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J Neuropathol Exp Neurol
Copyright Ó 2012 by the American Association of Neuropathologists, Inc.
Vol. 71, No. 5
May 2012
pp. 449Y466
ORIGINAL ARTICLE
A Brain Aggregate Model Gives New Insights Into the
Pathobiology and Treatment of Prion Diseases
Krystyna Bajsarowicz, MS, Misol Ahn, PhD, Larry Ackerman, BA, Bernadette N. DeArmond, MD, MPH,
George Carlson, PhD, and Stephen J. DeArmond, MD, PhD
From the Departments of Pathology (KB, MA, BND, SJD) and Anatomy
(LA), University of California, San Francisco; McLaughlin Research
Institute, Great Falls, Montana (GC); and Institute for Neurodegenerative
Diseases, University of California, San Francisco (SJD).
Send correspondence and reprint requests to: Stephen J. DeArmond, MD, PhD,
1855 Folsom Street, Suite 505, San Francisco, CA 94143-0803; E-mail:
[email protected]
Krystyna Bajsarowicz and Misol Ahn contributed equally to the work.
This study was supported by Grant No. NS041997 from the National Institutes of Health and by the Stephen and Patricia Schott Family Fund.
CNS, including astrocytes, oligodendrocytes, and microglia. We
adopted the BrnAggs model from Dr Lynn Pulliam who refined
a technique first described by DeLong to generate reaggregating cell cultures from fetal mouse isocortex and hippocampus
(1). Dr Pulliam used BrnAggs to study human immunodeficiency virus and herpes simplex virus encephalitis (2Y5). Ours
is the first study of prion disease in BrnAggs.
Brain aggregates differ from neurospheres that are composed of neural stem cells (6, 7). Two of the authors of this
article (S.D. and G.C.) were coauthors of the first report of the
use of neurospheres to study prion disease (8). Neurospheres are
not constantly rotated, not exposed to FBS, and are often grown
as monolayer cultures (9). They have been used for neural stem
cell assays, as models of neurogenesis, and to expand clones
of neural stem cells for transplantation purposes (10). Cultured
neurospheres that express cellular prion protein (PrPC) can be
infected with prions to study pathogenic prion protein (PrPSc)
in a limited manner. Our earlier study showed that the susceptibility of neurosphere cultures to prions mirrored that of
the parental mice and that neurospheres from Tg4053 mice,
which overexpress mouse PrPC, provide a sensitive bioassay
for mouse prions (8). Herva et al (11) reported that they were
not able to duplicate prion inoculation of undifferentiated neurospheres but did achieve successful prion amplification in
neurospheres that contained more differentiated cells.
Only a few cell lines have been infectible with prions
(8). The mouse neuroblastoma cell line (N2a) has been most
useful for investigating the biology of prion diseases, but only
a small percentage of N2a cells replicate prions when exposed
to scrapie (12); moreover, prion replication in scrapie-infected
N2a cells (ScN2a) has been unstable (13). Treatment of dividing
ScN2a cells with quinacrine (Qa) cleared PrPSc in 5 days (14);
however, Qa treatment of patients with Creutzfeldt-Jakob disease and scrapie-infected mice failed to reduce PrPSc or increase
survival time. These data suggest that ScN2a cells are not a good
model for drug screening (15Y18). Other prion-infectible cell
lines have also shown limitations, including PC12 rat pheochromocytoma cells (19), spontaneously immortalized hamster
brain cells (20), the GT1 immortalized hypothalamic neuronal
cell line (21), and immortalized cells of the CNS and peripheral
nervous system (22, 23). Primary cultures of neurons have also
failed to support prion replication (22).
The structure of BrnAggs, with their neuronal and microglial cells, resembles the organization in brain tissue. The neurons of uninfected control BrnAggs form lush dendritic trees
that permit the study of prion-induced dendritic degeneration.
J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
449
Abstract
Brain aggregates (BrnAggs) derived from fetal mouse brains contain mature neurons and glial cells. We determined that BrnAggs are
consistently infected with Rocky Mountain Laboratory scrapie strain
prions and produce increasing levels of the pathogenic form of the
prion protein (PrPSc). Their abundant dendrites undergo degeneration
shortly after prion infection. Treatment of prion-infected BrnAggs
with drugs, such as a F-secretase inhibitors and quinacrine (Qa), which
stop PrPSc formation and dendritic degeneration, mirrors the results
from rodent studies. Because PrPSc is trafficked into lysosomes by endocytosis and autophagosomes by phagocytosis in neurons of prion
strainYinfected BrnAggs, we studied the effects of drugs that modulate
subcellular trafficking. Rapamycin (Rap), which activates autophagy,
markedly increased light-chain 3-II (LC3-II)Ypositive autophagosomes and cathepsin DYpositive lysosomes in BrnAggs but could
not eliminate the intracellular PrPSc within them. Adding Qa to Rap
markedly reduced the number of LC3-IIYpositive autolysosomes. Rap
+ Qa created a competition between Rap increasing and Qa decreasing
LC3-II. Rapamycin + Qa decreased total PrPSc by 56% compared with
that of Qa alone, which reduced PrPSc by 37% relative to Rap alone.
We conclude that the decrease was dominated by the ability of Qa to
decrease the formation of PrPSc. Therefore, BrnAggs provide an efficient in vitro tool for screening drug therapies and studying the complex biology of prions.
Key Words: Autophagosomes, Brain aggregates, Exocytosis, Prion
disease, Quinacrine, Rapamycin, Scrapie.
INTRODUCTION
We used an in vitro brain aggregate (BrnAgg) model for
the study of prion disease. The BrnAggs are made from 15-day
gestational whole mouse brains grown in media supplemented
with fetal bovine serum (FBS) in constantly rotated flasks. After
15 days in culture, they form spheres of 750 to 1,000 Km diameter. Brain aggregates contain mature neurons that form dendrites, axons, synapses, and all the cellular elements of the
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Bajsarowicz et al
J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
In Rocky Mountain Laboratory (RML, Hamilton, MT) scrapie
prion strainYinfected BrnAggs, neurons show dendritic loss
after 1 week, as seen in mice (24). Because dendritic degeneration in mice follows local PrPSc accumulation and truncation
of Notch-1 to the Notch-1 intracellular domain (NICD) by the
F-secretase complex (24), we explored the prevention of dendritic degeneration by treating mice with F-secretase inhibitor LY411575 (GSI) plus Qa (GSI + Qa) (17). Treatment with
GSI alone produces very low NICD levels but no recovery
of dendrites, which we attributed to simultaneous inhibition
of F-secretase stimulators and inhibitors of dendritic growth.
Treatment with GSI + Qa prevented dendritic degeneration and
prevented the formation of PrPSc. Quinacrine ‘‘destabilizes’’
and fragments membrane rafts where the F-secretase complex,
PrPSc, Notch-1, and other F-secretase substrates reside (25).
Presumably, destabilization selectively separates some raft components from each other and permits others to interact. The GSI
+ Qa has its primary effect on raft domains that contain the
Notch-1 inhibitor of dendritic growth and not on F-secretase
substrates such as ErbB-4 and EphA4 that simulate dendritic
growth (26Y28).
Endocytosis of PrPSc into lysosomes and phagocytosis
of PrPSc into autophagosomes are attempts to degrade and
clear PrPSc from neurons. When these mechanisms fail, nerve
cell dysfunction and death result. Therefore, we used BrnAggs
to examine drug treatments that modify PrPSc trafficking pathways. Rapamycin (Rap) is reported to decrease PrPSc in vitro
(29) and to activate autophagy by inhibiting the mammalian
target of rapamycin (mTOR) (30, 31). Beneficial effects of Rap
have been reported for spinocerebellar ataxia (32), Parkinson
disease (33), frontotemporal lobar degeneration (34), Huntington
disease (35), and amyotrophic lateral sclerosis (36). To enhance
the efficacy of Rap, we added Qa (Rap + Qa), which inhibits
PrPSc formation in cultured cells and in mice (14, 17). Quinacrine increases the number of lysosomes in mouse liver cells
(37) but inhibits light-chain 3-II (LC3-II) turnover in mammary
cells (38). Here, we used confocal microscopy and Western
blot analysis for PrPSc to explore the effects of Rap alone and
Rap combined with Qa on RML-infected BrnAggs.
