Journal of Neuropathology and Experimental Neurology
Copyright q 2002 by the American Association of Neuropathologists
Vol. 61, No. 1
January, 2002
pp. 85 90
Accumulation of b-Amyloid Precursor Protein in Axons Correlates with CNS Expression
of SIV gp41
J. L. MANKOWSKI, DVM, PHD, S. E. QUEEN, BS, P. M. TARWATER, PHD, K. J. FOX, MS,
V. H. PERRY, MA, DPHIL
AND
Abstract. Axonal damage represented by accumulation of b-amyloid precursor protein (b-APP) develops in numerous central
nervous system (CNS) diseases including human immunodeficiency virus (HIV) infection. To study the underlying mechanisms of axonal damage associated with HIV CNS infection, the amount of axonal b-APP immunostaining in the corpus
callosum of 24 simian immunodeficiency virus (SIV)-infected macaques and 3 control macaques was measured by computerized image analysis. The amounts of b-APP accumulation were then compared with time post-inoculation, extent and
character of CNS inflammation, and viral load in the CNS measured by the amount of immunohistochemical staining for the
viral transmembrane protein gp41. Significant increases over control values were present in 10 of 24 SIV-infected animals.
SIV encephalitis was present in 9 of the 10 animals with elevated b-APP. Increases in b-APP correlated most strongly with
levels of SIV gp41 in the brain (p 5 0.005), but significant associations with macrophage infiltration and microglial activation
(p 5 0.04) and infiltration by cytotoxic lymphocytes (p 5 0.05) also were identified. These data demonstrate that b-APP
accumulation in the white matter of SIV-infected macaques develops during SIV infection in close correlation with levels of
viral replication and may serve as a sensitive marker of neuronal/axonal damage mediated by viral proteins.
Key Words:
b-Amyloid precursor protein; Axonal damage; SIV gp41.
INTRODUCTION
head trauma and ischemia secondary to cerebrovascular
disease (9–11). b-APP accumulation also has been documented in chronic neurologic diseases, including multiple sclerosis, HTLV-I myelopathy, and HIV encephalitis
(6, 12, 13). In these conditions, an association has been
suggested between CNS inflammation and b-APP accumulation. Normally, b-APP is transported by fast anterograde transport along axons and cannot be detected readily by immunohistochemical staining. In contrast,
accumulation of b-APP within axons reflects impairment
of axonal transport mechanisms indicative of axon damage (10). Although elevated axonal b-APP has been
linked with the presence of inflammatory cells, an understanding of the underlying mechanisms of this axonal
damage remains to be established.
The SIV/macaque model has proven valuable for examining the pathogenesis of HIV CNS infection. Pigtailed macaques co-inoculated with the neurovirulent SIV
clone SIV/17E-Fr and an immunosuppressive strain, SIV/
DeltaB670, develop SIV encephalitis in a rapid, reproducible fashion facilitating studies of HIV neuropathogenesis (14). To examine the underlying mechanisms of
axonal damage in HIV CNS infection, the relationship
between axonal accumulation of b-APP and both CNS
inflammation and viral load were examined in this wellcharacterized SIV/macaque model of HIV CNS disease.
Human immunodeficiency virus (HIV) infection of the
central nervous system (CNS) is often manifest clinically
as AIDS dementia, affecting approximately 20% of individuals with AIDS (1). Despite this high prevalence of
neurological disease in HIV-infected people, the mechanisms contributing to the pathogenesis of CNS disease
are not well understood. Although neuronal damage and
loss are features of HIV infection in the CNS, particularly
at end-stage AIDS, neuronal infection is not believed to
play a significant role in HIV encephalitis. Rather, neurotoxic products, including viral proteins and host immune factors produced by activated macrophages and microglia, are believed to cause the neuronal damage (2).
