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American Journal of Pathology, Vol. 146, No.

5, May 1995

Copyright X) American Societyfor Investigative Pathology


Apoptosis in the Brain

Physiology and Pathology

Dennis W. Dickson
From the Department of Pathology, Albert Einstein College
of Medicine, Bronx, New York

The report by Petito and Roberts1 in this issue of

American Journal of Pathology is the first documentation of apoptosis in the AIDS brain. The authors use
an in situ end-labeling (ISEL) method to detect DNA
fragmentation2 coupled with immunocytochemistry
with cell type-specific markers. They report that glial
and neuronal cells are positive in AIDS. Although
many of the ISEL-positive cells were unidentified
small cells, positive nuclei were detected in glial fibrillary acid protein (GFAP)-immunoreactive astrocytes
and neurofilament-immunoreactive neurons. The authors suggest that apoptosis may be the mechanism
of neuronal loss in HIV infection. This is a reasonable
hypothesis, given that neuronal loss in AIDS is not
readily apparent with routine methods of analysis because of the individual nature of neuronal cell death
and the absence of significant inflammatory reaction.
Apoptosis is a form of "physiological" cell death, also
referred to as programmed cell death, in which cells
die and are engulfed by phagocytes without discharging cytosol contents into the extracellular space
and without initiating an inflammatory reaction.3 Interestingly, a recent combined clinicopathological
and unbiased stereological study of cortical neuronal
loss in AIDS has suggested that neuronal loss may not
correlate with evidence of dementia.4 The clinical significance of the observed neuronal loss in AIDS is thus
open to debate. Nevertheless, the current consensus
is that AIDS dementia, like more common neurodegenerative diseases such as Alzheimer's disease
(AD), is due to neuronal loss or synaptic pathology, or
In addition to the report of Petito and Roberts describing apoptosis in the brain in AIDS, Lassmann et


al5 have recently used essentially an identical ISEL

procedure to study apoptosis in postmortem brains of
elderly controls and AD cases. Similar to Petito and
Roberts,1 Lassmann et al.5 found that the ISEL
method was not vulnerable to postmortem delay, and
that it could be performed in formalin-fixed routinely
processed paraffin tissue sections from cases with
even relatively long postmortem delays without excessive nonspecific labeling of cells due to postmortem DNA fragmentation. Lassmann et al5 also found
ISEL-positive cells, mostly glial cells, in control brains.
Only very rarely were ISEL-positive neurons detected.
In the Lassmann et a15 study the number of labeled
cells, both glia and neurons, was markedly (23-fold
for glia and 55-fold for neurons) increased in AD, and
the positive cells followed the anatomical distribution
of vulnerability in this disorder. In addition, some neurons with neurofibrillary degeneration were ISELpositive. Even though a direct comparison between
the two studies is not possible since the method of
enumerating the number of positive cells was different, the major point that emerges is that apoptotic cell
death can be detected in glial cells in control brains
and that apoptosis (including glial and neuronal) is
increased in disorders where neuronal loss has been
Lassmann et al5 found less than one cell per
square millimeter in normal brains. Even this small
number of ISEL-positive cells would seem to be
greater than in the control cases used by Petito and
Roberts.1 The average age of the control cases was
clearly different in the two studies, being older in the
Lassmann et al5 study, which raises the possibility
that apoptosis may increase with age. On the other
hand, two elderly control cases and a single AD case
Accepted for publication March 19, 1995.
Address reprint requests to Dennis W. Dickson, Department of
Pathology, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461.

