MS. Label- free quantication (LFQ) intensities were normalized on the individual baseline LFQ inten-
sity. Values are plotted in log2 scale. Data are presented as means ± SEM, and a one- tailed Student’s t test was performed (*P < 0.05). (E) Plasma concentrations of NF- L,
UCH- L1, and GFAP in plasma samples from patients with NPC (n = 8) and age- matched controls (CTRL, n = 8) were measured by ELISA. Data are presented as means ±
SEM, and a two- tailed unpaired Student’s t test was performed (*P < 0.05).
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
11 of 14
in astrocytes or neurons, contributing to region- specic microglial
pathology at later stages. e regional overlap of TSPO- PET and
MAOB- PET signals supports pathological cross- talk between mi-
croglia and astrocytes. A similar pathological cross- talk of brain
cells is described in microglial regulator colony stimulating factor
1R (CSF1R)–related leukoencephalopathy, a disease in which mi-
croglia are primarily aected and cause prominent axonal spheroid
pathology (71). Modeling Csf1r deciency in mice results in axonal
spheroids, astrogliosis, and region- specic neurodegeneration, with
the thalamus being a pathological hotspot (72,73). However, further
mechanistic studies are warranted to understand how global mi-
croglial dysfunction may provoke selective regional vulnerabilities.
Of translational relevance is also a reduction in the FDG- PET sig-
nal in Cre+ mice that is a phenotype occurring already in patients car-
rying a heterozygous NPC1 mutation (74), supporting early neuronal
dysfunction that precedes neurodegeneration. Similar to our results,
activated microglia were capable of inducing non–cell- autonomous
inhibition of neuronal autophagy (75), underscoring the pathological
role of microglia- to- neuron cross- talk in neurodegeneration.
We show that microglial hyperactivity in NPC is reected by a
massive increase in TSPO- PET signals and increased microglial
phagocytic function. We furthermore found a lowering of phago-
cytic activity and TSPO in blood- derived macrophages from patients
with NPC aer NALL treatment, a drug that emerges as a benecial
therapeutic for NPC (24). ese new data underscore the potential of
monitoring phagocytic alterations as a response to therapy and pro-
vides a functional correlate for microglial hyperactivation measured
by the increased TSPO expression. is work suggests the translational
potential for TSPO as a monitoring biomarker in NPC and suggests
that hyperactive microglia lost their supportive functions, leading to
the observed astrogliosis, neuronal dysfunction, and, eventually, neu-
rodegeneration. Furthermore, as now witnessed in other neurodegen-
erative diseases such as AD (76) or Parkinson’s disease (77), biological
disease classication develops toward a valuable tool in clinical routine.
Biomarkers capable of capturing microglial (TSPO), astrocytic (GFAP),
and neuronal (NF- L and UCH- L1) (MAN) pathology could be
combined and tested for their utility for disease monitoring in NPC
clinical trials.
One limitation of our study is that we cannot exclude the contribu-
tion of NPC1 loss in Cx3cr1- expressing peripheral myeloid cells to dis-
ease phenotypes in mice. An additional limitation is the low number of
NALL- treated patients. Aqneursa (NALL) has just been approved by
the US Food and Drug Administration for NPC (https://www.fda.gov/
news- events/press announcements/fda-approves-new-drug-treat-
niemann-pick-disease-type-c), which is a major clinical advancement
enabling future studies to link benecial clinical outcomes with bio-
markers capturing reduced MAN pathology in NPC.
MATERIALS AND METHODS
Study design
e aim of this study was to delineate the role of NPC1 in myeloid
cells and the contribution of neuroinammation to neurode generation.
Our study includes characterization of mice with myeloid cell–
specic NPC1 depletion, using primary cell culture, PET imaging,
and immunohistological, proteomic, and lipidomic analyses comple-
mented with analysis of patients with NPC and hiPSC- derived
models. For animal experiments, sample sizes were determined a
priori by applying G*power soware (V3.1.9.7) and on the basis of
our previous experimental data in rodent models of neurodegenera-
tion and neuroinammation. e animals were assigned to experi-
mental groups randomly. e P value for a priori calculation had
been set to 0.05 and power to 0.8. Analysis of all experiments was
performed blinded and with a randomized order. All animal experi-
ments complied with the ARRIVE guidelines and the German ani-
mal welfare law and were approved by the government of Upper
Bavaria (license numbers ROB- 55.2- 2532.Vet_02- 17- 075 and ROB-
55.2- 2532.Vet_02- 22- 125). All experiments using human material
were authorized by the local Ethics Committee of the University of
Munich (ethics- application: 20- 350) and University of Leipzig (ethics-
application: 370- 21- ek) and executed according to the 1964 Decla-
ration of Helsinki. PET acquisition (ethics- applications: 17- 569,
17- 755) was approved by the local institutional ethics committee
(LMU Munich) and the German radiation protection authorities
(BfS- application: Z 5- 22464/2017- 047- K- G). All participants pro-
vided written informed consent before the PET scans. Consider-
ing that NPC is a rare disease, all human patient data that we were
able to collect during the duration of the study were included. No
samples were excluded from the study. e n numbers of indepen-
dently performed experiments are reported in the gure legends.
Detailed Materials and Methods are outlined in the Supplementa-
ry Materials.