HIV/simian immunodeficiency virus (SIV)-associated
neuronal damage ranges from loss of dendritic arborization and neuronal atrophy to overt neuronal loss and cortical atrophy (3–5). Axonal damage also has been reported in HIV CNS infection but its cause and
consequences have not been defined (6–8).
Axonal accumulation of b-amyloid precursor protein
(b-APP), a protein constitutively produced by neurons,
serves as a sensitive marker of acute axonal damage in
From the Division of Comparative Medicine (JLM, SEQ) and Department of Pathology (JLM), Johns Hopkins University School of
Medicine, Baltimore, Maryland; Department of Epidemiology (PMT,
KJF), Johns Hopkins University School of Hygiene and Public Health,
Baltimore, Maryland; and The CNS Inflammation Group (VHP), University of Southampton, Southampton, United Kingdom.
Correspondence to: J. L. Mankowski, DVM, PhD, Johns Hopkins
University School of Medicine, Division of Comparative Medicine,
Ross 464, 720 Rutland Ave., Baltimore, MD 21205.
This work was supported by a Burroughs Wellcome Travel Grant
APP #3298 and by NIH RR000116.
MATERIALS AND METHODS
Animals
24 pig-tailed macaques (Macaca nemestrina) were intravenously inoculated with SIV/DeltaB670 (50 AID50), and SIV/
17E-Fr (10,000 AID50) as previously described (14). Infected
animals were killed at 3 different time points post-inoculation
(PI): 3 wk (6 animals), 8 wk (6 animals), and 3 months (12
85
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MANKOWSKI ET AL
animals). Three additional macaques were mock-inoculated
with media alone and served as virus-negative controls. The
animal procedures in this study were performed according to
the principles set forth by the Institutional Animal Care and
Use Committee at Johns Hopkins University and the National
Research Council’s Guide for the care and use of laboratory
animals.
Immunohistochemical Staining and Histopathology
To identify b-APP accumulation in axons, coronal sections
of brain tissue including basal ganglia, frontal cortex, and corpus callosum were immunohistochemically stained with the
monoclonal antibody anti-b-amyloid precursor protein 695 that
detects 3 forms of b-APP: b-APP695, b-APP751, and b-APP770
(Clone LN27, Zymed, South San Francisco, CA). For detection
of viral protein, a monoclonal antibody directed against the SIV
transmembrane portion of the SIV envelope, kk41, was used
(diluted 1:400, AIDS Reagent Program). Primary antibodies
against the following antigens were also used to assess CNS
cellular alterations: CD68, a marker of microglial activation and
macrophage infiltration (KP-1, diluted 1:2,000, DAKO, Carpinteria, CA), CD3 to detect T lymphocytes (diluted 1:400,
DAKO), TIA-1, a component of the cytotoxic granules of both
cytotoxic T lymphocytes and natural killer cells (diluted 1:100,
Coulter, Hialeah, FL), and GFAP for evaluation of astrocyte
activation (diluted 1:4,000, DAKO). All brain tissue sections
were stained by an automated immunostainer (Optimax Plus,
BioGenex, San Ramon, CA) for uniformity. Streck-fixed, paraffin-embedded brain tissue sections were deparaffinized, rehydrated, and then post-fixed in Streck tissue fixative (Streck
Laboratories, Omaha, NE) for 20 min. After rinsing in water,
tissues were heated in a microwave in sodium citrate buffer
(0.01 M, pH 6.0) for 8 min to retrieve antigen. Endogenous
peroxidase was quenched with 3% H2O2 for 10 min and then
sections were blocked with buffered casein for 5 min. Primary
antibody was applied to tissue sections for 60 min at room
temperature, the tissues were washed in buffer, and then secondary biotinylated multilink antibody (Biogenex) was added
for 20 min. After washing, streptavidin-horseradish peroxidase
was applied for 20 min, followed by diaminobenzidine tetrahydrochloride in buffer containing H2O2 for 10 min. Sections were
then washed, dehydrated, and mounted. To establish the relative
severity of encephalitis, sections of frontal and parietal cortex,
basal ganglia, thalamus, midbrain, and cerebellum from each
animal were scored in a blinded fashion. If sections contained
more than 30 perivascular macrophage-rich cuffs on average,
the animals were classified as having severe encephalitis. Animals averaging 10 to 30 perivascular cuffs were scored as moderate encephalitis, and those with less than 10 perivascular cuffs
were considered to have mild encephalitis.
