Schenck et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03124-5(2024) 21:127

Journal of Neuroinflammation

Open Access

RESEARCH

Extracellular vesicles produced by HIV‑1
Nef‑expressing cells induce myelin impairment
and oligodendrocyte damage in the mouse
central nervous system
Jessica K. Schenck1, Molly T. Karl1, Cheryl Clarkson‑Paredes1, Ashley Bastin1, Tatiana Pushkarsky1,
Beda Brichacek1, Robert H. Miller1 and Michael I. Bukrinsky1*

Abstract
HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affectapproximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy(ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlateswith the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicatedin HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo,
ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin
sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injectionof Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes
in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression
and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor
cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment.
These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin
maintenance in the pathogenesis of HAND.
Keywords HIV-1, Nef, Extracellular vesicles, HAND, Myelin, Oligodendrocyte, Astrocyte, Microglia
Introduction
HIV-associated neurocognitive disorders (HAND) represent a spectrum of cognitive impairments commonly
observed in people living with HIV (PLWH), affecting
up to half of all HIV-positive individuals. Despite theadvent of anti-retroviral therapy (ART), which has substantially reduced the incidence of severe HIV-associated*Correspondence:
Michael I. Bukrinsky
mbukrins@gwu.edu
1School of Medicine and Health Sciences, The George Washington
University, 2300 I St NW, Ross Hall 624, Washington, DC 20037, USA

dementia, milder forms of HAND persist even with sustained virologic control [1–6]. Notably, individuals withhigh CD4 T-cell counts [7–12] and those in the acute andearly periods of HIV infection are also at risk of neurocognitive impairment, as damage to the central nervous
system (CNS) occurs shortly after viral infection [13].
In the era of ART, the clinical manifestations of HIVassociated neuropathology have shifted away from severe
acute disease defined by encephalopathy and neurodegeneration towards more subtle neuroinflammatorychanges and sublethal alterations in neuronal functions[14], with white matter abnormalities persisting. Studies
utilizing neuroimaging techniques have identified white© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Schenck et al. Journal of Neuroinflammation(2024) 21:127

matter damage, including increased white matter hyperintensity and corpus callosum thinning, in HIV-infectedindividuals on ART [15–18]. Transcriptome analyses ofpatients with HAND on ART have revealed downregulation of genes associated with oligodendrocyte maturationand myelin metabolism [19, 20]. Importantly, the severity of white matter damage correlates with the degree ofneurocognitive impairment in patients with HAND [21–25], though the mechanisms underlying HIV-associatedwhite matter damage remain incompletely understood.
Oligodendrocyte injury is a hallmark of white matter damage and demyelination, processes demonstrated
in both cell culture and animal models of HIV-associated neuropathology [26, 27]. Oligodendrocyte-specificexpression of Nef was shown to affect appropriate differentiation of oligodendrocytes in vitro [28]. However,HIV does not directly infect oligodendrocytes; microglia and astrocytes are recognized HIV reservoirs within
the CNS and can produce viral proteins, including Nef,even during successful ART [29–31]. The release of theseviral proteins via extracellular vesicles may mediate the“bystander” effects of HIV infection on uninfected cells.
The HIV protein Nef is responsible for many pathogenic
effects of HIV infection [32] and has been implicatedas a key player in HAND pathogenesis [33–35]. Recent
animal studies have shown that astrocytic expression of
Nef induces deficits in spatial and recognition memory,which is correlated with increased CNS infiltration of
peripheral macrophages and neuroinflammation [36, 37].
Astrocytes are known to be essential for the development
and maintenance of oligodendrocytes, and Nef-mediated white matter damage likely involves perturbation ofastrocyte–oligodendrocyte–microglia crosstalk, which
is important for myelin formation and maintenance,
and aberrant astrocyte or microglial function has major
consequences for oligodendrocyte physiology and whitematter integrity [38, 39].
Previous research has demonstrated that Nef impairs
cellular cholesterol homeostasis in macrophages bydecreasing the abundance and functional activity of thecritical cholesterol transporter ATP-binding cassettetransporter A1 (ABCA1) and inhibiting cholesterol efflux[40]. This ultimately alters the lipid composition of themacrophage cellular membrane, potentiating inflammatory responses [41]. Importantly, Nef-containing extracellular vesicles (Nef EVs) impair ABCA1-mediatedcholesterol efflux in uninfected cells [42], similar to theeffect of Nef in HIV-infected cells. This underscores
the potential significance of Nef and Nef EVs in HAND
pathogenesis in ART-treated patients, as Nef can bedetected in the plasma of aviremic individuals [43], indicative of its production by latent HIV reservoirs despite
effective antiretroviral therapy. Additionally, the presence

