Download Neuropathology of NFHgp160 Transgenic Mice

Journal of Neuropathology and Experimental Neurology
Copyright q 2001 by the American Association of Neuropathologists
Vol. 60, No. 6
June, 2001
pp. 574 587
Neuropathology of NFHgp160 Transgenic Mice Expressing HIV-1 Env Protein in Neurons
JEAN MICHAUD, MD, FRCPC, RAUL FAJARDO, GUY CHARRON, PHD, ANNIE SAUVAGEAU, FOUAD BERRADA, PHD,
DJAMEL RAMLA, HUGO DILHUYDY, YVES ROBITAILLE, MD, FRCPC, AND ALLÉGRIA KESSOUS-ELBAZ, PHD
Key Words:
gp160; HIV-1; Transgenic mice.
INTRODUCTION
The acquired immunodeficiency syndrome (AIDS) is
caused by the human immunodeficiency virus (HIV),
with HIV-1 predominating over HIV-2. This member of
the Lentivirinae subfamily of retroviruses targets the immune and nervous systems (1). As, observed early in the
AIDS epidemic (2), neurological diseases became a significant part of the clinical picture (3, 4). The most frequent neurological disease of the central nervous system
(CNS) is HIV-1 associated cognitive/motor complex (5)
also referred to as AIDS dementia complex (6) or HIV1 associated dementia (HIVD) (7). According to various
estimates, its incidence ranges from 15% to 20% of patients with AIDS (8, 9) and it is now the leading cause
of dementia in the 20–59 age group (10). Morphologically, 2 major entities are consistently associated with
HIVD: HIV-1 encephalitis (HIVE) and HIV-1 leukoencephalopathy (HIVL) (7, 9–13). Yet, the lack of correlation between the severity of the dementing illness and
the inflammatory changes was recognized early (11).
Morphological abnormalities in early stages of HIV-1 infection were defined (14–16). The neuronal population,
From the Department of Pathology and Cellular Biology (JM, RF,
GC, AS, FB, DR, HD, YR, AK-E), University of Montreal, Montreal,
Canada.
Correspondence to: Allegria Kessous-Elbaz, Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, C.P. 6128, Succ. A, Montréal, Canada H3C 3J7.
Present address for Jean Michaud: Department of Pathology and Laboratory Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, K1H 8M5 Canada. Present address for Fouad Berrada: Al Akhawayn University, Avenue Hassan II, PO Box 1832, Ifrane 53000,
Morocco.
This study was supported by the Medical Research Council of Canada (MT-11282).
remarkably normal with conventional studies, was found
to be normal or decreased (17–22) by quantitative analysis. When neuronal loss was identified, it was not generalized and neurochemical (23) or structural subpopulations were involved (24, 25). The consensus is that
there is no good correlation between the quantitatively
determined loss of neurons and the level of dementia.
Subcellular abnormalities, such as dendritic simplification
and loss of pre-synaptic terminals, have a better potential
for correlating with the neurobehavioral abnormalities
(21, 26).
The search for the physiopathogenic mechanisms involved in the AIDS dementia complex was further complicated by the lack of evidence of the HIV-1 virus in the
neuronal cells. Using different approaches, several laboratories demonstrated the replication and/or expression of
HIV-1 mainly in macrophages, multinucleated giant cells,
microglial cells (27–32), and vascular endothelial cells
(31, 33) but not in neurons. Additionally, Teo et al (32)
showed that the presence of unintegrated circular HIV-1
DNA and the active replication and expression of the
virus correlated with the presence of multinucleated giant
cells and dementia. Thus, the previous reports (34, 35)
that showed by in situ RT-PCR hybridization the presence
of DNA and RNA in neuronal cells remain to be confirmed.
As the neuron is apparently not the primary target of
the HIV-1 CNS infection, several indirect multicellular
pathways were proposed and studied in a variety of in
vitro and in vivo experimental models (9, 12, 36, 37).