Millipore, Billerica, MA), A-3-tubulin (AB9354; Millipore), LC3II N-terminal epitope, APG8B (AP1802a; Abgent, San Diego,
CA), Iba-1 (019-19741; Wako, Richmond, VA), cathepsin-D
([Cat-D] Sc 6486; Santa Cruz Biotechnology, Santa Cruz, CA),
glial fibrillary acidic protein ([GFAP] 20334; Dako, Carpinteria,
CA), and NeuN (MAB377, Millipore). Antibodies to PrP included R2, D18, D13, and HumP were gifts from Dr Stanley
Prusiner, University of California, San Francisco. We used
the following secondary antibodies for immunohistochemistry:
donkey anti-rabbit IgG (711-175-152), donkey anti-human IgG
(709-295-149), donkey anti-goat IgG (705-485-003), and donkey
anti-chicken IgG (703-295-155). The above secondary antibodies are fluorophore conjugated and were purchased from
Jackson ImmunoResearch Laboratories, Inc (West Grove, PA).
For dot blot analysis, we used antibodies to PrP and biotinylated goat anti-human J chain (BA-3060; Vector Laboratories,
Burlingame, CA) followed by streptavidinYhorseradish peroxidase (P0397; Dako). For nuclear staining, we used Hoechst
33342 (Invitrogen, Carlsbad, CA).
MATERIALS AND METHODS
Animals
Pregnant wild-type FVB and FVB PrP knockout (Prnpo/o)
mice were used to make BrnAgg cultures. Adult CD1 mice that
have been inoculated intrathalamically with the RML strain of
mouse prions were used for additional studies of the brain. All
experiments were carried out in accordance with the Institutional
Animal Care and Use Committee/Laboratory Animal Research
Center protocol of the University of California, San Francisco.
Reagents
Drugs used in this study included Rap (R-5000; LC Laboratories, Woburn, MA); quinacrine (69-05-6; Sigma, St Louis,
MO); F-secretase inhibitor LY411575 (gift from Drs Todd
Golde and Pritam Das, Mayo Clinic, Jacksonville, FL), sodium
butyrate (B5887, Sigma-Aldrich). Primary antibodies included
were microtubule-associated protein 2 ([MAP-2] AB5622;
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Preparation of BrnAgg Cultures
Brain aggregates were prepared from embryonic day 15
gestation embryos from pregnant wild-type and Prnpo/o FVB
mice. Brain cells were dissociated through 2 nylon meshes from
a pool of embryonic brains. After 2 washes with Dulbecco’s
modified Eagle medium (D-MEM H21) (UCSF Cell Culture
Facility, San Francisco, CA) containing glucose (12 g/L), fungizone (2.5 mg/L), and gentamicin (50 mg/L), the dissociated
neural cells were resuspended in growth medium D-MEM H21
supplemented with glucose (6 g/L), gentamicin (50 mg/L), fungizone (2.5 mg/L), and 10% FBS at a density of 1 107 cells/mL.
Four milliliters of cells were placed in 25-mL DeLong flasks
at constant rotation (37-C, 10% CO2). The next day, 1 mL of
exchange medium D-MEM H21 supplemented with glucose
(6 g/L), gentamicin (50 mg/L), and 15% FBS was added to the
flask. After 2 to 3 days, the rotating BrnAggs were transferred
to 50-mL DeLong flasks to which 5 mL of exchange medium
was added for a total of 10 mL. Exchange media were refreshed every 2 to 3 days by removing 5 mL of conditioned
medium and replacing it with 5 mL of fresh exchange medium.
At 6 to 8 days, the growing BrnAggs were transferred from
rotating flasks to a 24-well culture plate, and 1 to 2 BrnAggs
per well were placed in 500 KL of exchange medium and
rotated constantly. The medium was changed every 2 to 3 days
by discarding 500 KL of conditioned medium and replacing an
equal volume of exchange medium. Approximately 15 BrnAggs
were obtained from the initial 4 107 neural cells. At 15 days
in culture, BrnAggs were exposed for 10 days to a 1:50 dilution of RML prions derived from RML-infected CD1 mouse
brains. Drug treatments began at day 25 and continued for
10 or more days. We routinely use the aggregates for 45 days.
After 50 days, necrosis develops in the center of aggregates,
and they are discarded.
Drug Treatments
Brain aggregates infected with RML prions were treated
with 2.5 KM of Qa, 0.5 KM of Rap, 5.0 KM of GSI, or the
combination of GSI + Qa or Rap + Qa for 10 or more days.
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
Prion-Infected Brain Aggregate Model
When using drugs in combination, we used the same concentration of each drug as when they were applied alone. At the
end of treatment, BrnAggs were washed with PBS, harvested,
and either lysed with lysis buffer (100 mM of Tris-HCl, pH
8.0; 150 mM of NaCl 0.05% Np-40, 0.5% of Na deoxycholate)
and stored at j80-C for Western blot analyses or fixed with
4% formaldehyde for immunostaining.
groups was quantified with the BioQuant Life Science software
(Bioquant Image Analysis Corporation, Nashville, TN).
Phosphotungstic Acid Precipitation of PrPC
and PrPSc
Fifteen BrnAggs were pooled and sonicated in lysis buffer for each of 2 uninfected control BrnAggs, 2 RML-infected
BrnAggs, and 2 RML-infected BrnAggs treated with GSI + Qa
groups. Total protein was measured by bicinchoninic acid assay
(Pierce, Rockford, IL), and 20 Kg of protein was loaded per well
on a Novex NuPAGE 4% to 12% Bis-Tris gel for total PrP. For
phosphotungstic acid (PTA) precipitation, 50 Kg of lysate protein was incubated with 0.8% PTA in 2% sarkosyl/PBS buffer
with complete protease inhibitor cocktails (Roche, Indianapolis,
IN) for 2 hours at 37-C with continuous shaking at 350 rpm.
Samples were centrifuged at 14,000 rpm for 1 hour. The entire
supernatant (PTA Sup) was collected and precipitated with
methanol at j20-C overnight. The methanol-precipitated PTA
Sup samples were centrifuged at 14,000 rpm for 1 hour, and the
PTA Sup pellets were collected and resuspended in 15 KL of
lysis buffer. After adding 4 SDS sample buffer and reducing agent (Invitrogen), the samples were loaded on the gel and
tested for PrPC. For PrPSc, the PTA pellet was washed with
0.2% sarkosyl/PBS, and samples were centrifuged at 14,000 rpm
for 1 hour. The collected PTA pellets were resuspended in lysis
buffer, and 4 SDS sample buffer with a reducing reagent
was added. Samples were run on gel and transferred to a polyvinylidene fluoride membrane using iBlot (Invitrogen). After
blocking with 5% milk for 30 minutes, the polyvinylidene
fluoride membrane was incubated with the PrP-specific D13Y
horseradish peroxidase antibody overnight. The next day, the
membrane was washed with Tris-buffered salineYTween buffer
for 5 minutes (3) and developed with an enhanced chemiluminescence plus Western blotting detection reagent (Pierce).
For studies in mice, the neocortex was collected from uninfected and scrapie-sick CD-1 mice and homogenized using a
Percellys bead beater (Percellys, Basking Ridge, NJ). One hundred micrograms of protein was used for PTA precipitation,
and Western blot analysis was performed as described above.
Drug Treatment of Stationary CL3.ScN2a Cells
CL3.ScN2a cells were maintained at 37-C in minimum
essential media (Invitrogen) supplemented with 5% FBS, 1%
GlutaMax (Invitrogen), and fed with fresh media every 2 days.