Quantitative Image Analysis
To standardize sampling from animal to animal, coronal brain
tissue sections from all animals were prepared from the same
location in the basal ganglia, 5 mm posterior to the head of the
caudate nucleus. For each animal, 20 adjacent fields in the corpus callosum from sections immunostained for b-APP were
captured at 3200 magnification (an area of 2.8 mm2) using a
J Neuropathol Exp Neurol, Vol 61, January, 2002
Sensys 2 digital camera (Photometrics, Tucson, AZ) then analyzed by IP Lab imaging software (Scanalytics, Vienna, VA).
Image analysis transects of the corpus callosum started on the
midline (truncus) and proceeded laterally, encompassing the
identical region of corpus callosum in all animals. Similarly,
twenty 3200 fields were captured in subcortical white matter
subjacent to cingulate gyrus for measurement of viral protein,
cytotoxic lymphocytes, macrophage/microglial activation, and
astrocytic activation. In addition, GFAP immunostaining was
also measured in cortical grey matter of the cingulate gyrus on
the same tissue section. Images were binarized and the total
area occupied by immunopositive pixels calculated to measure
the total area of immunostaining.
Statistical Analysis
For each animal, the mean total area of b-APP immunostaining per 3200 field was calculated and then compared with the
mean of the 3 control animals by use of the 2-sample t-test to
determine which infected animals had significant increases in
b-APP accumulation. This analysis separated the SIV-infected
animals into 2 groups: those with b-APP immunostaining similar to control animals (b-APP like controls) and those with
increased b-APP immunostaining (b-APP unlike controls).
These 2 groups were then compared with the mean total immunostaining for viral antigen, macrophage/microglial activation, T cells, cytotoxic lymphocytes, and astrocyte activation in
white matter and cortical grey matter by the 2-tailed MannWhitney test to determine which viral and cellular parameters
in the brain were associated with increased axonal b-APP accumulation. Finally, the animals with elevated b-APP were individually ranked against the various CNS viral and cellular
parameters to measure the strength of the association between
b-APP and both viral and cellular markers by calculating Spearman’s Correlation Coefficients.
RESULTS
Patterns of Axonal APP Accumulation
Axonal accumulation of b-APP was detected by immunohistochemical staining in the corpus callosum and
diffusely throughout subcortical white matter in frontal,
temporal, parietal, and occipital lobes from macaques infected with neurovirulent SIV. Morphologically, the immunostaining pattern for b-APP was consistent with axonal accumulation represented by distinct oval structures
and long, thin fibrillar processes (Fig. 1). Axonal b-APP
immunostaining was not consistently associated topographically with infiltrates of macrophages or lymphocytes, multinucleated giant cells, microglial nodules,
presence of viral antigen within macrophages, or perivascular regions. The 4 animals with the highest b-APP
levels in corpus callosum also had the highest b-APP
levels in subcortical white matter subjacent to cingulate
gyrus, suggesting that similar axonal damage develops in
parallel in corpus callosum and centrum semiovale. However, in this study, measuring b-APP levels in corpus callosum proved more sensitive for the animals with smaller
b-AMYLOID PRECURSOR PROTEIN ACCUMULATION AND SIV GP41
Fig. 1. Photomicrograph of immunohistochemical staining
for b-APP (dark brown immunoreaction product) in damaged
axons in the corpus callosum of SIV-infected macaques reveals
2 morphologic patterns: long, thin fibrillar processes (arrows)
and distinct ovoid structures (arrowheads and inset). Hematoxylin counterstain, original magnification 3200, scale bar 5 50
mM.