Activation of macrophage/microglial cells following infection by HIV-l appeared to induce secretion of excitotoxins and/or neurotoxins such as glutamate, cytokines of
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Abstract. The physiopathology of HIV-1 dementia remains largely hypothetical. Although several sets of evidence point
towards an indirect multicellular inflammatory pathway, gp120, one of the HIV-1 env products, was shown to be very cytotoxic
for neurons in vitro. To explore a direct pathway in the physiopathology of dementia in AIDS, we developed transgenic
mouse models carrying the HIV-1 env proteins gp 120 and gp 41 (gp 160) under the control of the human light neurofilament
and murine heavy neurofilament promoters. To date, this is the first mouse model in which the HIV-1 env protein can be
detected in neurons by immunohistochemistry. The expression is found in several brainstem and spinal cord gray structures
and in the cerebellum in one of the mouse lines bearing the NFHgp160 transgene. The morphological findings at 3 months
are subtle and are dominated by a watery, dendritic degeneration and a reactive gliosis. At 12 months, the evidence of neuronal
degeneration and loss is present along with various degenerative phenomena involving synapses, dendrites and axons, including axonal swellings. Cytoskeletal abnormalities were found by immunohistochemistry. Chronic inflammation was also
observed in the leptomeninges of the spinal cord and brainstem and in the cerebellar white matter. These models thus offer
an exciting sequence of morphological findings initiated by the neuronal expression of the HIV-1 env proteins and offer a
different tool to explore the neuronal dysfunction in AIDS.
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HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
TABLE
CNS Distribution of Neuronal Perikarya
Immunoreactive for HIV-1 Env in Transegenic NFH
1932 As Compared to NFLgp160Xba*
Reactivity in mouse line
Anatomical structures**
Fig. 1. Structure and restriction map of NFHgp160 plasmid. A: The plasmid Bluescript pSKNFH harboring the 2.5-kb
NFH promoter was modified by introducing a Sal I site at the
unique Not I restriction site. The 4.8-kb Sal I-Xba I gp160
fragment of HIV-1 HXBc2 strain was introduced in the pUC18
at Sal I and Xba I sites as previously described (56). The 2.5kb Sal I segment containing the murine NFH promoter was
inserted in Sal I site at the 59 end of the HIV-1 4.8-kb fragment.
The NFHgp160 transgene was deleted from the vector pUC18
by EcoR I digestion before microinjection. B: The KS-Sty construct was derived to provide the 2.2-kb HindIII subfragment
of the HIV-1 env gene as a probe for Southern hybridization
analyses.
several types, arachidonic acid, quinolate, plateletactivated factor, nitric oxide synthetase and others (38),
potentially targeting the neurons, astrocytes and oligodendrocytes (37, 39). Apoptosis (40–44) and necrosis
(38) were also put forward as mechanisms of neuronal
loss. Finally, axonal damage in close relationship with
blood vessels (45, 46), axonal swellings (47), or focal
accumulation of amyloid precursor protein (48) were observed in HIVE, and even in HIV-1-positive patients
without AIDS (49).
Direct pathways involving HIV-1 replication or gene
products are still possible. In vitro, it has been demonstrated that the gp120 subunit of the HIV-1 env product
was neurotoxic even at low concentration (50). This direct effect of gpl20 was associated with an intracellular
increase in free calcium (36), which seemed mediated by
an excessive activation of N-methyl-D-aspartate
(NMDA) receptor-operated channels (36, 51–54). In vitro
also, HIV-1 and gp120 induced apoptosis of human central nervous system neurons through the activation of cJun N-terminal and p-42 extracellular-regulated kinase
(55).
NFLgp160Xba
1
1
1
2
1
1
1
1
1
2
1
2
1
1
1
2
* Described in reference 56. ** The anatomical references
and nomenclature were determined using the atlas of the mouse
brain and spinal cord (59) and the stereotaxic atlas for the rat
(58). Neuroantomical nomenclature: Mesencephalon: (R) red
nucleus; 3, oculomotor; (Sc) superior colliculus; 4, trochlear
nucleus; (DpMe) deep mesencephalic nucleus; (Pcom) nucleus
of the posterior nucleus; (VP) ventral pallidum, (mlf) medial
longitudinal fasciculus, (RMC) red magnocellular nuclei; (RPC)
red parvocellular nuclei. Pons: (PnO) pontine reticular nucleus,
oral part; (PnC) pontine reticular nucleus, caudal part; (5) motor
trigeminal nuclei; (Sp5) spinal trigeminal tract nucleus; (Acs5)
accessory trigeminal nucleus; (Me5) mesencephalic trigeminal
nucleus; (7) facial nuclei 7; (Acs7) accessory facial nucleus;
(Lve) lateral vestibular nucleus; (SpVe) spinal vestibular nuclei;
(Mve) medial vestibular nuclei; (RtTg) reticulo-tegmental nuclei pons; (DPGi) dorsal paragigantocellular nuclei; (Y) nucleus
Y; (Pr5) principal sensory trigeminal nucleus; (VCA) ventral
cochlear nuclei anterior. Medulla: (12) hypoglossal nuclei;
(Amb) ambiguous nucleus; (MdV/Gi) medullary reticular nucleus, ventral part; (GiA, GiV) gigantocellular reticular nuclei;
(LRt) lateral reticular nuclei; (sol) solitary tract; (RLV) rostroventrolateral reticular nuclei; (cu) cuneate fascicles; (PrH) prepositus hypoglossal nuclei; (tz) trapezoid body; (Li) linear nuclei of medulla; (MdV) medullary reticular nuclei ventral.