Page 2 of 21

of Nef EVs in the brains of ART-treated PLWH may predict neurocognitive impairment status [44].
The intricate relationship between cholesterol metabolism and myelin synthesis underscores the importance ofunderstanding how Nef-mediated disruption in cholesterol homeostasis may contribute to HAND. Oligodendrocytes, responsible for myelin formation, rely heavily
on cholesterol for the maintenance of myelin sheaths,
the fatty insulation surrounding axons [45]. In the CNS,
where the blood‒brain barrier restricts the entry of LDL
particles, cholesterol homeostasis depends on recycling
and de novo biosynthesis [46]. While oligodendrocytes
contribute to the synthesis of cholesterol incorporated
into myelin membranes during development [47, 48],
astrocytes primarily produce cholesterol in the adult
CNS [47–49]. Furthermore, cholesterol recycling by
phagocytic astrocytes and microglia is crucial for myelin
repair and overall cholesterol homeostasis. ABCA1, present in both astrocytes and mature oligodendrocytes,
plays a pivotal role in myelination by facilitating cholesterol transport [50]. However, the function of ABCA1mediated cholesterol transport may be compromised by
Nef-containing EVs, thereby disrupting myelin formationor maintenance by oligodendrocytes. In this study, we
employed multiple methodologies to explore the impact
of Nef EVs on myelin sheaths, aiming to elucidate a novel
mechanism underlying CNS demyelination in HAND.
We hypothesize that Nef-induced downregulation andinactivation of ABCA1 disrupt the intricate process ofmyelin formation or maintenance by oligodendrocytes.
This proposed mechanism may provide insights into why
mild forms of cognitive impairment persist despite undetectable viral loads in ART-treated HIV patients.

Methods
Animals

C57BL/6J mice (Jackson Laboratory) were maintained
under standard mouse husbandry conditions, including a
12:12 h light:dark cycle and ad libitum access to food andwater. All the experimental procedures were approved by
the George Washington University Institutional Animal
Care and Use Committee.
Isolation and purification of extracellular vesicles (EVs)

EVs were produced as previously described [51]. Briefly,
HEK293T cells were transfected with either a pcDNA3.1
vector expressing Nef from HIV-1 NL4-3 to produce
Nef EVs or an empty vector to produce control (Ctrl)
EVs; the cells were subsequently grown in DMEM supplemented with 10% heat-inactivated exosome-depleted
fetal bovine serum (FBS) (Thermo Fisher, A2720801). At
48 h post transfection, the medium was collected, andthe EVs were isolated via differential centrifugation [52].