Statistical analysis
Measured values from LC- MS lipidomic analysis were log2 trans-
formed. Dierences between Npc1+/+ or Cre− and Npc1−/− or Cre+
in selected lipids were estimated using a robust linear model adjust-
ing for covariates of sex, age, and the interaction between age and
genotype in the model (78). e P values were adjusted for multiple
comparisons using the Benjamin- Hochberg procedure (adjusted
P value). Statistical analysis of the LC- MS proteomic data was per-
formed as described in the respective section. For each experiment,
the used sample size and statistical test are denoted in the gure
legends. A P value of P < 0.05 was considered as signicant [not
signicant (ns) = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001].
Supplementary Materials
The PDF le includes:
Materials and Methods
Figs. S1 to S10
References (80–101)
Other Supplementary Material for this manuscript includes the following:
Data les S1 and S2
MDAR Reproducibility checklist
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Acknowledgments: We thank C. Haass for the support with the NF- L analysis and S. Wagner
for support with PET imaging. Funding: This work was supported by an Alzheimer’s
Association grant through the AD Strategic Fund to S.T. (ADSF- 21- 831226- C), the DFG (DFG
Research Unit FOR2858–project numbers 403161218: GR 4403/7- 1 and GR 4403/7- 2 to A.G.; TA
551/3- 2 to S.T.; HE 2328/14- 1 to J.H.; PA 3450/4- 1 to L.P.; DFG Priority Programme SPP2395–
project numbers 500118375, TA 551/2- 1 to S.T. and HE 2328/13- 1 to J.H.), the Centers of
Excellence in Neurodegeneration (CoEN6005), the Bundesministerium für Bildung und
Forschung (BMBF, FKZ: 01ED2402A) under the aegis of JPND and by the Cure Alzheimer’s Fund
to S.F.L., the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under
Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems
Neurology (EXC 2145 SyNergy–ID 390857198) to M. Schierer, M. Simons, J.H., S.F.L., N.F., A.L.,
and M.B. S.A.S. was supported by the LMU Clinician Scientist Excellence Programme. Author
contributions: L.D., S.T., and M.B. supervised the study. L.D., M.P., M.B., and S.T. wrote the
manuscript with the input of all coauthors. L.D., V.Z., and Y.T. isolated primary cells and
performed in vitro experiments. L.D. and J.R.N. collected brain tissue and performed
immunohistochemical analysis. L.D. and A.C. performed the phagocytosis assay with human
blood samples. J.H.S., T.L., and G.D.P. performed the lipidomic analysis. S.R. and A.L. performed
the rotarod test. L.P. and J.H. performed the Purkinje cell analysis. P.H., O.B., and A.G. performed
the retina analysis. A.S., S.A.M., and S.F.L. performed the proteomic analysis. L.D. and V.Z.
performed Western blot analysis. M.P. performed IPA analysis. M. Schierer and M. Simons
performed the EM analysis. S.H. performed small- animal PET with the help of L.S., L.H., and
S.T.K. P.B., N.F., J.G., and M.B. performed human PET and human PET analyses. L.M.B., K.W.- M.,
and S.T.K. performed scRadiotracing. S.L. synthesized all radiotracers. L.D., V.Z., M.M., L.S.M.,
M.P., and D.P. established and analyzed the hiPSC lines. B.N. performed mouse and human
ELISA. T.B.- E., C.C., S.B., C.B., S.A.S., M. Strupp, and M.B. recruited patients with NPC for blood
sampling and PET. Competing interests: M. Strupp is joint chief editor of the Journal of
Neurology, editor- in- chief of Frontiers of Neuro- otology, and section editor of F1000. He has
received speaker honoraria from Abbott, Auris Medical, Biogen, Eisai, Grünenthal, GSK,
Henning Pharma, Interacoustics, J&J, MSD, NeuroUpdate, Otometrics, Pierre- Fabre, TEVA, UCB,
and Viatris. He receives support for clinical studies from Decibel, USA; Cure within Reach, USA;
and Heel, Germany. He distributes “M- glasses” and “Positional vertigo App.” He acts as a
consultant for Abbott, AurisMedical, Bulbitec, Heel, IntraBio, Sensorion, and Vertify. He is an
investor, shareholder, and patent holder of IntraBio. T.B.- E. received speaker’s honoraria and
consultant fees from Actelion and Sano- Genz yme. She acts as a consultant for Azafaros and
machineMD. She received AB honoraria from Zevra and ScenicBio. She acts as a blinded video
rater for Intrabio. Her research has been supported by Baasch- Medicus Foundation, grant- no.
20211001 and InnoSuisse grant- no. 55424.1 IP- LS. M.B. received speaker honoraria from GE
HealthCare IBA, Roche, and Life Molecular Imaging and is an adviser for GE HealthCare, MIAC,
and Life Molecular Imaging, all outside the submitted work. The other authors declare that
they have no competing interests. Data and materials availability: The proteomics data have
been deposited with the ProteomeXchange Consortium via the PRIDE (79) partner repository
with the dataset identiers PXD056188 and PXD056249. C57BL/6- Npc1tm1.2Apl mice were
provided by A. Lieberman (University of Michigan, Ann Arbor, MI, USA) and B6.
Cx3cr1tm1.1(cre)Jung/N mice by S. Jung (Weizmann Institute of Science) under a material
transfer agreement. hiPSCs and the NPC1 depletion mouse model will be shared by S.T. upon
request under a material transfer agreement that will be provided by the DZNE Technology
Transfer Oce (techtransfer@ dzne. de). All data associated with this study are present in the
paper or the Supplementary Materials.
Submitted 19 October 2023
Resubmitted 12 July 2024
Accepted 11 November 2024
Published 4 December 2024
10.1126/scitranslmed.adl4616
Downloaded from https://www.science.org at Deutsches Zentrum fr Neurodegenerative Erkrankungen on December 05, 2024