Spinal cord: (DRG) dorsal root ganglia; motoneurons of layers
4-8. Cerebellum: (Int A) interposed cerebellar nuclei anterior;
(Med DL) medial cerebellar nuclei; (Lat:PC) lateral cerebellar
nuclei.
In patients, however, whether neuronal lesions are directly related to gp160 remains undetermined because of
the HIV-1 infection process and its systemic effects. In
order to better investigate the pathological effects of
gp120 on the neuronal cells in vivo we developed transgenic mouse models carrying the HIV-l env gene under
the control of 2 neuron-specific promoters: the human
neurofilament light (NFL) and the murine neurofilament
heavy (NFH) genes. We described the NFLgp160Xba
transgenic mice, the expression pattern of the env protein
in their CNS, and their pathological evaluation at 4
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Mesencephalon: (R, 3, Sc, 4, DpMe,
Pcom)
(VP, mlf, RMC, RPC)
Pons: (PnO/PnC, Mo5, Sp5, Acs5,
Me5, 7, Acs7, LVe)
(SpVe, Mve, RtTg, DPGi, Pr5, Y,
VCA)
Medulla: (12, Amb, MdV/Gi, LRt)
(sol, RLV, cu, PrH, tz, Li)
Spinal cord: (DRG, motoneurons of
layers 4-8)
Cerebellum: (Int A, Med DL, Lat:
PC)
NFH
1932
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MICHAUD ET AL
months of age (56). In the present report, we describe the
pathological changes in the NFHgp160 transgenic mice
with emphasis on the neuropathological lesions in older
animals, at approximately 1 yr of age.
MATERIALS AND METHODS
Transgene Construct and Transgenic Mouse
Development
The plasmid harboring the NFHgpl60 transgene was obtained
as follows: the 4.8 kb Sall-Xbal HIV-l fragment containing the
sequence encoding the env protein of HXBc2 provirus was excised from the env expressor plasmid psvlIIexE7 and inserted
in the pUC18 vector, as previously described (56). The pSKNFH plasmid (gift of Dr. J. P. Julien, McGill University), harboring the NFH gene promoter was modified by replacing the
Not1 site with a Sal1 restriction site. This modified plasmid
was used to excise the 2.5-kb Sal1 segment that contains the
NFH promoter. The NFH promoter was then inserted in the Sal1
site at the 59 end of the env sequence (Fig. 1A). The 7.2-kb
NFHgp160 transgene was deleted from the plasmid construct
by EcoRI digestion and then purified and microinjected, as previously reported (56, 57). The integration of the transgene into
the mouse genome was assessed by Southern blot hybridization
of mouse tail DNA using, as probe, the env 2.2-kb HindIII
fragment of the KS-Sty construct (Fig. 1B) and conditions previously reported (56). All transgenic mice were developed and
maintained in a pathogen-free facility.
J Neuropathol Exp Neurol, Vol 60, June, 2001
In Vivo Expression of the Env Protein
Since the expression of the Env proteins has been targeted
to the CNS tissue, its presence was primarily sought in the
nervous tissue. This was assessed by immunohistochemistry,
using vibratome serial sections and monoclonal antibodies
against gp41 and gp120 (Dupont NEN Canada, Mississauga,
Ontario, Canada) and HIV-1 patient serum. The anesthetic treatment and the fixative transcardiac perfusion of the control and
transgenic animals as well as the immunoreactions were carried
out as previously described (56). The stereotaxic atlas of Paxinos et al (58) for the rat and the atlas of the mouse brain and
spinal cord of Sidman et al (59) were used as anatomical references; the nomenclature used in Paxinos et al (58) was adopted for the present description.
Neuropathologic Assessment
Histopathology: After transcardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, CNS tissues
of 3- and 12-month-old transgenic mice were immersed in 2%
paraformaldehyde in the same buffer for 24 h, then embedded
in paraffin blocks. Serial 5-mm sections were used for the following stains: hematoxylin-phloxin-safran (HPS), Luxol-cresyl
violet, modified Bielschowsky and Holzer.
Immunohistochemistry: The indirect immunoperoxidase technique of Sternberger (PAP) was applied on 5-mm sections using
the following antisera: glial fibrillary acidic protein (GFAP)
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Fig. 2. Immunodetection of HIV-1 Env proteins in CNS sections of NFH 1932 transgenic mice with human anti-HIV-1 serum.