Schenck et al. Journal of Neuroinflammation(2024) 21:127

First, culture supernatants were pre-clarified to remove
cells and cellular debris by centrifugation at 500×g for
10 min at 4 °C and subsequently clarified to remove any
remaining debris and large apoptotic bodies by centrifugation at 3000×g for 30 min at 4 °C. Nef and Ctrl EVs
were pelleted via a final centrifugation step at 100,000×g
for 75 min at 4 °C. The pellet was resuspended in sterile
saline supplemented with 10% mouse serum (Innovative
Research, IGMSBCSER) for in vivo brain injections or in
DMEM/F-12 (Thermo Fisher, 11320033) supplemented
with 1% FBS for in vitro and ex vivo culture, aliquoted,
and frozen at − 70 °C. The utilization of frozen samples
was indispensable to ensure consistency of Nef concentrations in the EV preparations utilized across various
experiments. This approach also facilitated the validation of the biological activity of EV preparations in vitro
before undertaking more complex in vivo experimental
procedures. Recent studies have shown that freezing of
EVs leads to particle loss and fusion [53, 54]. However,considering the primary focus of our study on Nef, we
ensured consistent Nef concentrations as a priority.
Additionally, since we consistently compared Nef EVs tocontrol EVs prepared concurrently, any potential damage
incurred during freezing was anticipated to affect bothtypes of EVs equally, thus not influencing the outcomeof comparisons. Ultimately, any potential damage to EVs
could only attenuate the observed effects, rather thanalter the conclusions drawn from this study. To ensure
consistency, we aliquoted the EVs, froze them, and utilized the aliquots in all in vitro and in vivo experiments,
thereby avoiding repeated freeze–thaw cycles or the useof EV batches with varying Nef concentrations. The concentrations of the Nef and Ctrl EVs were estimated using
Tunable Resistive Pulse Sensing technology on the Exoid
instrument (Izon Sciences Ltd) (Supplemental Fig. S1A).
Specifically, 3.2 × ­109 particles in 10 µL were used totreat ex vivo slice cultures and 1 × ­105 cells in vitro, while
4.3 × ­109 particles in 5 µL were injected into mouse brainsin vivo. Nef EVs carried 0.67 ng Nef per 4.4 × ­1010 EV particles, as measured by a home-made Nef ELISA (Supplemental Fig. S1B).
Nef ELISA

An in-house Nef ELISA was developed to quantify
Nef associated to EVs. First, 96-well plates were coatedwith 0.25 μg of a mouse monoclonal anti-Nef antibody(Abcam, Waltham, MA; #ab42358) per well overnight.
The coating antibody was diluted in coating buffer (BioRad, Hercules, CA). After incubation overnight at room
temperature, all wells were washed with ELISA washing buffer (Bio-Rad) and saturated with 300 μL of BSA
blocking buffer (Bio-Rad) for 2 h at room temperature.
Standards and samples were resuspended in 50 μL ofPage 3 of 21

Nef ELISA buffer (PBS 1X, 20 mM TRIS, 0.05% Tween
20, 0.2% BSA) supplemented with 0.5% Triton X-100.
Samples were run in duplicate and quantified using therespective standard curve. The range of Nef (recombinant HIV-1NL4-3 Nef from Abcam, #ab63996) concentrations for standard curve was 1000 to 0.98 ng/mL.
Following three washes with 300 μL of washing buffer,
50 μL of primary antibody (rabbit polyclonal anti-Nef
antibody (Abcam, #ab63918) diluted in blocking buffer)
was added to each well for 1 h at room temperature withgentle agitation. Wells were washed and 50 μL of secondary antibody [anti-rabbit horse peroxidase-conjugatedantibody (SBI Biosciences, Palo Alto, CA)] was added
to each well and incubated for 1 h at room temperature
under gentle agitation. Wells were washed and 50 μL of
TMB substrate (ThermoFisher Scientific, Waltham, MA)
was added to each well. After incubation in the dark for
30 min under gentle agitation, the reaction was stopped
by adding 50 μL of 2 N sulfuric acid in each well. OD wasread immediately at 450 nm and 540 nm using a 96-well
Synergy microplate reader (BioTek, Winooski, VT). For
each sample, Nef concentrations were determined using
4P regression analysis of OD values in Prism.
In Vivo injections