increases in b-APP accumulation because of the predominantly longitudinal orientation of axons in corpus callosum in coronal sections. In contrast, the variable sectioning of axons (including oblique and cross-sections as
well as occasional longitudinal sections through axons)
in centrum semiovale makes it more difficult to measure
b-APP accumulation in those axons. Myelin pallor was
not identified by Luxol fast blue staining in any brain
sections of SIV-infected animals consistent with previous
reports noting the lack of white matter pallor in SIVinfected macaques (15, 16).
Axonal b-APP Accumulation and SIV Encephalitis
No animals (0/6) euthanized at 3 wk PI developed encephalitis, whereas 1 of the 6 animals killed at 8 wk PI
had mild encephalitis. In contrast, 11 of the 12 animals
euthanized at 12 wk PI developed encephalitis. Of these
11 animals with encephalitis, 2 were classified as mild,
5 were moderate, and 4 were severe. Characteristic encephalitis consisted of multifocal and perivascular aggregates of macrophages and multinucleated giant cells.
In total, 10 of 24 SIV-infected animals had significant
elevations in axonal b-APP in the corpus callosum (Fig.
2). Nine of the 10 animals with elevated b-APP levels
also had SIV encephalitis. Of these 9 macaques, 8 animals were euthanized 12 wk PI; the other animal with
elevated b-APP and encephalitis was killed 8 wk PI. The
only animal with elevated b-APP that did not have encephalitis was killed 3 wk PI. In addition, 3 of the animals euthanized 12 wk PI without elevated b-APP levels
did develop encephalitis, suggesting that encephalitis
87
Fig. 2. A scattergram of b-APP accumulation in corpus callosum versus time post-inoculation (PI) with SIV demonstrates
that although elevated axonal b-APP may develop as early as
3 wk PI, most animals with increased b-APP levels (D) are in
the 12 wk PI group. Most, but not all, animals with increased
b-APP levels have encephalitis. Animals with levels of b-APP,
like control animals, are denoted by a circle (O). The presence
or absence of SIV encephalitis (SIVE) is denoted by 1/2 symbols, respectively.
alone was not sufficient to induce b-APP accumulation.
The sole animal without encephalitis euthanized 3
months PI did not have elevated b-APP levels. As
amounts of b-APP accumulation did not correlate with
the severity of the encephalitis score (a semi-objective
assessment), studies of the relationship between b-APP
accumulation and activated inflammatory cells in the
CNS were performed by immunohistochemical staining
and quantitative image analysis as detailed in the following section.
An advantage of this particular SIV/macaque model of
HIV CNS disease is that over 90% of inoculated macaques develop CNS disease by 3 months PI. Thus, the
majority of animals euthanized at earlier time points in
disease (including 3 wk PI and 8 wk PI) are destined to
develop CNS disease and therefore enable evaluation of
early CNS alterations that precede development of overt
encephalitis. Given this consistent progression to CNS
disease in this model, the lack of elevated b-APP in axons in most animals examined at 3 and 8 wk PI indicates
that axonal b-APP is not increased throughout infection
in most animals that ultimately will develop CNS disease.
Rather, axonal damage develops at later stages of infection.
CNS Parameters Associated with Elevated Axonal
b-APP
The 2 groups of animals (either with b-APP scores
similar to control animals or with b-APP scores elevated
J Neuropathol Exp Neurol, Vol 61, January, 2002
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MANKOWSKI ET AL
Fig. 3. Histogram depicting the relationship between
groups of animals with b-APP immunostaining similar to controls (gray on left) and groups of animals with b-APP levels
increased over controls (black on right) versus CNS parameters.
Viral antigen reflects amount of immunostaining for SIV gp41.