A: Immunostaining of cerebellar and brainstem structures. B: Immunostaining of cerebellar structures. C: Immunolabeling of red
magnocellular (RMC) and red parvocellular (PRC) nuclei. D: Immunostaining of neurons in the anterior gray horns of the spinal
cord. Magnifications: A, C, 3200; B, D, 3400.
HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
RESULTS
Development of the NFHgp160 Transgenic Mice
Prior to microinjection, the NFLgpl60Xba and
NFHgpl60 transgenes were tested in transient expression
assays. As previously reported (56), the data showed that
both constructs expressed and correctly processed the
Env precursor and the resulting gp120 and gp41 subunits
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(Dako, Glostrup, Denmark, 1: 1,000), synaptophysin (Boehringer Mannheim Canada, Laval, Quebec, Canada, 1: 20), neuronal specific enolase (NSE) (Dako, 1: 500). The neurofilament
proteins were immunolabeled with antibodies directed against
the phosphorylated heavy subunit present in axons (NF-a) and
the nonphosphorylated heavy subunit mostly present in the neuronal perikaryon (NF-n) using, respectively, the monoclonals
SMI 31 and SMI 32 (Sternberger Monoclonals, Baltimore, MD,
1: 500) and the indirect PAP method. All sections were deparaffinized and treated for 20 min in 3% hydrogen peroxide before incubation for either 1 h (NF-a, NF-n, synaptophysin) or
20 min (all other antibodies). HRP (Zymed Laboratories, San
Francisco, CA) was used to label NF-a and NF-n antibodies,
while the streptavidin-peroxidase method (Lipshaw Immunon,
Detroit, MI) was used for all the other markers.
Finally, in order to screen for co-localization of neuronal loss
and reactive gliosis, immunoreactions with monoclonal antibodies against GFAP (Boehringer Mannheim, Canada, 1: 500)
and the microtubule-associated protein-2 (MAP-2) (Amersham
Pharmacia Biotech, Baie d’Urfé, Quebec, Canada, 1: 400) and
FITC-labeled goat anti-mouse IgG (Jackson ImmunoReseach
Laboratories, West Grove, PA, 1: 200) were performed on 5mm step-sections from the same blocks as for immunoperoxidase studies. Because MAP-2 is mostly expressed in the dendrites it was chosen as a marker to quantitatively evaluate the
changes in the dendritic tree. Field selection and image capture
and storage were done with a confocal microscope (LSM 410,
Zeiss, Germany). Quantitative analyses were carried out with
an image analysis system (IBAS, Kontron Elektronik, Germany). Briefly, for each animal the area percentage occupied by
the labeling was averaged from 8 microscopic fields of frontocentral cortex, 4 fields from trigeminal (5), facial (7), and hypoglossal (12) brainstem nuclei, and 4 fields from cervical and
lumbar spinal anterior horns. These quantitative analyses were
performed blindly by 3 different examiners on 5 transgenic
mice and 5 age-matched normal control mice.
Ultrastructure: It was carried out as previously described
(60). Briefly, for transgenic as well as normal mice, 4 animals
(2 at 3 months old and 2 at 12 months) were perfused with 20
ml of 3.5% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Tissues were immersed in the
same fixative solution for 4 h at 48C. Tissues were then washed
in 0.1 M sodium phosphate buffer (pH 7.4) overnight at 48C,
postfixed in 2% OsO4 for 1 h, dehydrated in graded ethanol
solutions, immersed in propylene oxide, and embedded in epon
resin (J.B. EM.Services, St-Laurent, Quebec, Canada). Sections
of 60–90 nm thickness from representative regions of the CNS
were mounted onto grids, counterstained with uranyl acetate
and lead citrate, and blindly examined by 2 observers with a
Philips EM201 microscope.
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Fig. 3. Immunoreactions with anti-synaptophysin antibody.
Synaptophysin immunostaining of the lumbar anterior gray
horns of 3-month-old NFH 1932 transgenic mice (A) and normal control mice (B). Synaptophysin immunostaining of a
megasynapse in the anterior gray horn of the spinal cord (C).
Magnifications: A, B, 3250; C, 3400.
maintained their biological properties (56). The transgenes were then used for microinjection and their integration in the mouse genome was determined by Southern blot hybridization as described (56). The mouse lines
carrying NFLgpl60Xba have been phenotypically and genetically characterized (56). With the NFHgpl60 transgene (Fig. 1A), 5 out of 33 animals tested (NFH 354,
NFH 1932, NFH 1243, NFH 2016, and NFH 2917) were
found to harbor 2 to 10 copies of full-length copies of
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MICHAUD ET AL
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HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
the transgene. All these transgenic founders except for
NFH 354, which was infertile, reproduced normally and
transmitted the transgene to their progeny. The mouse
lines NFH 1932, NFH 1243, NFH 2016, and NFH 2917
derived from these founders, as well as the normal control animals, were maintained in a pathogen-free facility
and have remained healthy and free of infections.