Sixteen- to twenty-week-old C57BL/6J male and female
mice were anesthetized using isoflurane, and stereotaxic
intracranial injection was performed. Five microliters of
Nef or Ctrl EVs in sterile saline containing 10% mouseserum was infused at a rate of 7 nL/s into the right hemisphere corpus callosum 1.1 mm anterior and 1 mm lateral to bregma at a depth of 1.8 mm. At three days post
injection, the mice were deeply anesthetized with avertin
and then perfused with fixative, after which the tissue
was processed for immunohistochemistry (IHC) or electron microscopy as described previously [55].
For immunohistochemical analysis, animals were perfused with 4% paraformaldehyde (PFA; EMS, 15710);
brains were dissected, postfixed at 4 °C, equilibrated in
30% sucrose, embedded in Tissue-Tek® O.C.T. Compound (Sakura), and frozen at − 80 °C. Cryosections(11 µm) were prepared on a Leica CM1950 cryostat
microtome.
For ultrastructural analysis via electron microscopy,
mice were perfused with 4% PFA and 2% glutaraldehyde (EMS, 16010) in 0.1 M sodium cacodylate (EMS,
11653) adjusted to pH 7.4; 200 μm coronal sections
were obtained with a vibratome (Campden Instruments,
5100 mz), postfixed in 1% ­OsO4 (EMS, 19192), dehydrated through a series of graded ethanol, incubated insaturated uranyl acetate, and infiltrated/embedded in apoly/Bed812 resin (EMS, 14900). One micrometer thickcoronal sections were stained with toluidine blue, andSchenck et al. Journal of Neuroinflammation(2024) 21:127

areas for ultrathin EM sectioning were selected. Ultrathin
coronal sections (120 nm) were placed on silicon wafersand carbon-taped in aluminum stubs for subsequent
SEM imaging.
Cerebellar slice culture

Primary cerebellar cultures were prepared from C57BL/6J
mice at postnatal day 10 as previously described [55,56]. Briefly, cerebella were embedded in 1% agarose andsagittally sectioned in ice-cold DPBS (Sigma‒Aldrich,
G8769) supplemented with 5% glucose (Sigma‒Aldrich,
G8769) at 300 μm using a Leica VT1000S vibrating
microtome. Slices were immediately placed in 24-wellplates on individual 0.4 μm 12 mm diameter MillicellCM cell culture inserts (Millipore, PICM01250) andgrown in 350 μL of cell culture medium containing 50%
BME (Thermo Fisher, 21010046), 15% heat-inactivatedhorse serum (Thermo Fisher, 16050114), 25% Hank’ssolution (Sigma‒Aldrich, H4641), 0.5% glucose, 1% GlutaMAX™ Supplement (Thermo Fisher, 35050061), 1%
penicillin‒streptomycin (Corning, 30-002-CI), N2, and10 ng/mL PDGF-AA (Sigma‒Aldrich, P3076). The slices
were incubated at 37 °C in 5% ­CO2. Half-volume media
changes were made two days after plating. After 4 days,
mouse cerebellar slices were treated with 10 µL of Nef orCtrl EVs once daily for 2 or 4 days. The slices were fixedwith 4% PFA for 15 min, delipidated in ice-cold 2.5% acidmethanol for 15 min at − 20 °C, and subsequently processed for immunohistochemistry as described below.
Mixed cell cultures