CL3.ScN2a cells that were approximately 70% confluent were
plated and treated with Rap (1 KM) or Rap + Qa (1 KM each)
in the presence of 10 mM sodium butyrate. The cells were
washed with PBS, harvested, and lysed, with lysis buffer daily
for 7 days. Protein concentrations were measured with bicinchoninic acid assay, and 100 Kg of protein was digested with
protease K (PK) at 37-C for 1 hour. The PK-digested pellets
were collected for Western blot analysis. The level of PKresistant PrPSc in untreated, Rap-treated, and Rap + QaYtreated
Dot Blot Analysis of PrPSc
Single BrnAggs were washed with PBS, immersed in
lysis buffer, sonicated, and exposed to PK to digest PrPC. The
remaining PK-sensitive proteins were precipitated with methanol in j20-C overnight. After centrifugation at 14,000 rpm
for 20 minutes, the methanol-precipitated pellet was washed
with PBS and reconstituted with lysis buffer. Serial 2-fold dilutions were applied to nitrocellulose membranes where PrPSc was
denatured with 3M guanidinium isothiocyanate and detected
with D18 antibody, followed by biotinylated goat anti-human
IgG and streptavidinYhorseradish peroxidase. Horseradish peroxidase was revealed by enhanced chemiluminescence. The
concentration of PrPSc in the dot blots was estimated by
densitometry.
Measurement of Dendritic Degeneration
We used 2 methods to estimate the lengths of dendrites
that had been stained with antiYMAP-2 antibodies. In the first
method, 3-dimensional images of dendrites selected at random
were obtained with a Zeiss LSM 510 confocal microscope that
captured stacks of 1-Km-thick serial sections. To avoid detection of MAP-2Ystained cell bodies, we traced dendrites on
acetate membranes, and BioQuant software was used to estimate the total dendritic load. To normalize the dendritic measurements, the density of dendrites per 100 Km2 was divided
by the total number of confocal images obtained at one site and
multiplied by the number of slices containing more than 3%
dendrites. In the second method, lengths of dendrites were
obtained from 3-dimensional images obtained with a 63 objective lens. The images were reconstructed using the IMARIS 3.0
software (Bitplane AG, Zurich, Switzerland), and the total
length of dendrites was generated using the Filament Tracer
module. Three sites were chosen for the highest density of
dendrites in each BrnAgg, and 3 stacks of approximately 35 to
50 sections each were collected from each BrnAgg.
Immunocytochemistry for Confocal Microscopy
The BrnAggs were fixed in 4% formaldehyde followed
by 3 washes with PBS. For detection of LC3-II, Cat-D, PrP,
and Iba-1, the BrnAggs were autoclaved (10 minutes at 121-C
in 0.01 M Na citrate buffer, pH 6.0), washed with PBS containing 0.3% Triton X-100 and 0.1% Tween 20, blocked
overnight with 2% bovine serum albumin (BSA) and 10%
normal donkey serum (NDS) and then incubated for 3 days at
4-C with antibodies specific for autophagy (LC3-II, diluted
1:75), lysosomes (Cat-D, diluted 1:100), prions (R2 and HumP,
diluted 1:250), and microglia (Iba1, diluted 1:250). After incubation with primary antibodies, BrnAggs were washed with
PBS containing 0.3% Triton X-100 and 0.1% Tween 20 and
incubated with fluorophore-conjugated anti-human IgG (1:800),
anti-goat IgG (1:800), and anti-rabbit IgG (1:800) at room
temperature (RT) for 12 hours. All antibodies were diluted in
PBS with 0.3% Triton X-100, 0.1% Tween 20 containing 2%
BSA, and 10% NDS. For detection of dendrites, axons, and
astrocytes, the BrnAggs were washed with PBS and blocked
with PBS containing 0.3% Triton X-100, 2% BSA, and 10%
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451
Bajsarowicz et al
J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
NDS for 90 minutes at RT. Primary antibodies, antiYMAP-2
for dendrites (1:1000), antiYA-3-tubulin for axons (1:100), and
anti-GFAP for astrocytes (1:250) were diluted into blocking
buffer and incubated for 3 days at 4-C. After 3 washes with
PBS, the fluorophore-conjugated secondary antibodies corresponding to the primary antibodies were added and incubated
452
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
Prion-Infected Brain Aggregate Model
for 12 hours at RT. All antibodies were diluted into PBS-0.3%
Triton X-100 containing 2% BSA and 10% NDS. To identify
nuclei, the BrnAggs were incubated for 30 minutes at RT with
Hoechst 33324 (1:5000 in PBS). Three to 4 stacks of approximately forty 1-Km-thick serial sections were captured by 2
confocal microscopes at different times (Zeiss LSM 510, Jena,
Germany, or Leica Microsystems, Wetzlar, Germany).
inconsistent. Equal amounts of protein were loaded on the
polyacrylamide gels. No normal or abnormal PrP signal was
found in the homogenates of BrnAggs derived from Prnpo/o
knockout mice whether they had been exposed to RML prions
(Fig. 1D, lanes 1Y3). A single band of PrPC was present in uninfected control aggregates (Fig. 1D, lane 4). The PrPSc bands
corresponding to variations of the 2 N-linked glycosylation
appeared in RML-infected aggregates (Fig. 1D, lane 5). Treatment of the RML-infected BrnAggs with GSI + Qa eliminated
the PrPSc signal (Fig. 1D, lane 6). The absence of any detectable PrPSc in BrnAggs made from Prnpo/o mice after exposure
to RML prions argues that any residual PrPSc introduced during
the infection of the aggregates remained below detection or
was eliminated from BrnAgg with changes of media.
The sensitivity of PrPSc detection was increased with
PTA precipitation of the homogenates, which selectively separate PrPSc from PrPC (41, 42). For these experiments, 90
BrnAggs were separated into 3 groups of 30 aggregates per
group: uninfected controls, RML infected, and RML infected
treated with GSI + Qa. Each group was further divided into
2 pools of 15 BrnAggs each for homogenization. The pool was
analyzed for total PrP (PrPC and PrPSc) in the lysate, PrPC in
the PTA supernatant, and PrPSc in the PTA pellet. None of the
samples were digested with PK. The BrnAgg lysates showed
a single PrPC band in uninfected control aggregates (Fig. 1E,
Lysate, 1a,b). Three major PrPSc bands at 34, 29, and 25 kDa
and a minor band at 20 kDa were seen in RML-infected aggregates (Fig. 1E, Lysate, 2a,b). A single band located at
34 kDa, which represents PrPC, was seen in RML-infected
aggregates treated with GSI + Qa for 12 days (Fig. 1E, Lysate,
3a,b). In the PTA supernatant, only a single PrPC band was
seen in all groups (Fig. 1E, PTA Sup). In the PTA pellet, no
PrPC was identified in the control as expected (Fig. 1E, PTA
Pellet, 1a,b). A very intense PrPSc signal was revealed in the
untreated RML-infected PTA pellet (Fig. 1E, PTA Pellet 2a,
2b). Treatment with GSI + Qa completely eliminated conversion of PrPC to PrPSc (Fig. 1E, PTA, Pellet 3a, 3b).
The intensity of the PrPC bands in RML-infected BrnAggs
(Fig. 1E, PTA Sup [PrPC] 2a,b) was decreased by approximately 70% compared with uninfected controls, as measured
RESULTS
Growth Kinetics of BrnAggs
When constantly rotated in a culture flask 15-day gestational embryonic brain cells that formed spherical aggregates
from 50 to 170 Km in diameter within 72 hours (Fig. 1A; 3 days).
The outer surface of the BrnAggs contained a layer of GFAPimmunopositive astrocytes (red), with their processes projecting
into the interior of the aggregates. Neurons stained positively
with the neuron-specific antibody NeuN (green) filled the interior of the aggregates. Brain aggregates grow by accretion
of clusters of individual neural cells and by fusion with other
BrnAggs (Fig. 1A; 10 days). On the sixth to eighth day of culture, BrnAggs reached 200 to 700 Km in diameter and were
transferred from the flasks to constantly rotated 24-well plates.