Expression and Distribution of Env Proteins in the CNS
of NFH 1932 Mice
Neuropathologic Phenotype
Because of the extended expression pattern observed
in the NFH 1932 mice, the neuropathological changes
and their eventual progression were investigated at 3 and
12 months of age in transgenic and normal control mice.
In the 3-month-old transgenic animals, the results and
findings obtained with the histochemical and immunohistological studies with the neurofilaments (NF-a, NF-n)
and GFAP antibodies were similar to those previously
reported for the NFLgpl60Xba transgenic mice (56). Further investigations on the brain tissue from these animals
using the anti-NSE and synaptophysin antibodies suggested a slight enhancement of the density of dendritic
projections in the neuropil of the anterior horns of the
spinal cord (Fig. 3A). In these structures, synaptophysin
immunostaining showed grain irregularities when compared to controls; occasional aggregates of larger grains
were also present (Fig. 3C), which signals a disturbance
in distribution and/or size of the synapses. The GFAP and
MAP-2 (Fig. 4) analyses of the fronto-central cortex, trigeminal, facial, and hypoglossal nuclei as well as anterior
gray horns of the spinal cord by confocal microscopy
showed a statistically significant reactive gliosis in the
anterior horns of the spinal cord (Fig. 4A). The GFAP
reaction was also increased in the cortex, the cranial and
brainstem motor nuclei, but this increase was not statistically significant. The MAP-2 was also found slightly
augmented for the transgenic animals in each sampled
CNS area, but it reached statistical significance only in
the cortex (Fig. 4B, C).
At 12 months of age the NFH 1932 transgenic and
normal control mice were similarly investigated. With the
HPS staining, the transgenic mice demonstrated chronic
perivascular inflammation in the leptomeninges of the
spinal cord (Fig. 5A) and brainstem and also in the cerebellar white matter (Fig. 5B). In the cerebellum, the antisynaptophysin antibody reacted positively only with the
neurons of the internal granular layer, indirectly confirming the inflammatory nature of these perivascular white
matter infiltrates dominated by lymphocytes (Fig. 5B).
The anterior gray horns of the spinal cord and the cranial
nerve nuclei 5, 7, and 12 showed mild neuronal loss, with
occasional degenerated or retracted acidophilic neurons
(Fig. 5C, D). These areas demonstrated acidophilic
amorphous or fibrillary-like deposits that do not seem to
react with any immunohistochemical stains that have
been used. Many neuronal intracytoplasmic acidophilic
inclusion bodies and intravascular calcifications were
more frequently observed in the thalamus of transgenic
than in control mice. Additionally, a variety of dendritic
and axonal swellings were detected with HPS staining
(Fig. 6A, arrows) and also by immunoreactions with the
HIV-l antiserum, synaptophysin, NF-a, as well as NF-n
antibodies, with NF-n giving a much stronger signal (Fig.
6B). They were observed mostly in the motor cranial
nerve nuclei 5, 7, and 12, in the gray matter of the spinal
←
Fig. 4. Confocal microscopy evaluation of the GFAP (A) and MAP-2 (B) in the cortex, the motor trigeminal (Mo5), facial
(7) and hypoglossal (12) nuclei, and in the anterior gray horns of the spinal cord of control and 3-month-old NFH 1932 transgenic
mice. C: Laser confocal images of MAP-2-stained cortex, hypoglossal nuclei (12), and spinal cord in normal (N) and transgenic
mice (T).
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Expression of the transgene was assessed in CNS tissues by immunostaining of serial sections from brain and
spinal cord of 3- and 12-month-old transgenic and normal
control mice using monoclonal antibodies against gp41,
gp120, or HIV-1 patient serum, as described (56). As
detailed in the Table and illustrated (Fig. 2A–D), all the
CNS structures that were immunoreactive in
NFLgp160Xba mice (56) were similarly found expressing the transgene in NFH 1932 mice. In addition, immunopositive perikarya were also observed in the following structures: Mesencephalon: ventral pallidum (VP),
medial longitudinal fasciculus (mlf). Pons: spinal vestibular nuclei (SpVe), medial vestibular nuclei (MVe), reticulo-tegmental nuclei pons (RtTg), dorsal paragigantocellular nuclei (DPGi), nucleus Y (Y), ventral cochlear
nuclei anterior (VCA). Medulla: solitary tract (sol), rostroventrolateral reticular nuclei (RLV), cuneate fascicles
(cu), prepositus hypoglossal nuclei (PrH), trapezoid body
(tz), linear nuclei of medulla (Li). Finally, the expression
of the transgenic proteins was detected in the cerebellum,
in the interposed cerebellar nuclei anterior (Int A), medial
cerebellar nuclei (Med DL) and lateral cerebellar nuclei
(Lat:PC) (Fig. 2A, B) that always tested negative in
NLFgp160Xba mice. Thus, with the exception of the cerebral and the cerebellum cortices, all the structures normally positive for the neurofilament proteins were found
expressing the transgene in NFH 1932 mice.