Primary mouse brain cells were obtained from the brainsof C57BL/6J mice at postnatal day 3 using the Miltenyi
Biotec Neural Tissue Dissociation Kit (Miltenyi Biotec,
130-093-231) according to the manufacturer’s instructions. Cells were plated on 12 mm coverslips coated with
poly-L-lysine (PLL) (Sigma Aldrich, P1274) at 70,000
cells per well for immunostaining and on 6-well plates
at 3 × ­105 cells per well for western blot and cytokine
expression analysis. The culture media consisted of
DMEM/F-12 supplemented with 1% FBS, 1% penicillin‒streptomycin, N2, and 10 ng/mL PDGF-AA. Cultures
were grown for 5 days before treatment with 10 µL of Nef
or Ctrl EVs for 48 h. For drug treatment, cells were cultured for 7 days prior to receiving once-daily treatment
with 1 µM of AMS-55 (generously provided by Dr. Amol
Kulkarni) or TO-901317 (Sigma‒Aldrich, 575310) for
3 consecutive days. Subsequently, the cells were treatedwith EVs for 48 h. Post-treatment, the media were collected for cytokine analysis, while cells underwent analysis via western blotting or immunohistochemistry, as
described below.

Page 4 of 21

A2B5 + enriched cell cultures

Enriched populations of A2B5 + cells were isolated fromthe brains of C57BL/6J mice at postnatal day 5 via magnetic activated cell sorting with anti-A2B5 microbeads(Miltenyi Biotec, 130-093-392) according to the manufacturer’s instructions. The cells were plated on PLL-coatedcoverslips and differentiated into O4 + oligodendrocytes
or GFAP + astrocytes. To promote OPC proliferation,
A2B5 + cells were initially seeded at 10,000 cells per welland cultured for 24 h in medium supplemented with
DMEM/F12 and 2X B-27 supplement (Thermo Fisher,
12587010), 2 mM l-glutamine (Gibco, 25030081), 20 ng/
mL PDGF-AA and 20 ng/mL bFGF (PeproTech, 10018B). The cells were then switched to oligodendrocyte
differentiation medium containing DMEM/F12 supplemented with 2X B-27 supplement, 2 mM l-glutamine,
10 ng/mL PDGF, 30 ng/mL T3 (Sigma‒Aldrich, T63-97),
and 10 ng/mL NT-3 (PeproTech, 450-03). Cultures weredifferentiated for 2 or 6 days before treatment with 10 µL
of Nef or Ctrl EVs for 48 h and then analyzed via immunohistochemistry. To promote astrocyte differentiationand proliferation, A2B5 + cells were initially seeded at
10,000 cells per well and cultured in “astrocyte media”,
DMEM/F-12 supplemented with 5% FBS, 1% penicillin‒
streptomycin, N2, and 10 ng/mL PDGF-AA. After 3 days,
the cells were treated with 10 µL of Nef or Ctrl EVs for
48 h and then analyzed via immunohistochemistry.
Immunohistochemistry

Brain cryosections were processed for immunohistochemistry as described previously [55], frozen sections
were dried at room temperature for 1 h and rehydrated
in 1 × PBS for 5 min. Samples subjected to MBP analysis
were treated for 20 min at − 20 °C with ice-cold 2.5% acidmethanol. All the samples were then incubated for 1 h atroom temperature in 10% normal goat serum (NGS) and
0.3% Triton™ X-100 (Sigma‒Aldrich) in 1 × PBS and incubated overnight at 4 °C with primary antibodies (Supplementary Table 1), followed by incubation with Alexa
Fluor-conjugated secondary antibodies (1:500) for 1 h atroom temperature (both primary and secondary antibodies were diluted in 10% NGS and 0.3% Triton in 1 × PBS).
Sections were counterstained with DAPI (1:3000; Thermo
Fisher, 46190) and mounted in ProLong™ Gold Antifade
Mountant (Invitrogen, P36934).
Processing samples from slice cultures was performed
as described previously [57]. After the slices were fixedwith 4% PFA and delipidated in 2.5% acid methanol, theslices were blocked first with a primary blocking solution(0.1% Triton, 15% NGS, 10% bovine serum albumin (BSA;
Fisher, BP9706100), diluted in PBS) for 1.5 h at room
temperature and then with a secondary blocking solution