By 15 days in culture, many of the BrnAggs were as large as
1,000 Km (Fig. 1A; 15 days).
Measurement of PrPC and PrPSc in BrnAggs
For dot blot analysis, 3 BrnAggs exposed to RML scrapie
prion strain on day 15 in culture were chosen for detection of
PrPSc at 5, 20, and 35 days postinfection (dpi) (Fig. 1B). Serial
dilutions of single BrnAgg lysates were treated with PK (40 Kg)
to eliminate PrPC and preserve PrPSc and dotted onto nitrocellulose membranes. Densitometry of the 1:4 dilution showed
that levels of PrPSc increased each dpi, as they do in scrapieinfected mice (39, 40) (Fig. 1C).
For Western blot analysis, BrnAggs exposed to RML
scrapie prions were homogenized in lysis buffer. The samples
were not treated with PK for this analysis. Fifteen pooled
BrnAggs consistently yielded detectable levels of PrPSc, whereas
PrPSc signals from 5 and 10 pooled aggregates were weak and
FIGURE 1. Growth characteristics of brain aggregates (BrnAggs) and measurements of pathogenic form of the prion protein (PrPSc).
(A) Brain aggregates grow by progressive accretion of single cells and smaller aggregates. A confocal image immunostained for glial
fibrillary acidic protein ([GFAP], red) and NeuN (green) shows the location of astrocytes and neurons. In the left panel at 3 days in
culture, scale bar = 30 Km. In the right panel at 15 days in culture, scale bar = 100 Km, and applies also to the middle panel from day
10. (B) The PrPSc is detected by dot blot analysis of 3 single aggregates; 4 dilutions from 3 time points in culture are shown. (C) The
mean and SD of the 1:4 dilution of PrPSc were measured by densitometry (arbitrary units). (D) Western blot analysis was used to
measure cellular prion protein (PrPC) and PrPSc in the pools of 15 BrnAggs made from PrP knockout (Prnpo/o) (lanes 1Y3) and wild-type
FVB mouse embryos (lanes 4Y6). Neither PrPC nor PrPSc is detected in the Prnpo/o samples. In BrnAggs derived from FVB embryos, PrPC
is detected in uninfected BrnAggs (lane 4); PrPSc is detected in aggregates exposed to Rocky Mountain Laboratory (RML) scrapie prion
strain prions (lane 5); little PrPSc is detected in BrnAggs exposed to RML prions and treated with F-secretase inhibitor (GSI) quinacrine
(Qa) for 10 days (lane 6). (E) Phosphotungstic acid (PTA) precipitation of homogenates from 2 pools of 15 BrnAggs yields characteristic the PrPC and PrPSc bands after 35 days in culture. Lanes 1a and 1b are uninfected BrnAggs; lanes 2a and 2b are BrnAggs
exposed to RML prions on day 15 in culture; lanes 3a and 3b are BrnAggs infected with RML prions at 15 days and treated with GSI +
Qa for 10 days starting at day 25. (F) Phosphotungstic acid precipitation of CD-1 mouse neocortex uninfected (lanes 1Y3) and
infected with RML prions (lanes 4Y6); mice were killed at 130 days postinfection (dpi). The conversion of PrPC to PrPSc reduces PrPC.
Intense PrPC bands are found in PTA supernatant (S) in the neocortex of the 3 uninfected control mice. The intensity of the PrPC bands
(S) is weaker when the PrPSc is abundant in the PTA pellet (P). (G) Densitometry shows that PrPC was significantly reduced by 95%
when PrPSc is formed. **t-test probability of a decrease p G 0.01.
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
by densitometry (data not shown). The decrease was associated with the intense PrPSc bands in RML-infected BrnAgg
(Fig. 1E, PTA Pellet, 2a,b). Such a decrease in PrPC was consistent with data showing a 95% depletion of PrPC in RML-
infected CD-1 mice, which was caused by the rapid conversion
of PrPC to PrPSc (Figs. 1F, G).
After GSI + Qa treatment of RML-infected BrnAggs,
PrPC levels were approximately 58% lower than in uninfected
FIGURE 2. (AYC) The number of microtubule-associated protein 2 (MAP-2)Ypositive dendrites per given volume (dendritic density)
was estimated in 3 uninfected (AYC) and 3 Rocky Mountain Laboratory (RML) scrapie prion strainYinfected brain aggregates
(BrnAggs) (D, E). (F) To estimate dendrite densities, MAP-2Ystained dendrites in each confocal slice were drawn onto acetate
membranes, and the density in each slice was measured by BioQuant morphometry. **Probability of a difference between control
and RML-infected BrnAggs at 25 days, p = 0.028; at 35 days, p = 0.032 (n = 3 BrnAggs each). (G) The responses of dendritic
lengths to 10-day treatments with F-secretase inhibitor (GSI), quinacrine (Qa) and GSI + Qa were measured. Control represents
uninfected BrnAggs. RML (UnTx), RML-infected BrnAggs without treatment. The lengths of dendrites immunostained with MAP-2
were measured with IMARIS 3 software using confocal microscopy to generate a 3-dimensional image. n = number of BrnAggs
analyzed. (H) Immunocytochemistry for pathogenic form of the prion protein (PrPSc, red) and cathepsin-D (Cat-D, green) in
a BrnAgg neuron early in RML infection (10 days postinfection) showing prominent plasma membrane immunostaining for PrPSc
and Cat-DYimmunopositive lysosomes with a minimum number of merged PrPSc (yellow). Nuclei are stained with Hoechst (blue).
Scale bars = (E) 30 Km; (AYD, H) 10 Km.
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
Prion-Infected Brain Aggregate Model
controls, which indicates that the GSI + Qa treatment did not
restore PrPC levels, although it reduced PrPSc levels by 100%
(Fig. 1E, PTA Lysate 3a, 3b).
dritic degeneration was detectable approximately 1.5 weeks
after PrPSc began to form in the brain region. When BrnAggs
were infected with RML prions on culture day 15, degeneration of dendrites was detectable within 5 days after PrPSc accumulation. To measure dendritic densities, dendrites were
immunostained by MAP-2 and traced with a felt-tipped pen on
an acetate membrane on the computer screen. The drawings
were quantified with BioQuant Morphometry software to estimate dendritic loads. Three 40 confocal stacks were obtained from 3 to 4 BrnAggs at 15, 25, and 35 days in culture.
In uninfected control BrnAggs, the density of dendrites doubled from 15 to 25 days (Figs. 2A, B). At 35 days, the density of
dendrites decreased to the 15-day level presumably as a result
of pruning back of the dendrites with maturation (Fig. 2C).
In contrast, BrnAggs infected with RML prions showed a
progressive decrease in the length and number of dendrites
at 25 and 35 days (Figs. 2D, E). BioQuant measurement of
dendritic densities showed a significant decrease in RMLinfected BrnAggs at 25 and 35 days (10 and 20 dpi) versus
uninfected controls (p G 0.03, n = 3) (Fig. 2F).
Dendritic Degeneration in Prion-Infected
BrnAggs
Among the earliest neurodegenerative changes seen in
experimental RML scrapie are shortening of dendrites and a
decrease in the number of dendritic branches (24). In mice and
BrnAggs, dendritic degeneration occurs in response to PrPSc
accumulation in plasma membranes (Fig. 2H) (43). Therefore,
we measured the growth rate of dendritic density in uninfected
control and in RML-infected BrnAggs. In mouse studies, den-
Dendritic Loss Is Not Caused by Loss of Neurons
We evaluated the MAP-2 staining as a function of the
number of neurons at 20 dpi to RML prions (35 days in culture) and compared them with uninfected cultures. Differentiated neurons were identified by MAP-2 staining of their
cytoplasm (44). In 3 uninfected cultures, nerve cell bodies with
multiple attached dendrites showed intense perinuclear MAP-2
staining and deposition of MAP-2 throughout the remainder
of the enlarged nerve cell body (Fig. 3A). In contrast, 3 RMLinfected BrnAggs showed very few MAP-2Ypositive dendrites
and lack of enlargement of neuronal cell bodies, but they did
show prominent perinuclear MAP-2 staining (Fig. 1B, arrows).