Macrophage/microglial activation was determined by measuring CD68 immunostaining. Similarly, values for T lymphocytes
and cytotoxic lymphocytes were obtained by measuring immunostaining for CD3 and TIA-1, a component of cytotoxic
granules, respectively. Astrocyte activation in white and gray
matter was evaluated by quantifying GFAP immunostaining.
TABLE
Analysis of Viral and Cellular Parameters Associated
with Elevated b-APP
Mean total area of immunostaining
(Pixels)
b-APP Simi- b-APP
lar to
Increased
Control Over control p value*
Viral antigen
Macrophages/Microglia
T cells
Cytotoxic lymphocytes
Astrocytes (White
matter)
Astrocytes (Grey matter)
0
2,633
190
44
219,850
1,687
16,395
1,479
407
153,645
0.003
0.001
0.001
0.001
0.613
714
57,074
0.001
* p values for analysis of the group of macaques with b-APP
levels similar to control animals versus the group of macaques
with b-APP values elevated over control animals determined by
2-tailed Mann-Whitney test.
over control animals) were then compared with measurements of CNS viral load and markers of CNS activation
and infiltration to determine which CNS parameters were
associated with axonal b-APP accumulation. The group
of 10 animals with elevated APP levels had significant
concurrent increases in viral gp41, anti-CD68 immunostaining (representing both infiltrating macrophages and
activated microglia), and both T lymphocytes and cytotoxic lymphocytes versus the group of 14 SIV-infected
animals that had b-APP levels similar to controls (Fig. 3;
J Neuropathol Exp Neurol, Vol 61, January, 2002
Table). The group of macaques with elevated b-APP also
had significant concurrent increases in GFAP immunostaining in cortical grey matter, but there was no significant difference between the 2 groups of macaques with
respect to GFAP immunostaining in subcortical white
matter, suggesting that b-APP elevations were unrelated
to astrocyte activation in white matter.
To examine the strength of the association between elevated axonal b-APP and the specific viral and cellular
parameters that were found to increase concurrently with
b-APP in the preceding group analysis, Spearman’s Correlation Coefficients were calculated. The strongest significant positive correlation was found between b-APP
accumulation and the amount of immunostaining for the
viral transmembrane protein gp41 (r 5 0.81, p 5 0.005).
Significant positive correlations were also identified between elevated b-APP and CD68 immunostaining (r 5
0.065, p 5 0.04) and cytotoxic lymphocyte infiltration (r
5 0.062, p 5 0.05), although at lower levels of statistical
significance than found for gp41. In contrast, a significant
correlation was not found between elevated b-APP and
either T lymphocytes (r 5 0.46, p 5 0.44) or GFAP immunostaining (r 5 0.66, p 5 0.16) for grey matter astrocyte activation.
DISCUSSION
This study demonstrated significant accumulation of
axonal b-APP in SIV-infected macaques with high CNS
viral burdens. In many neurologic diseases ranging from
acute CNS trauma to multiple sclerosis, accumulation of
b-amyloid precursor protein in axons is believed to represent disrupted anterograde transport (9, 12). The degree
of axonal damage in multiple sclerosis is related to the
intensity of the cellular inflammatory response but the
molecular mechanisms have not been defined (12, 17,
18). Similar associations between b-APP accumulation
and inflammation have been shown in HTLV-I myelopathy (13). Axonal damage also has been reported in people infected with HIV, including seropositive, asymptomatic individuals lacking encephalitis, as well as those with
HIV encephalitis accompanying AIDS (8). This finding
suggests that there may be other causes of axonal damage
in addition to neuroinflammation.