Apoptosis in the Brain


AJP May 1995, Vol. 146, No. 5

were included in the Petito and Roberts1 study, and

apoptosis was not detected in any of these cases.
The apparent discrepancy between the two reports
awaits additional confirmatory studies.
Lassmann et a15 found that most of the labeled glia
in aging and AD were microglia and oligodendroglia;
ISEL-positive astrocytes were not detected. This
raises the possibility that increased apoptosis of astrocytes may be characteristic of AIDS. If the results
can be confirmed, the reason for astrocytic vulnerability in AIDS needs to be determined. In AIDS it has
been a matter of some controversy whether astrocytes are infected by HIV. Several recent studies
would tend to support this contention,6'7 including a
recent report showing HIV genome in GFAP-positive
astrocytes with sensitive in situ polymerase chain reaction.8 That the infection is different from the productive infection that is more characteristic of brain
macrophages and microglia seems increasingly
likely. In particular astrocyte infection is associated
with increased production of HIV regulatory molecules, including nef, but few structural proteins (eg,
gpl20). This form of infection has been referred to as
"restricted".7 The Petito and Roberts1 study would
suggest that restricted infection of astrocytes may be
associated with susceptibility to apoptotic cell death.
Obviously, additional studies are needed combining
in situ end labeling and immunocytochemistry with
antibodies to nef to prove this speculation.
With respect to ISEL-positive astrocytes in control
brains, Petito and Roberts1 speculated that apoptosis
may be a means of controlling astrocyte cell numbers
during gliosis. As an aside it should be noted that not
all gliosis is necessarily associated with increased
numbers of astrocytes, but rather with increased astrocyte size and GFAP-immunoreactivity.9 Nevertheless, a similar physiological role of apoptosis has
been invoked to explain control of cell number in development10 and other disease states, including
resolution of acute inflammation.11'12 Apoptosis may
be a mechanism for maintaining cell numbers at a
critical set point determined by a complex interaction
of cellular and extracellular factors, including growth
factors and cytokines. Its role in reactive cellular
changes in the brain is an area of investigation that is
only in its infancy.
In the Petito and Roberts1 report most of the cells
that were positive with ISEL were negative for cell
type-specific markers. The positive cells were small
cells in the perineuronal and perivascular spaces of
both gray and white matter. This distribution is consistent with either oligodendroglial or microglial cells.
The perivascular region is of particular interest in
AIDS, because this is a location of a particular popu-

lation of monocyte-derived macrophages, so-called

perivascular microglia, that have a relatively rapid
turnover rate and that appear to be replenished by
circulating monocytes.13 The turnover of perivascular
microglia is enhanced in a variety of disease states,
including HIV infection, although this is not easily determined in human tissue. This is a potential route of
HIV entry into the brain via infected monocytes (the
so-called "Trojan horse hypothesis"). The perivascular microglial cell is also one of the few cell types that
may express CD4 in detectable levels in normal
brain.14 Since CD4 is a receptor for HIV, this population of microglia is a prime target for apoptosis in
AIDS (see below).
Although the failure of Petito and Roberts1 to detect
macrophage markers in these cells may at first glance
suggest that the cells were not of microgliamacrophage origin, it should be noted that the study
was based upon analysis of paraffin sections. Currently there are no markers for microglia that are entirely satisfactory for paraffin sections.15 The marker
used by Petito and Roberts1 was a monoclonal antibody to CD68. Inspection of the photomicrographs
reveals that only a subpopulation of microglia were
immunolabeled with their procedure.
The presence of apoptosis in microglia may seem
counterintuitive in a disorder associated with diffuse
microglial activation and focal microglial proliferation.
On the other hand, activation and proliferation of microglia has recently been shown to be associated
with induction of apoptosis.16 Brain macrophage (microglial) apoptosis has also been demonstrated in experimental allergic encephalomyelitis, an animal
model for multiple sclerosis.17 That microglia are activated in AIDS cannot be disputed, because this is
the cell type that is the primary target of HIV infection
in the brain. The perivascular microglia appear to be
unusually susceptible, while detection of viral antigens in ramified parenchymal microglia follows a
more restricted distribution.18
The other possibility is that the unidentified cell type
in the Petito and Roberts1 study is oligodendroglial.
Again, no truly ideal marker for oligodendroglia has
been identified for retrospective studies of archival
formalin-fixed and paraffin-embedded tissue. Lassmann et a1l used an antibody to myelin oligodendrocyte glycoprotein (anti-MOG) to identify apoptotic oligodendrocytes. No such marker was used by Petito
and Roberts.1 That the oligodendroglial cell may be
the target of apoptosis in AIDS is reasonable. White
matter pathology is one of the most consistent findings in AIDS brains, but satisfactory explanations for
white matter changes are lacking. Oligodendroglial
apoptosis has also been demonstrated in experimen-