We counted all neurons with perinuclear MAP-2 in each confocal stack whether or not they had attached dendrites. The
RML-infected BrnAggs had 30% to 40% more neurons fulfilling the minimum MAP-2 criteria (Fig. 1C). This increase in the
numbers of neurons was significant (p G 0.03, n = 3). These
data indicate that dendritic degeneration occurred without loss
FIGURE 3. Degeneration of dendrites during scrapie infection
is not associated with a decrease in the number of neurons.
Dendrites and nerve cell bodies are stained with microtubuleassociated protein 2 (MAP-2, red). Nuclei are stained with Hoechst
(blue). (A) Representative of 3 uninfected brain aggregates
(BrnAggs) at 35 days in culture showing many neurons with
long and branched dendrites, dendrite-bearing neurons that
have enlarged MAP-2Ypositive cell bodies, and neurons without
dendrites that have perinuclear MAP-2 staining. (B) Brain aggregates at 35 days in culture (n = 3) that were infected with
Rocky Mountain Laboratory (RML) scrapie prion strain prions for
20 days contain a few neurons with short and unbranched dendrites, atrophied cell bodies, and many neurons with perinuclear
MAP-2 staining. Arrows indicate neurons without dendrites but
with perinuclear MAP-2 staining. (C) Number of all neurons
identified with perinuclear MAP-2 staining are significantly increased in RML-infected BrnAggs versus control (p G 0.05 by t-test).
Analysis is based on stacks of 1-Km-thick confocal images.
Scale bar = (A, B) 30 Km.
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of neurons. This conclusion is consistent with the fact that
dendritic loss in prion disease is caused by PrPSc activation
of Notch-1 and can be prevented by treatment of mice and
BrnAgg with GSI + Qa. One possibility that more neurons
displayed MAP-2 staining may be caused by the existence
of neuronal stem cells in the BrnAggs that do not express
MAP-2. Another possibility is that to rebuild dendrites lost
because of RML infection, neurons released dendritic growth
factors from presynaptic nerve endings that act on all neurons
to induce MAP-2.
FIGURE 4. Confocal 1-Km-thick images of uninfected brain aggregates (BrnAggs). (A, B) Light-chain 3-II (LC3-II, red), cathepsin-D
(Cat-D, green) and Hoechst stain (blue) at 25 (A) and 37 (B) days in culture are shown. Yellow color indicates colocalization of
LC3-II and Cat-D. The rectangle in (A) corresponds to the images in (C) and (D). The inside of the BrnAggs are marked with an ‘‘i’’
and outside the BrnAggs with an ‘‘o.’’ (C) Higher magnification of the image in (A) shows that LC3-II (red) localized in neuronal
nuclei (lavender color), in the nerve cell body (oval), and in the neuropil away from the cell body (arrows). (D) Same field as in (C)
but stained for Cat-D (green). Cell body lysosomes are distributed in numbers, and distribution is similar to that in Figure 2H.
Scale bars = (A, B) 30 Km; (C, D) 20 Km.
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
Prevention of Dendritic Degeneration
Given that dendrites degenerate in BrnAggs as they do
in vivo, we tested whether treatment with GSI, Qa, or GSI + Qa
would prevent dendritic degeneration as in mice (17). The effects of each drug treatment were evaluated on 4 to 7 BrnAggs.
The 3 sites chosen in each BrnAgg contained the highest density of dendrites. Treatment with GSI (5 KM), Qa (2.5 KM), or
GSI + Qa (same doses) was begun 10 days after the BrnAggs
had been exposed to RML prions and continued for 10 more
days. Mean total lengths of dendrites and standard deviations
were measured (Fig. 2G). Untreated RML infection reduced
the lengths of dendrites by approximately 40% compared with
the lengths in uninfected control BrnAggs. Treatment with GSI
had virtually no effect on dendritic lengths. Treatment with Qa
preserved dendritic lengths 75% compared with those of controls. In the GSI + QaYtreated BrnAggs, dendritic lengths were
similar to those in uninfected controls. Thus, GSI + Qa effects
on PrPSc levels (Figs. 1D, E) and on dendrites verify that the
BrnAgg model accurately reproduces treatment effects in mice.
Confocal Microscopy
We used confocal microscopy in most to localize PrPSc,
Cat-DYimmunopositive lysosomes, and LC3-IIYimmunopositive
autophagosomes in groups of 3 to 5 uninfected control, RMLinfected untreated, and RML-infected and Rap-, Qa-, or Rap +
QaYtreated BrnAggs. In each of these categories, the 3 to 5
BrnAggs yielded different but characteristic distributions of
PrPSc, lysosomes, and autophagosomes. Representative confocal images emphasize the characteristic similarities and differences, although the confocal data are not quantitative.
Subcellular Compartments in Uninfected BrnAggs
At day 25 in culture, many nuclei in uninfected control
BrnAggs exhibited LC3-II (red) immunoreactivity that combines
with Hoechst (blue) to produce a lavender color (Figs. 4A, C).
At this time point, punctate deposits of LC3-II filled the neuronal cell body (Fig. 4C) and most of the neuropil between nerve
cell bodies (Fig. 4C, arrows). A nuclear form of LC3 that regulates levels of fibronectin mRNA has been described (45). The
presence of soluble LC3 in the nucleus or cytoplasm becomes
less obvious as levels of LC3 become associated with autophagosomes. LC3 was originally identified in the cytoplasm as a
microtubule-associated protein (46, 47). LC3-II interacts with the
dendrite-specific Ca2+-sensing protein, caldendrin (48). These
latter 2 associations indicate that some of the cell body immunostaining and the neuropil immunostaining of LC3-II were
within dendrites and were likely participating in the pruning back
of dendrites between 25 and 35 days in culture (Figs. 2AYC, F).
Large numbers of lysosomes in the nerve cell bodies at 25 days
were also probably contributing to pruning back the dendritic
trees (Fig. 4D). At 37 days, the number of LC3-II particles and
Cat-D-immunopositive lysosomes were greatly diminished.
In neurons of uninfected BrnAggs at 25 days in culture,
lysosomes filled the neuronal cytoplasm and tend to surround
their nuclei (Fig. 4D). Small numbers of LC3-II autophagosomes were scattered among the lysosomes, and some of the
lysosomes and autophagosomes were merged (Fig. 4A, yellow).
At day 37 in culture, most of the LC3-II had migrated out of
nuclei and appeared to be associated with autophagosomes
Prion-Infected Brain Aggregate Model
based on the size of the LC3-II particles and their subcellular
location scattered among lysosomes (Fig. 3B). Other LC3-II
deposits were scattered in the neuropil. Very few LC3-II particles appeared merged with Cat-D (Fig. 4B).
PrPSc in Subcellular Compartments
The temporal stages of subcellular PrPSc accumulation in
BrnAggs infected with RML prions were similar to those found
in vivo (43, 49). In the rodent brain, PrPC was converted to
PrPSc in the plasma membrane and/or in early endosomes (50).
Accumulation of PrPSc in the neuronal plasma membrane occurred rapidly and caused a dysfunction of membrane components followed by degeneration of the plasma membrane and
synaptic structures (51, 52). In the following 3 to 6 weeks, PrPSc
accumulated in sufficient amounts in lysosomes and autophagic
vacuoles, where it triggered further nerve cell dysfunction and,
with the help of microglia, nerve cell death (49, 53, 54).
At 5 and 10 dpi, PrPSc was confined to the neuronal
plasma membrane in some RML-infected BrnAggs (Fig. 2H).