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cord, particularly in the gray commissures and in the thoracic nuclei and also in the posterior or anterior funiculi.
The immunostainings with NSE, NF-n, NF-a, GFAP, and
synaptophysin led to the following observations: 1) an
increase of the reactive immunohistochemical densities in
the neuropil of the anterior gray horns of the spinal cord
comparatively to controls; 2) clusters of larger synaptic
grains and even megasynapses (Fig. 3C), with some
merging the size of axonal swellings; 3) a significant
number of perikarya reacting positively with the NF-a
antibody (Fig. 6D) in the transgenic mice while they remain negative in the controls (Fig. 6C); 4) a moderate
but significant gliosis in the anterior gray horns of the
spinal cord (Fig. 7A) when compared to controls (Fig.
7B). The GFAP was also moderately positive in the cortex of the cerebellum (Fig. 7C). In the molecular layer
there was patchy Bergmann gliosis. Mild fibrillary gliosis
was also present in the internal granular layer. In the deep
white matter and nuclei of the cerebellum, the reactive
gliosis was present but the reaction was closer to that
seen in the control mice. The control mice showed only
focal and mild areas of gliosis in the molecular layer (Fig.
7D). No gliosis was observed in the cerebral cortex or in
the deep structures of the cerebral hemispheres.
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Ultrastructural Analysis
Ultrastructural studies done essentially on the anterior
gray horn of the cervical, thoracic and lumbar spinal cord
and on the seventh cranial nerve nuclei revealed in transgenic animals several findings largely confined to their
dendrites and axons. An early change regularly observed
in 3- and 4-month-old transgenic animals was a watery
swelling of dendrites with preservation of a few organelles like mitochondria or neurotubules. Complex membranes could be present but these were more frequently
found at 12 months (Fig. 8A). At this late age, watery
degeneration was still very much present with occasional
aberrant mitochondria in the abnormal dendrites (Fig.
8B). The most frequent changes involved the cristae and
their orientation, and the matrix, which was sometimes
watery, was sometimes replaced by a fibrillar or granular
substance. Axonal swellings were observed regularly
with myelin sheath degeneration (Fig. 9A) of variable
severity. These lesions could be associated with an abnormal accumulation of intermediate filaments. In addition, some myelinated axons were, without being swollen, focally replaced by granular material or dense
granular bodies (Fig. 9B). Vacuolar degeneration was observed in certain neuronal projections and it was also
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Fig. 5. Pathological evaluation of the NFH 1932 transgenic mice at 12 months of age. A: Inflammation in the leptomeninges
with HPS staining and (B) in the cerebellum white matter with synaptophysin immunostaining that positively reacted with the
ectopic granular neurons in the neighboring structures but not with perivascular inflammatory lesion. C: HPS-stained, retracted
acidophilic and (D) degenerated neurons. Magnifications: A, C, D, 3640; B, 3500.
HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
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present focally in neuronal nuclei and the adjacent cytoplasm. Moreover, the neuronal perikarya seemed to be
spared although occasional neurons demonstrated a vesicular hyperplasia in the Golgi area. Finally, some dendrites harbored occasional microtubuloreticular inclusions (Fig. 10).
DISCUSSION
Transgenic mice harboring and expressing the HIV-1
env gene under the control of neurofilament promoters
were successfully generated (56). So far these models are
unique in that they are the only ones in which both Env
proteins, gpl20 and gp41, are produced in neurons at levels detectable by immunostaining technique. The topography of the protein expression in both NFLgp160Xba
and NFH 1932 transgenic mice included several structures in the brainstem, cerebellum, and the spinal cord,
but spared the cerebral cortex, the limbic system and essentially all the subcortical supratentorial structures. Explanation for this topography remains as yet unclear. We
can reasonably exclude developmental intra-uterine death
of Env producing neurons as the expression of the protein
appears at day 8 of the postnatal period. In addition, these
spared structures do not show any reactive gliosis that
could signal a postnatal neuronal loss, even if enhanced
intra-uterine or neonatal developmental apoptosis presumably would leave no trace. Nevertheless, these mice
allowed us to demonstrate at the cellular and subcellular
levels some of the pathological changes induced by the
transgene products in absence of the effects related to
systemic HIV-1 infection.