AJP May 1995, Vol. 146, No. 5

tal allergic encephalomyelitis.19 Oligodendroglial

cells appear to be increased in white matter in AIDS
cases with mild myelin pallor, but depleted in cases
with more extensive white matter disease.20 Apoptosis may be the mechanism for oligodendroglial depletion in advanced stages of HIV infection. In vitro studies have shown that cultured oligodendroglia can be
killed by apoptotic mechanisms with lymphotoxin.21
What is the mechanism of apoptosis in AIDS? Are
similar mechanisms involved in apoptosis of neurons
and glia? These are areas that require further investigation. One interesting avenue of research with respect to apoptosis of astrocytes and possibly microglia is the role of cell surface receptors that mediate
apoptosis, in particular APO-1/Fas. APO-1/Fas
(CD95) is a 45-kd membrane-associated protein that
is a member of the nerve growth factor and tumor
necrosis factor (TNF) receptor superfamily.22 Upon
binding to its ligand, a TNF-like membraneassociated factor,23 or its cross-linking by antibodies
to Fas,24 cells bearing APO-1/Fas undergo apoptosis. Fas has been implicated in bystander apoptosis
that appears to be an increasingly important mechanism of cell death in HIV infection.25 In particular, recent studies suggest that Fas may be involved in CD4
lymphocyte depletion in AIDS.2627 The signaling
pathway for Fas is not known with certainty, but some
evidence suggests that Fas activation may lead to
activation of a sphingomyelinase and production of
ceramide.28 Ceramide in turn activates phosphatases29 that further amplify the signal. The method of
cell killing appears to be distinct from apoptosis mediated by TNF.30 Although Fas has not been demonstrated in the brain parenchyma of normal humans,31
it is present in gliomas of astrocytic origin, where it has
been shown to be upregulated in response to
interferon-y and TNF-a.32 Insufficient information is
available about whether Fas is expressed in activated
astrocytes or microglia in diseased brains, but further
studies would seem to be warranted given its potential role in apoptosis in AIDS.
Of potential further relevance to AIDS, APO-1/Fas
shares sequence homology with HIV-1 gp120.33 A
humoral immune response to HIV infection generates
antibodies to gpl 20, some of which may theoretically
cross-react with Fas. Antibody-mediated crosslinking of Fas leads to apoptosis.34 Interestingly, in
lymphocytes gpl 20 appears to play a role in inducing
apoptosis.27 In CD4+ lymphocytes gp120 binds to
CD4, and cross-linkage of CD4 yields increased
interferon-y and TNF-a production and upregulation
of Fas, which then promotes apoptosis.26 It is unknown whether microglia, particularly CD4+ perivascular microglia, also express APO-1/Fas. The cellular

labeling in the only published immunocytochemical

study did show APO-1/Fas immunoreactivity in the
vicinity of blood vessels.31 This was interpreted as
endothelial labeling, but differentiation of perivascular microglial staining from endothelial staining, particularly in cryostat sections, may be difficult. In fact,
misidentification of perivascular microglia as endothelia led to the erroneous assumption for several
years that class 11 major histocompatibility antigens
were expressed in brain endothelial cells.35 Careful
studies are needed to address this issue, because
APO-1/Fas expression in microglia may be important
for the pathogenesis of central nervous system manifestations of HIV infection.
The mechanism of apoptosis in neurons in AIDS is
likely to be different from that for glia. In primary cortical cultures, gp12O induces apoptosis in neurons,
but not in astrocytes.36 Neuronal death mediated by
gp12O shares similarities with glutamate-mediated
neurotoxicity.37 In addition undefined microglialderived factors may be involved in gp120-mediated
neurotoxicity.38 Nitric oxide has been implicated as
one of the potential neurotoxins, and it has also been
implicated in apoptotic cell death of other cell types,
including macrophages.39 Clearly, these are different
pathways from those discussed above.
Apoptosis may explain several of the apparent
paradoxes of HIV infection. With respect to systemic
immune deficiency, CD4 depletion occurs in the setting of relatively low levels of productive infection of
CD4 cells. In the brain, HIV-related pathology, eg,
white matter pathology, is associated with relatively
low levels of productive HIV infection. In both of these
situations HIV-derived molecules, such as gp12O or
antibodies to gpl 20, may lead to apoptosis. Defining
the mechanism of apoptosis in neurons and glia in
AIDS may have potential therapeutic significance;
however, apoptosis is likely to be a two-edged sword.
Inhibitors of apoptosis may eventually be developed
to prevent neuronal or oligodendroglial cell death, but
if it is determined that physiological cell death is essential in maintaining a normal balance in the number
or density of renewing cell populations, such as glial
cells in the brain, therapies will have to be directed to
specific mechanisms of induction of apoptosis.