By 22 dpi, PrPSc colocalized with the Cat-DYimmunopositive
(green) lysosomal compartment (Figs. 5A, C). Many of the
remaining small and large aggregates of PrPSc in the neuronal cell body were adjacent to the lysosomal compartment
(Fig. 5C). These intracellular aggregates of PrPSc colocalized with LC3-IIYimmunopositive autophagosome (yellow)
(Figs. 5B, D). Compared with the 37-day control (Fig. 4B),
there was no or only a small increase in the number of lysosomes; however, the RML-infected lysosomes at 22 dpi
(37 days in culture) tended to distribute abnormally in the nerve
cell body, where they formed adherent clusters, in which some
contained PrPSc and displayed yellow colocalization with green
Cat-D (Fig. 5C). The PrPSc bearing Cat-D and LC3-II compartments did not colocalize (data not shown).
In RML-infected BrnAggs, LC3-II staining was also seen
in a large number of particles, 1 to 4 Km in diameter, scattered
throughout the neuropil and overlying some neuronal nuclei
(Figs. 5B, D, green). A few of these particles were seen in the
uninfected control BrnAggs at 37 days in culture (Fig. 4B). One
possibility is that they were populations of autophagosomes
that accumulated in axons and presynaptic boutons during
prion disease, as we will discuss below (52, 55).
Rap Effects
Rapamycin has been shown to decrease PrPSc in vitro
(29, 56) and to increase autophagy (30, 31). Rapamycin, also
known as sirolimus, has manageable side effects and has been
approved for chronic treatment in humans to prevent rejection
after renal transplantation (Prescribing Information for Rapamune [sirolimus], Pfizer, Inc, 2010).
We tested the ability of Rap and Rap + Qa to clear PrPSc
from CL3.ScN2a cells, in which cell division had been
arrested with sodium butyrate. Rapamycin alone did not clear
PrPSc from CL3.ScN2a cells (Fig. 6). Treatment with Rap
alone was identical to untreated CL3.ScN2a cells. In contrast,
Rap + Qa rapidly cleared PrPSc in 1 to 2 days and held the
markedly reduced PrPSc levels for another 5 days (Fig. 6).
Therefore, we used BrnAggs to explore the effect of Rap or
Rap + Qa on PrPSc levels.
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Prion-Infected Brain Aggregate Model
was examined with Cat-D (green), very little intracellular LC3II was identified, and the small amount of LC3-II that was
present showed little or no merging with Cat-D (Fig. 8C).
Rap + Qa Effects
FIGURE 6. Treatment of nondividing CL3.ScN2a cells with
rapamycin (Rap) alone had no effect on the pathogenic form of
the prion protein (PrPSc) levels; Rap + quinacrine (Qa) markedly
reduced PrPSc levels within 2 days of treatment. The Rap alone
curve is very similar to that of untreated CL3.ScN2a cells.
At 20 dpi with RML prions and after 10-day treatment
with Rap, BrnAggs had large increases in the number of
Cat-DYimmunopositive lysosomes (Fig. 7A) and LC3-IIY
immunopositive autophagosomes in neurons (Fig. 7B). Some
neurons appeared full of merged Cat-D and LC3-II, suggesting that a single autolysosome compartment had been formed
(Fig. 7C). The PrPSc colocalized with both the Cat-D compartment (Fig. 7D) and the LC3-II compartment (Fig. 7E) and
remained, for the most part, intracellularly within neurons.
Very little PrPSc accumulated at the periphery of the BrnAgg
(Figs. 7Di, Ei). Rapamycin alone treatment did not appear to
degrade PrPSc in lysosomes, autophagosomes, or autolysosomes,
consistent with its protease resistance.
Qa Effects
In nondividing CL3.ScN2a cells, continuous treatment
with Qa initially decreased PrPSc levels; but after 2 days, the levels rebounded (57). Quinacrine increased the number of lysosomes in mouse liver cells (37) but inhibited LC3-II turnover in
mammary cells (38). Similarly, treatment of BrnAggs with Qa
alone for 17 days dramatically eliminated LC3-II staining in
the cell bodies of neurons (Fig. 8B) and increased the number
of Cat-D lysosomes (Figs. 8A, C). It also appeared to decrease
the number of the individual punctate LC3-II particles in the
neuropil (Fig. 8B). The PrPSc colocalized with a few of the
punctate deposits of LC3-II (Fig. 8B). In evaluating PrPSc
treated with Qa in BrnAggs, panels A and B of Figure 8 show
an overall decrease of PrPSc in the nerve cell body but an increase of PrPSc at the periphery of the BrnAgg. Western blot
analysis showed a statistically significant 37% decrease in
PrPSc (p G 0.05, n = 3) (Figs. 9A, B). When the LC3-II (red)
Rapamycin + Qa treatment appeared to trigger a conflict
between the effect of Rap to increase LC3-II autophagosomes
(Fig. 7B) and the inhibitory effect of Qa on autophagosomes
(Fig. 8B). After 12 days of treatment with Rap + Qa, Qa dominated the inhibitory effect on LC3-II. It cleared most of the
LC3-II compartment from nerve cell bodies (Fig. 10B); however, the Rap effect on autophagosome left some LC3-II in the
cell bodies, which merged with Cat-D lysosomes (Fig. 10C).
The Cat-D lysosomes in the cell body tended to be more irregularly arranged with Rap + Qa (Fig. 10A); whereas with Qa,
the lysosomes tended to be the same size and evenly dispersed
in the cell body (Figs. 8A, C). Some PrPSc remained associated
with Cat-D lysosomes (Fig. 10A). Other deposits of PrPSc occurred in the cell bodies of cells that did not contain Cat-D
or LC3-II (Figs. 10A, B). These cells are most likely microglia
(see below). With Rap + Qa, Western blot analysis showed a
statistically significant 56% decrease of PrPSc (Fig. 9).
Extracellular PrPSc Is Ingested by Microglia
We examined the extracellular space for the presence of
microglia and evaluated their capability to ingest PrPSc. Microglia identified with the Iba-1 antibody (green) displayed yellow
immunolabeling after merging with PrPSc (red) (Fig. 11). The
number of double-immunolabeled yellow signals varied with
infection and treatment. Uninfected BrnAggs showed very few
resting microglia, with a very small amount of cytoplasm containing merged PrPC and Iba-1 (Fig. 11A). Untreated RML-infected
BrnAggs showed small amounts of PrPSc in the extracellular
space and relatively few microglia with the merged signals
(Fig. 11B). Treatment with Qa showed the least amount of
merged PrPSc and Iba-1 (Fig. 11C), which indicated that Qa did
not cause proliferation of activated microglia. Treatment with
Rap showed an intermediate level of merged signal (Fig. 11D),
indicating that larger numbers of activated microglia had ingested PrPSc. Treatment with Rap + Qa showed the largest
amount of ingested PrPSc and the highest amount of merged
PrPSc and Iba-1 (Fig. 11E). The increased volume of microglial
cytoplasm after GSI + Qa treatment indicates that the microglia
were activated.
DISCUSSION
Brain aggregates provided a clear view of the distribution of PrPSc in the plasma membrane, lysosomes, and
autophagosomes of neurons. They also allowed us to view the
release of PrPSc into the extracellular space and ingestion
FIGURE 5. Confocal images of untreated, Rocky Mountain Laboratory (RML) scrapie prion strainYinfected brain aggregates
(BrnAggs), 1 Km thick, 20 days postinfection (dpi). (A) Low-power view stained for pathogenic form of the prion protein (PrPSc, red),
cathepsin-D (Cat-D, green) and Hoechst (blue). Colocalization of PrPSc with Cat-D is yellow. (B) Low-power view of PrPSc (red), lightchain 3-II (LC3-II, green) and colocalization of PrPSc with LC3-II (yellow). The inside of the BrnAgg and the outside culture medium are
indicated by i and o, respectively. (C) Higher power of the neuron marked with an asterisk (A). The PrPSc (red) is not detected in the
plasma membrane of neurons. The overlap of PrPSc and Cat-D (Green) is yellow (merge). The remaining intraneuronal PrPSc is
adjacent to lysosomes (arrow). (D) Most of the PrPSc (red) adjacent to lysosomes are located in LC3-II (green)Ypositive autophagosomes (yellow) (arrow). Asterisks indicate the same neuron in all images. Scale bars = (B, D) 20 Km; ([A, C], respectively) 20 Km.