Schenck et al. Journal of Neuroinflammation(2024) 21:127

Page 5 of 21(0.1% saponin, 5% NGS, 5% BSA, diluted in PBS) for 3 h
at room temperature. The slices were incubated at 4 °C
overnight with primary antibodies, incubated with theappropriate Alexa Fluor-conjugated secondary antibodies (1:500) and DAPI (1:5,000) for 3 h at room temperature (primary and secondary antibodies diluted in 0.1%
saponin and 5% NGS with 1xPBS), and then mounted
in VECTASHIELD® PLUS Antifade Mounting Medium(Vector Laboratories, H-1900).
Cell cultures were stained either live or after fixation.
For live staining with anti-O4 IgM, coverslips were incubated at 37 °C with primary antibody for 30 min, followed
by incubation with secondary antibody for 15 min (bothprimary and secondary antibodies were diluted in 10%
NGS with warm DMEM). The cells were fixed with 2.5%
acid methanol for 20 min at − 20 °C and then labeled for
immunohistochemistry. Coverslips were then incubatedfor 1 h at room temperature with primary antibodies(Table 1) followed by incubation with Alexa Fluor-conjugated secondary antibodies (1:500) for 1 h at room temperature (both primary and secondary antibodies werediluted in 10% NGS and 0.3% Triton in 1xPBS). Sections
were counterstained with DAPI (1:3000) and mounted in
VECTASHIELD® PLUS Antifade Mounting Medium.
TUNEL assay

Apoptotic cells in mixed cultures were detected with the
Click-iT™ Plus TUNEL Assay Kit for In Situ Apoptosis
Detection (Invitrogen, C10617) according to the manufacturer’s protocol. Briefly, cells were grown on coverslips, fixed with 2.5% acid methanol for 20 min at − 20 °C
and permeabilized with 0.25% Triton X-100 for 20 min

at room temperature. Coverslips were then incubated at
37 °C with TdT reaction buffer for 10 min, followed byincubation with the TdT reaction mixture for 1 h. Afterrinsing with 3% BSA in PBS, the coverslips were incubated with Click-iT™ Plus TUNEL reaction cocktail for
30 min at 37 °C. After washing with 3% BSA in PBS, thecoverslips were counterstained with DAPI (1:3000) andmounted in VECTASHIELD® PLUS Antifade Mounting
Medium.
Electron microscopy and analysis

To determine the extent of the lesions and relevantalterations in myelin ultrastructure, axonal integrity, andperivascular astrocytes in the corpus callosum after Nef(n = 2) or Ctrl (n = 3) EV injections, scanning electron
microscopy (SEM) analysis was performed at The George
Washington University (GW) Nanofabrication and Imaging Center (GWNIC) using an FEI Helios NanoLab 660
DualBeam SEM (Thermo Fisher). To maximize the backscatter electron collection, we used a concentric backscattering detector (CBS) in immersion mode to acquire
high-resolution SEM images. The acquisition conditions
included 2 kV with a beam landing current ranging from
0.1 to 0.4 nA and a working distance of 4 mm. For imaging, we used a low-magnification tile area, including thecortex, corpus callosum, and hippocampus (600 ×), asa navigation map to identify the lesion location (MAPS
software). Then, high-resolution (12,000 ×) imaging
was performed in the focus area at a 5 ms dwell time,
34.5 mm horizontal field of view, and a pixel width of
11.2413 nm.

Table 1 Antibodies used for immunohistochemistry
Antibodies

VendorCatalog #Clone

Dilution

Use

Mouse anti-myelin basic protein (MBP)