Among the possible viral factors suspected for the neuronal dysfunction or loss in HIVE or HIVL, gpl20 became a prime suspect following the in vitro demonstration that, at low concentration, this protein was highly
neurotoxic to neurons. Several direct and indirect pathways were evoked to explain this gp120-induced toxicity
(38, 50–52, 61). Our transgenic mouse models allow selection of 1 specific hypothesis (direct pathway) out of
several others in which neurons are not directly affected
by the virus but are secondarily involved through different cellular reactions (37).
The morphological evaluation of these 2 transgenic
mouse lines strongly suggested an initial neuronal involvement. By ultrastructure, at 3 months of age the initial lesion was a segmental watery degeneration of the
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Fig. 6. Pathological evaluation of 12-month-old NFH 1932 transgenic mice. HPS staining of axonal swellings (arrows) in the
dorsal posterior and anterior funiculi of the spinal cord (A). Axonal swellings immunostained with the anti-NF-n antibodies (B).
Negative immunostaining of normal mouse neurons with the anti-NF-a antibodies (C). Positive immunostaining of neurons from
transgenic mice (line NFH 1932) with the anti-NF-a antibodies (D). Magnifications: A, 3500; B, 3125; C, 3640; D, 3312.
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neuronal dendritic tree. Its enhancement with some cytoskeletal markers (NF-a, NF-n, for example) without ultrastructural abnormalities of the cytoskeleton at that
stage, suggested a prefibrillogenesis derangement. At 3
months this watery change was found associated with
axonal swellings that were best revealed with the HIV-1
antiserum, indicating an axonal transport of the transgenic proteins. These swellings were not very numerous
when compared to our findings in the 12-month-old mice.
This very early change probably reflects the effect(s) of
the transgene (or part of it v.g. gp120) on the cellular
membrane of the neurons. However, whether these effects
could be related to an increase of intraneuronal free calcium by gp120 as reported for neuronal cell cultures (36,
51–54) remains to be determined. Further studies aimed
at measurements of intracellular calcium as well as the
state of the calcium dependent channels in the Env expressing neurons are needed to help determine the underlying mechanisms for these lesions. In addition, studies on 2 different calcium binding proteins, calbindin and
J Neuropathol Exp Neurol, Vol 60, June, 2001
parvalbumin, in the CNS of moderate and severe HIVE
patients suggested that different pathogenetic mechanisms could be involved and these may vary in different
regions of the brain (62).
Beyond the watery dendritic and axonal changes, the
morphometric analysis with the MAP-2 protein and the
synaptophysin immunostaining patterns suggested an increased density of the neuronal projections along with
irregularities of synapses. Moreover, the neuronal loss became obvious at 12 months of age as degenerating neurons were observed by conventional histology. The gliosis already detected at 3 months by conventional
histological and immunohistochemical techniques and
confirmed by morphometric analysis is likely secondary
to all these neuronal changes. However, the cellular reaction went beyond the reactive gliosis as chronic inflammation was found in the leptomeninges and white matter
of the cerebellum in the NFH 1932 line at 12 months of
age. The spleens of these animals also showed a reactive
hyperplasia (data not shown). We cannot exclude that this
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Fig. 7. Immunoreactions with anti-GFAP antibodies on 12-month-old NFH 1932 transgenic and normal control mice. This
antibody showed a moderate but significant gliosis in the anterior gray horns of the spinal cord of transgenic mice (A), similar
to that observed in younger animals of the same line. In the normal control (B), only rare astrocytes are observed in the same
areas. In the cerebellum, the GFAP antibodies demonstrated moderate gliosis in the cerebellar cortex of the transgenic mice (C).
The same antibodies show focal limited areas of gliosis in the molecular layer of the normal control (D). Magnifications: A, B,
3250; C, D, 3160.
HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
583
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Fig. 8. Ultrastructural analysis of the thoracic spinal cord from 12-month-old NFH 1932 transgenic mice. A: Pale swelling
with numerous membranous debris. B: Watery swelling with abnormal mitochondria. Magnifications: A, B, 313,700.
inflammation could influence the morphological picture
in the CNS and, more specifically, the neuronal damage
by the introduction of indirect cytotoxic pathways.