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Prion-Infected Brain Aggregate Model
of PrPSc by microglia. Using specific markers to identify
lysosomes and autophagosomes, we monitored 3 subcellular
mechanisms that control trafficking of PrPSc in neurons: endocytosis, phagocytosis, and exocytosis. Treatment with Rap
alone indicated that most of the PrPSc was not degradable
by cellular enzymes, meaning that PrPSc accumulated inside
neurons in the BrnAgg. We also have preliminary data suggesting that PrPSc was not released from the extracellular
space to the medium surrounding BrnAggs (unpublished data)
and speculate that decreases in PrPSc levels must result from
decreased formation of PrPC to PrPSc in membrane rafts.
Neurons in BrnAggs form luxuriant dendritic trees. The
response of dendritic degeneration to drug treatment during
prion disease was identical in BrnAggs to the results obtained
in mice in which dendritic degeneration was the result of
PrPSc accumulation in plasma membranes that activated the
F-secretase complex cleavage of Notch-1 to the NICD (17).
Accumulation of PrPSc in plasma membrane was verified in
BrnAggs at early dpi with scrapie (Fig. 2H). The NICD activates the HES and HERP families of inhibitor effecter proteins, which in turn inhibit proneuronal genes that maintain
the length and branching of dendrites (58). Because GSI + Qa
treatment prevented PrPSc formation and dendritic degeneration in both mice and BrnAggs, we conclude that BrnAggs
accurately mimic the in vivo condition.
Transport of PrPSc by endocytosis from the plasma
membrane to late endosomes and lysosomes is well established
(43, 49, 53, 59). In BrnAggs, subcellular trafficking of PrPSc
follows the same route as in animals. Cathepsin-D immunohistochemistry demonstrated aggregation and clustering of
lysosomes and colocalization with PrPSc as in scrapie-infected
mice (43, 49). In BrnAggs, extralysosomal PrPSc in neuron cell
bodies colocalized with LC3-IIYpositive autophagosomes. This
is also consistent with data from electron microscopy that showed
large aggregates of lysosomes, autophagosomes, and multivesicular bodies in the neuron cell bodies of rodents with prion
disease (60). We also detected LC3-IIYpositive autophagosomes
intermingled with lysosomes. The LC3-II immunoreactivity
was seen as 1- to 4-Km deposits scattered throughout the neuropil, suggesting that it is located in axons during prion disease.
By electron microscopy, autophagosomes with double membranes are located in axons, axon terminals, and presynaptic
boutons in prion diseases (52, 55).
In uninfected control BrnAggs, LC3-II immunopositivity
varied with time in culture and was different from that in RML
prion-infected BrnAggs. At 25 days in culture, LC3-II was
located in the nuclei of many neurons, in the cell body, and in
the neuropil away from the cell body. By 37 days in culture,
LC3-II in the cell body becomes associated with autophagosomes, and nuclear staining is largely absent. The large amount
of LC3-II in the neuropil between nerve cell bodies seen at
25 days in culture is probably not associated with autophago-
somes but with microtubule-associated protein and dendritespecific Ca2+-sensing protein caldendrin in dendrites (46Y48).
This correlates with the evidence that LC3-II expression is
increased during dendritic growth (61). The neuropil location
of LC3-II occurs at the peak of dendritic growth in uninfected
BrnAggs. The pruning back of dendrites during the succeeding 10 days correlates with the decrease of LC3-II in the neuropil. Immunocolocalization of LC3-II in dendrites requires
autoclaving to make the epitopes available to LC3-IIYspecific
antibodies. We found, however, that autoclaving precludes
detection of MAP-2Ypositive dendrites and A-3-tubulinY
positive axons (unpublished data). Therefore, we were unable
to verify that LC3-II was located in dendrites.
Quinacrine is reported to increase the number of lysosomes in liver cells (37) and to inhibit LC3-II turnover in
mammary cells (38). In addition to destabilizing membrane
rats, we found that Qa exerts a broad range of effects on
RML-infected BrnAggs, which increased the number of
Cat-DYimmunopositive lysosomes in neural cells and caused
an almost complete reduction of LC3-II in RML-infected
BrnAggs. Western blot measurements indicated that Qa treatment decreased PrPSc levels by 37% versus untreated and Rap
aloneYtreated infected BrnAggs (Fig. 9). Quinacrine increased
the number of Cat-D lysosomes, but they do not degrade PrPSc.
Very few activated microglial cells were present in the extracellular space, and they do not appear to degrade PrPSc
either. Therefore, the decrease of PrPSc was most likely caused
by effects of Qa on membrane rafts, which would prevent
PrPC from interacting with PrPSc and forming more PrPSc.
In the usual progression of autophagy in normal cells,
autophagosomes with double limiting membranes bind to
lysosomes to acquire acidic hydrolases and then mature into
autolysosomes with a single limiting membrane (62). In
BrnAggs, the discovery of PrPSc in LC3-IIYpositive autophagosomes is new evidence that this phagocytic pathway is
active in prion disease as has been reported in Alzheimer
disease (63, 64). Both PrPC and PrPSc are released from RMLinfected cells into the extracellular space by exocytosis (65,
66). The cellular mechanisms underlying this form of exocytosis have been uncovered in studies of lipid raft proteins,
including glycophosphatidylinositol-anchored proteins such as
PrPC and PrPSc (67). Developing autophagosomes presumably
sequester plasma membrane fragments containing PrPC and PrPSc
(68Y70). The PrPSc-containing autophagosomes fuse with the
multivesicular bodies derived from the endosomal compartment, and the resulting hybrid structure binds to the plasma
membrane to release its contents into the extracellular space.
Normal neurons contain very few autophagosomes, whereas in
prion diseases, neuron cell bodies contain large clusters of
a mixture of lysosomes, autophagosomes, and multivesicular
bodies that are partially fusing or fully fused (60). In prion
disease, distal axons and presynaptic boutons also contain
FIGURE 7. (AYE) In Rocky Mountain Laboratory (RML) scrapie prion strainYinfected brain aggregates (BrnAggs), Rapamycin (Rap)
alone increases the number and amount of cathepsin-D (Cat-D, green) (A) and light-chain 3-II (LC3-II, red) (B) in neurons and
merges the 2 compartments (yellow) (C). Most of the pathogenic form of the prion protein (PrPSc, red) colocalize with the Cat-D
(green) compartment (yellow) (D). The PrPSc (red) also colocalize with the LC3-II compartment (green) (E). See Figure 5 for
appropriate untreated control. ‘‘o’’ indicates outside the BrnAgg in (D) and (E). Scale bar = (AYE) 20 Km.
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FIGURE 8. In Rocky Mountain Laboratory (RML) scrapie prion strainYinfected brain aggregates (BrnAggs), quinacrine (Qa) alone
seems to increase the number of cathepsin-D (Cat-D, green) lysosomes (A), which was verified by merging the Cat-D signal
(green) with the light-chain 3-II (LC3-II, red) (C). Quinacrine mainly eliminates most of the LC3-II signal (green), except the
relatively small numbers of diffusely distributed LC3-II particles (B). Arrows in (B) indicate colocalization of punctate LC3-II particles
with the pathogenic form of the prion protein (PrPSc, red). In (A), (B) and (C), inside and outside the BrnAgg are indicated by ‘‘o’’
and ‘‘i.’’ Scale bar = (AYC) 30 Km.
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
FIGURE 9. Groups of 15 brain aggregates (BrnAggs) were subjected to Western blot measurements of total pathogenic form
of the prion protein (PrPSc). The groups were untreated (UnTx),
Rapamycin (Rap) aloneYtreated, quinacrine (Qa) aloneYtreated,
and Rap + QaYtreated BrnAggs. (A) Representative Western blot
not exposed to proteinase K shows PrPSc and A-actin. (B) Densitometry measurements of the 3 untreated Rocky Mountain Laboratory (RML) scrapie prion strainYinfected samples were given
the same value because the measurements varied considerably
for the 3 groups of data. The mean and standard deviations and
t-test were determined for the 3 treatments. *t-test probability
(p G 0.05, n = 3) for a decrease of PrPSc for Qa alone and
Rap + Qa versus Rap alone.
autophagosomes and no obvious multivesicular bodies adjacent
to disintegrating plasma membranes (52, 55). It is possible that
some autophagosomes can release their contents including PrPSc
into the extracellular space via local degenerating membranes.