1. Petito CK, Roberts B: Evidence of apoptotic cell death
in HIV encephalitis. Am J Pathol 1995, 146:1121-1130
2. Wijsman JH, Jonker RR, Keijzer R van de Velde CJ,
Cornelisse CJ, van Dierendonck JH: A new method to
detect apoptosis in paraffin sections: in situ end-

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labeling of fragmented DNA. J Histochem Cytochem

1993, 41 :7-12
Cohen JJ: Overview: Mechanisms of apoptosis. Immunol Today 1993, 14:126-130
Everall IP, Glass JD, McArthur J, Spargo E, Lantos P:
Neuronal density in the superior frontal and temporal
gyri does not correlate with the degree of human immunodeficiency virus-associated dementia. Acta Neuropathol 1994, 88:538-544
Lassmann H, Bancher C, Breitschopf H, Wegiel J, Bobinski M, Jellinger K, Wisniewski HM: Cell death in Alzheimer's disease evaluated by DNA fragmentation in
situ. Acta Neuropathol 1995, 89:35-41
Tornatore C, Meyers K, Atwood W, Conant K, Major E:
Temporal patterns of human immunodeficiency virus
type 1 transcripts in human fetal astrocytes. J Virol
1994, 68:93-102
Saito Y, Sharer LR, Epstein LG, Michaels J, Mintz M,
Louder M, Golding K, Cvetkovich TA, Blumberg BM:
Overexpression of nef as a marker for restricted HIV-1
infection of astrocytes in postmortem pediatric central
nervous tissues. Neurology 1994, 44:474-481
Nuovo GJ, Gallery F, MacConnell P, Braun A: In situ
detection of polymerase chain reaction-amplified
HIV-1 nucleic acids and tumor necrosis factor-a RNA
in the central nervous system. Am J Pathol 1994, 144:
da Cunha A, Jefferson JJ, Tyor WR, Glass JD, Jannotta FS, Vitkovic L: Gliosis in human brain: relationship to size but not other properties of astrocytes.
Brain Res 1993, 600:161-165
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y,
Jacobson MD: Programmed cell death and the control
of cell survival: lessons from the nervous system. Science 1993, 262:695-700
Haslett C: Resolution of acute inflammation and the
role of apoptosis in the tissue fate of granulocytes.
Clin Sci 1992, 83:639-648
Schmied M, Breitschopf H, Gold R, Zischler H, Rothe
G, Wekerle H, Lassmann H: Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis.
Evidence for programmed cell death as a mechanism
to control inflammation in the brain. Am J Pathol 1993,
Lassmann H, Schmied M, Vass K, Hickey WF: Bone
marrow derived elements and resident microglia in
brain inflammation. GLIA 1993, 7:19-24
Peudenier S, Hery C, Ng KH, Tardieu M: HIV receptors
within the brain: a study of CD4 and MHC-11 on human
neurons, astrocytes and microglial cells. Res Virol
1991, 142:145-149
Dickson DW, Mattiace LA, Kure K, Hutchins KD, Lyman WD, Brosnan CF: Biology of disease: microglia in
human disease, with an emphasis on the acquired immunodeficiency syndrome. Lab Invest 1991, 64:135156
Reid DM, Perry VH, Andersson PB, Gordon S: Mitosis
and apoptosis of microglia in vivo induced by an anti-












CR3 antibody which crosses the blood-brain barrier.