The immunohistochemical reactions on control and
transgenic animals with NF-n and NF-a antibodies revealed abnormal enhancement in the neuropil and/or aberrant reactions in the perikarya, all indicating profound
dysregulation in the processing of these cytoskeletal proteins. Recently, similar alterations of the neurofilaments
and severe cortical neuronal changes were reported in
cats infected with the feline immunodeficiency virus
(FIV), a lentivirus sharing many properties with HIV-1
(63). In this animal model, the SMI 32 antibodies used
for CNS investigations reacted with pyramidal neurons
J Neuropathol Exp Neurol, Vol 60, June, 2001
584
MICHAUD ET AL
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Fig. 9. Ultrastructural analysis of the anterior horns of the spinal cord from 12-month-old NFH 1932 transgenic mice. A:
Myelinated axon with bilobed axonal swelling and severe degeneration of the myelin sheath. B: Myelinated axonal swelling
focally filled with cellular organelles and granular or multilamellar bodies. Magnifications: A, 33,400; B, 313,700.
only in the frontal neocortex and showed a significant
increase in their number in the infected animals. In the
rat neonate model, dystrophic neurons were generated in
the neocortex following systemic injection of purified
gp120. This was associated with delayed developmental
J Neuropathol Exp Neurol, Vol 60, June, 2001
milestones and complex motor behavior (64). Thus, both
these studies and our findings clearly indicate early alterations of the neuronal cytoskeleton by viral infection
or gp120 alone. Following our findings on the transgenic
mice with NF-a and NF-n antibodies, we used the same
HIV-1 ENV PROTEIN IN NEURONS OF TRANSGENIC MICE
585
antibodies for preliminary studies on 5 HIVE patients
(including one already reported [47]) and 3 controls.
Striking changes were observed in some of these patients,
with marked increase and/or aberrant localization of the
immunoreaction in the neocortex. Although these changes were similar to those observed in the transgenic mice
it is premature to draw definite conclusions and establish
a firm parallel between these and HIVE patients because
of the possible artifacts associated with postmortem material. Nevertheless, these results underline the frequent
presence of neurofilament modifications in HIV-1-associated neurological diseases.
The watery vacuolization found in our transgenic mice
is reminiscent of the observations made in humans. The
vacuolization of the dendritic processes was observed in
patients by electron microscopy (49, 65) but it was never
illustrated, likely because autopsy material is not optimal
for electron microscopic studies. Indeed, the vast literature on AIDS remains silent on ultrastructural subcellular
neuronal changes. In vitro, by electron microscopy,
Yeung et al (66) described large cytoplasmic vacuoles in
neural cells with morphology consistent with neurons.
They used primary cultures of human brain cell aggregates that contained all cerebral cell types. The vacuoles
were obtained by addition of interleukin-6 to the culture
medium, while addition of gp120 produced changes that
were mostly nuclear. The neuronal dendrites and axons
were not described (66). In vivo, pathologic distension of
the smooth endoplasmic reticulum (SER) was found to
correlate with the dendritic vacuolization of pyramidal
neurons in the neocortex of gp120 transgenic mice (65).
The authors suggested that calcium toxicity could play a
role through aberrant activation of the inositol phospholipid pathway, which is linked to the glutamate receptors
known to be highly concentrated in dendritic SER.
In conclusion, the NFLgpl60Xba and NFHgpl60 transgenic mice demonstrate an exciting sequence of events
initiated with neuronal expression of HIV-1 Env proteins
in neurons at 8–10 days after birth. The subsequent morphological changes, although nonspecific, can lead to a
variety of cellular, biochemical, and molecular investigations. Prior to the development of chronic inflammation, it will be possible to explore a direct pathway linking gp120, the neuron, and its glial environment. With
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Fig. 10. Thoracic spinal cord of 12-month-old NFH 1932 transgenic mice. Para-neuronal dendrite with a microtubuloreticular
inclusion (34650).
586
MICHAUD ET AL
the presence of inflammation, the exploration of indirect
pathways becomes a possibility not only in the confinement of the CNS but also between the CNS and the extracranial compartment. This laboratory animal model could
contribute to the understanding of neuronal dysfunction
in AIDS and in other neurodegenerative disorders.
ACKNOWLEDGMENTS
Jean Michaud would like to thank Jean-Jacques Hauw and the medical staff of the Laboratoire de Neuropathologie R. Escourolle, Hôpital
La Salpêtrière, Paris, for stimulating discussions and academic support
during his sabbatical year. We also thank M. E. Weinreb for reviewing
the manuscript. We are grateful to F. Lacourse-Brière, K. Dupuis, F.
Saker, D. Rodrigue and G. Lambert for technical and photographic support.
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