In untreated RML-infected BrnAggs, most of the autophagosomes did not appear to bind to lysosomes; instead, they
were positioned adjacent to lysosomes and existed as large
and small aggregates of PrPSc that colocalized with LC3-IIY
positive autophagosomes. It has been reported that autophagosomes containing AA-42 peptide in Alzheimer disease
function abnormally and may be the origin of most of the AA42 in the brain (63, 64). Autophagosomes containing PrPSc
enter an ‘‘unconventional’’ exocytosis pathway that deposits
PrPSc in the extracellular space (68Y70).
It is assumed that protease-resistant PrPSc is not degradable by lysosomal enzymes. It is also assumed that the
BrnAgg is a closed system, that is, that PrPSc formed and
released by neurons stay within the BrnAgg. We need to
Prion-Infected Brain Aggregate Model
verify this by collecting media for 1 week from RML-infected
BrnAgg cultures and assaying for PrPSc. Rapamycin alone did
not produce a significant decrease of intracellular PrPSc as a
result of exocytosis when compared with PrPSc levels in untreated RML-infected BrnAggs. This was attributed to storage
of PrPSc intracellularly within increased numbers of Cat-D
and LC3-II compartments. However, if a sizable proportion
of total PrPSc were protease sensitive, a significant decrease
in total PrPSc would have been expected.
Treatment with Rap + Qa produced a significant decrease in PrPSc (Fig. 9). Confocal microscopy revealed competition between activation of autophagy by Rap (30, 31) and
inhibition of autophagy by Qa (38) (Fig. 10). Merging of the
Cat-D and LC3-II signals demonstrated a modest overlap of
lysosomes and autophagosomes (Fig. 10C). Treatment with
Rap alone led to a much larger overlap (Figs. 7AYC) that
contained undegraded PrPSc. Western blot analysis showed an
approximately 10% decrease in PrPSc levels compared with
untreated controls (Fig. 9). Together, these data support the
conclusion that most of the PrPSc accumulated in lysosomes,
autophagosomes, and autolysosomes and was not degraded.
The overlap of Cat-D lysosomes and LC3-II autophagosomes
with Rap + Qa accounted for 5% to 10% decrease of PrPSc,
as did with Rap alone. Therefore, Qa effects on membrane
lipid rafts likely caused the remaining approximately 46%
decrease in PrPSc.
The LC3-II autophagosomes predictably fuse with multivesicular bodies, which lead to unknown amounts of PrPSc
exocytosis. As a surrogate marker of exocytosis, we used Iba-1
antibodies to mark microglia and PrP antibodies to identify
ingested PrPSc. In scrapie-infected BrnAggs, activated microglia ingested PrPSc. They do not form significant amounts of
PrPSc de novo. Activated microglia with ingested PrPSc release
cytokines that cause nerve cell death of neurons expressing
PrPC (71). We found the highest numbers of activated microglia with the Rap + Qa treatment followed by Rap alone. We
believed that Rap activates microglia, although it is reported
to inhibit microglial activation (72). It is possible that the relatively large amount of PrPSc ingested by microglia caused
their activation; an increased volume of the microglial cell
body identifies activation. In RML-infected BrnAggs that
were untreated or treated with Qa only, we observed very low
numbers of activated microglia with a small amount of ingested PrPSc. Uninfected BrnAggs did not have recognizable
activated microglia. Therefore, we conclude that PrPSc was
transported to the extracellular space, particularly following
treatment with Rap + Qa and Qa alone.
The present results explain why Rap alone did not clear
PrPSc from CL3.ScN2a cells (Fig. 6). Rapamycin alone increased
LC3-II autolysosomes and Cat-D lysosomes in BrnAggs. The
PrPSc became stored in both of those compartments, and it
appeared that very little PrPSc was removed from neurons by
multivesicular bodyYfacilitated exocytosis. Western blot analysis showed an approximately 10% decrease of total PrPSc versus untreated controls. In contrast, Rap + Qa treatment of
CL3.ScN2a cells cleared PrPSc by 100% in 2 days. In BrnAggs,
Rap + Qa reduced total PrPSc by 56%. Confocal microcopy
showed that the Qa component reduced the number of LC3-II
autophagosomes and merged some indigestible PrPSc with
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FIGURE 10. (A, B) In Rocky Mountain Laboratory (RML) scrapie prion strainYinfected brain aggregates (BrnAggs), rapamycin (Rap)
+ quinacrine (Qa) treatment has little effect on cathepsin-D (Cat-D)Ypositive lysosomes (A) but markedly reduces the number of
light-chain 3-II (LC3-II)Ypositive autophagosomes (B). Pathogenic form of the prion protein (PrPSc, red) is located in neurons where
it variably merges with Cat-D (large arrows) (A) but not with LC3-II (B). The small arrows in (A) and (B) indicate cells that contain
PrPSc but not merged with Cat-D or LC3-II, respectively. Most of the PrPSc appears to be beneath the outer perimeter of the
BrnAggs. The region outside of the BrnAggs is marked with an ‘‘o.’’ (C) The LC3-II (red) and Cat-D (green) are merged (yellow) in
some neurons from a different field than shown in (A) and (B). Scale bar = (AYC) 20 Km.
464
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J Neuropathol Exp Neurol Volume 71, Number 5, May 2012
Prion-Infected Brain Aggregate Model
FIGURE 11. (AYE) Pathogenic form of the prion protein (PrPSc, red) is present in the extracellular space where is ingested by
microglia identified with Iba-1 (green). Merging the PrPSc and the Iba-1 stains (yellow) signifies ingestion. Scale bar = (AYE) 30 Km.
Cat-DYpositive lysosomes in neurons and with Iba-1Ypositive
microglia in the extracellular space. Because PrPSc is largely
indigestible, Rap + Qa decreased PrPSc formation by the effect
of Qa on membrane rafts. Most of the PrPSc remained in the
BrnAgg within neurons, within the extracellular space, and
within microglia. In CL3.ScN2a cells, we expect that PrPSc
formation was reduced by the Qa component of Rap + Qa, and
all PrPSc was discharged from the cells by exocytosis. Exocytosed PrPSc was removed from the culture with media change.
To confirm this hypothesis, we will collect media from the cell
culture to test for the presence of PrPSc.
In summary, we examined the behavior of RML prions
in BrnAggs in detail. Brain aggregate cultures are a suitable
model because their neurons and glia interact with each other
as much as they do in the CNS. The neuronal-glial relationship most likely fostered prion infection, prion propagation,
and neuronal dysfunction. The results explain why Rap alone
treatments of many genetic disorders and prion disease have
had limited success. In our preliminary study in CD-1 mice,
Rap + Qa did not increase survival from RML infection (Ahn et
al, unpublished data). We will use BrnAggs to explore the
behavior of multiple other scrapie prion strains and, in appropriate transgenic mice expressing human PrPC, test CreutzfeldtJakob disease prions. Our first goal will be to determine
whether, unlike neurospheres, BrnAggs are susceptible to different prion strains. The ultimate goal is to use an in vitro
system to test anti-prion drugs and gene therapies for human
prion diseases. Brain aggregates will elucidate cellular and
molecular pathobiology of prion diseases and other neurodegenerative disorders. Because it is possible to obtain meaningful biology and drug data in approximately 40 days with
BrnAggs, this is more efficient than obtaining comparable data
from mice that take 100 to 200 days to obtain.
ACKNOWLEDGMENTS
The authors thank Dr Stanley Prusiner for animals and
PrP antibodies and Dr Lynn Pulliam for her help with learning
the BrnAgg culture methods.
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