Neuroscience 1993, 56:529-533
Nguyen KB, McCombe PA, Pender MP: Macrophage
apoptosis in the central nervous system in experimental autoimmune encephalomyelitis. J Autoimmun 1994,
Kure K, Weidenheim K, Lyman WD, Dickson DW: Morphology and distribution of HIV-1 gp-41-positive microglia in subacute AIDS encephalitis: pattern of involvement suggestive of a multisystem degeneration.
Acta Neuropathol 1990, 80:393-400
Pender MP, Nguyen KB, McCombe PA, Kerr JF: Apoptosis in the nervous system in experimental allergic
encephalomyelitis. J Neurol Sci 1991, 104:81-87
Esiri MM, Morris CS, Millard PR: Fate of oligodendrocytes in HIV-1 infection. AIDS 1991, 5:1081-1088
Selmaj K, Raine CS, Farooq M, Norton WT, Brosnan
CF: Cytokine cytotoxicity against oligodendrocytes.
Apoptosis induced by lymphotoxin. J Immunol 1991,
Oehm A, Behrmann I, Falk W, Pawlita M, Maier G,
Klas C, Li-Weber M, Richards S, Dhein J, Trauth BC:
Purification and molecular cloning of the APO-1 cell
surface antigen, a member of the tumor necrosis
factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen. J Biol Chem
1992, 267:10709-10715
Suda T, Takahashi T, Golstein P, Nagata S: Molecular
cloning and expression of the Fas ligand, a novel
member of the tumor necrosis factor family. Cell 1993,
Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S,
Sameshima M, Hase A, Seto Y, Nagata S: The
polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991,
Schnittman SM, Fauci AS: Human immunodeficiency
virus and acquired immunodeficiency syndrome: an
update. Adv Int Med 1994, 39:305-355
Oyaizu N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, Pahwa S: Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by
inducing interferon-y and tumor necrosis factor-a secretion. Blood 1994, 84:2622-2631
Laurent-Crawford AG, Krust B, Riviere Y, Desgranges
C, Muller S, Kieny MP, Dauguet C, Hovanessian AG:
Membrane expression of HIV envelope glycoproteins
triggers apoptosis in CD4 cells. AIDS Res Hum Retroviruses 1993, 9:761-773
Cifone MG, De Maria R, Roncaioli P, Rippo MR,
Azuma M, Lanier LL, Santoni A, Testi R: Apoptotic
signaling through CD95(Fas/Apo-1) activates an
acidic sphingomyelinase. J Exp Med 1994, 180:1547-

29. Hannun YA, Linardic CM: Sphingolipid breakdown
products: anti-proliferative and tumor-suppressor lipids. Biochim Biophys Acta 1993, 1154:223-236
30. Schulze-Osthoff K, Krammer PH, Droge W: Divergent



AJP May 1995, Vol. 146, No. 5





signalling via APO-1/Fas and the TNF receptor, two

homologous molecules involved in physiological cell
death. EMBO J 1994, 13:4587-4596
Leithauser F, Dhein J, Mechtersheimer G, Koretz K,
Bruderlein S, Henne C, Schmidt A, Debatin KM, Krammer PH, Moller P: Constitutive and induced expression of APO-1, a new member of the nerve growth
factor/tumor necrosis factor receptor superfamily, in
normal and neoplastic cells. Lab Invest 1993, 69:415429
Weller M, Frei K, Groscurth P, Krammer PH, Yonekawa
Y, Fontana A: Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and
modulation of sensitivity by cytokines. J Clin Invest
1994, 94:954-964
Zagury JF, Lachgar A, Achour A, Chams-Harvey V,
Cho YY, Le Coq H, Bizzini B, Feldman M, Burny A,
Zagury D: Pathogenic disorders involved in immunosuppression and T cell depletion characterizing AIDS.
Biomed Pharmacother 1994, 48:11-16
Kobayashi N, Hamamoto Y, Yamamoto N, Ishii A,






Yonehara M, Yonehara S: Anti-Fas monoclonal antibody is cytocidal to human immunodeficiency virusinfected cells without augmenting viral replication.
Proc Natl Acad Sci USA 1990, 87:9620-9624
Mattiace LA, Davies P, Dickson DW: Detection of
HLA-DR in human microglia is dependent on clinical
and technical factors. Am J Pathol 1990, 136:11011114
Muller WE, Schroder HC, Ushijima H, Dapper J, Bormann J: gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by memantine. Eur J Pharmacol 1992, 226:209-214
Lipton SA: HIV-related neurotoxicity. Brain Pathol
1991, 1:193-199
Giulian D, Wendt E, Vaca K, Noonan CA: The envelope glycoprotein of human immunodeficiency virus
type 1 stimulates release of neurotoxins from monocytes. Proc Natl Acad Sci USA 1993, 90:2769-2773
Albina JE, Cui S, Mateo RB, Reichner JS: Nitric oxidemediated apoptosis in murine peritoneal macrophages. J Immunol 1993, 150:5080-5085