To help define the mechanisms of axonal damage in
HIV CNS infection, this study examined the relationships
between axonal accumulation of b-APP and CNS levels
of SIV gp41 as well as CNS inflammatory responses in
a well-characterized SIV/macaque model of HIV CNS
disease. This study was aided by the use of image analysis techniques to obtain objective measurements of immunostaining rather than relying on semi-objective scoring systems. A previous study in the SIV/macaque model
has demonstrated a statistically significant correlation between the amounts of viral antigen (SIV gp41) measured
by image analysis and the levels of viral RNA measured
b-AMYLOID PRECURSOR PROTEIN ACCUMULATION AND SIV GP41
by real-time RT-PCR in the brain (19). In this study, a
strong association (p 5 0.005) was identified between
elevated axonal b-APP levels and the amount of SIV
gp41 present in white matter, implicating HIV/SIV gp41
as a mediator of axonal damage. Interestingly, immunohistochemical assessments of CNS viral load in HIV-infected individuals employing antibodies directed against
HIVgp41 also have been correlated with neocortical dendritic and synaptic damage (20).
Viral proteins are key candidates for inducing neurodegeneration. The HIV/SIV envelope protein gp160 is
cleaved by proteases into gp120, the surface glycoprotein, and gp41, the transmembrane domain. Both of these
proteins are believed to play roles in neuronal damage.
While gp120 has been implicated as an excitotoxic mediator of neuronal damage via binding to the NMDA receptor, the relationship between CNS gp120 expression
and development of dementia has not been characterized
(21). In contrast, levels of HIV gp41 in the brain detected
by immunoblotting have been shown to correlate with the
severity and progression rate of HIV-associated dementia
(22, 23). Studies demonstrating that neuronal cultures established from iNOS -/- mice are resistant to gp41-mediated neurotoxicity suggests that gp41 may exert neurotoxic effects via iNOS induction (23, 24). Another
study demonstrated that staining for iNOS and HIV gp41
increased linearly with the severity of AIDS dementia
(25). In our report, the strength of the correlation identified between axonal b-APP accumulation and SIV gp41
suggests that axonal damage may be a mechanistic link
between gp41 production in the brain and HIV dementia.
In this study, increases in axonal b-APP levels also
were associated with the presence of CNS inflammation,
including both macrophage infiltration/microglial activation (p 5 0.04) and cytotoxic T cell infiltration (p 5
0.05). These relationships, although statistically significant, were not as strong as the demonstrated association
between b-APP and SIV gp41. In addition, 3 animals
with SIV encephalitis examined 12 wk PI did not have
b-APP levels higher than uninfected control animals.
These data suggest that although activated inflammatory
cells and their secreted products may contribute to axonal
damage, other factors such as HIV/SIV envelope proteins
may play a more significant role in axon damage.
Previous studies of MS and HIV have suggested an
association between accumulation of b-APP within axons
and myelin pallor or demyelination (7, 26). In contrast,
in this study, SIV-infected macaques with increased bAPP immunostaining did not show morphologic evidence
of myelin pallor. Although this suggests that production
of myelinotoxic substances may not be necessary for
axon damage to develop, further detailed studies to detect
subtle myelin damage are needed to clarify this relationship.
89
Although neuronal loss is characteristic of terminal
AIDS patients with HIV dementia, the progressive stages
of neuronal damage that precede overt neuronal loss have
not been well characterized. Morphometric analyses have
suggested that loss of dendritic complexity may be a morphologic feature of HIV/SIV-induced neuronal damage,
but the relationships between such complex assessments
of neuronal damage and the underlying causes of neuronal degeneration and loss have not been clearly established (5, 27). In contrast, measurement of axonal damage may offer a ready means of detecting CNS damage,
thereby facilitating study of mechanisms of neuronal
damage throughout various stages of HIV CNS infection.
ACKNOWLEDGMENTS
The antibody kk41 used to detect viral antigen was provided by the
NIH AIDS Research and Reference Reagent Program. We would like
to acknowledge the excellent assistance provided by Dr. R. J. Adams
in animal studies and M. K. Brooks in manuscript preparation. We appreciate the helpful comments on this manuscript provided by Dr. M.
C. Zink.
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Received June 7, 2001
Revision received September 27, 2001
Accepted October 1, 2001