Biolegend

836504

SMI-991:500

Detection of myelin

Mouse anti-APC

Millipore

MABC200

CC1

1:500

Identify mature oligodendrocytes

Rat anti-GFAP

Invitrogen13-0300

2.2B101:500

Detection of astrocytes

Rabbit anti-Vimentin

Abcam

ab92547

EPR3776

1:500

Detection of astrocytes, glial precur‑sors, and ependymal cells

Rabbit anti-Iba1

Wako Chemicals

019-19741

polyclonal

1:500

Detection of microglia/macrophages

Chicken anti-Neurofilament M

Biolegend

822701

Poly28227

1:500

Detection of axons

mouse anti-O4 IgM

R&D Systems

MAB132604

1:50

Identify oligodendrocytes

Goat anti-mouse Alexa 594

Invitrogen

A11032–

1:500

Secondary

Goat anti-mouse Alexa 488

Invitrogen

A11029–

1:500

Secondary

Goat anti-rabbit Alexa 594

Invitrogen

A11012–

1:500

Secondary

Goat anti-rabbit Alexa 488

Invitrogen

A11008–

1:500

Secondary

Goat anti-rat Alexa 594

Invitrogen

A11007–

1:500

Secondary

Goat anti-rat Alexa 488

Invitrogen

A11006–

1:500

Secondary

Goat anti-chicken Alexa 647

Invitrogen

A21449–

1:500

Secondary

Goat anti-mouse IgM Alexa 488

Invitrogen

A21042–

1:300

SecondarySchenck et al. Journal of Neuroinflammation(2024) 21:127

Fluorescence microscopy and analysis

Fluorescence and phase contrast microscopy were performed by an individual blinded to the experimental
conditions using a Leica DM 5500 microscope with a
Hamamatsu ORCA-R2 camera, and the results were analyzed with ImageJ (NIH). In vivo 20 × tile scan images of
the ipsilateral and contralateral corpus callosum wereobtained from 3 to 5 mice per condition. Regions of interest (ROIs) of the ipsilateral hemisphere were identified as400-to-500-micron wide sections of the corpus callosum
centered at the injection site; corresponding ROIs werethen made in the contralateral hemisphere. For GFAP
and vimentin expression analysis, single 40 × images of
the corpus callosum were obtained directly at the injection site, and Mander’s correlation and Pearson’s overlap
coefficients were estimated employing the ImageJ Fiji
JaCoP plugin.
Ex vivo slice culture images were captured at 20 × from
2 to 3 sections per treatment condition. All the images
were processed with the Leica Blind Deblur tool. The
NF + /MBP- axon length was measured with the ImageJ
line tool. The MBP-stained area was calculated as a percentage of the total tissue area.
In vitro images were captured at 20 × or 40 ×, and thecells were counted in ImageJ using a cell counter tool.
For experiments involving O4 + oligodendrocytes, only
live cells with intact nuclei and cell bodies were counted.
“Normal branching” was defined as the presence of complex branching processes without disrupted or severely
diminished immunostaining. Cell damage in purified
oligodendrocyte cultures was indicated by the presenceof DAPI + chromatin fragments, which are hallmarks ofapoptosis [58], overlaying injured cells (when analyzed
by phase contrast microscopy) and disrupted oligodendrocytes (by O4 + immunofluorescence) (Supplemental Fig. 2). DAPI + fragments were identified in ImageJusing watershed separation and particle tools with anarea up to 5 microns. For IBA1 + microglia, highly ramified cells were defined as those possessing one or more
projections with a length greater than 1.5 × that of thecell body. GFAP + astrocytes were categorized based onmorphology as follows: (i) fibrous with an amorphousshape, (ii) bipolar with a single defined projection fromthe cell body, or (iii) multipolar with two or more projections from the cell body. Images for analysis by TUNEL
were captured at 10 ×, and randomized ROIs with an areaof 0.04 ­mm2 were generated to count TUNEL + cells. Allin vitro experiments were run on coverslips in duplicate
or in triplicate, with 2–3 images per replicate.
Cytokine measurement

The cell culture supernatant was collected, andcytokine protein expression measured with a mouse

Page 6 of 21

atherosclerosis antibody array (Abcam, #ab169807)
according to the manufacturer’s instructions. The membrane array can simultaneously detect 22 factors implicated in atherosclerosis, including those commonly
upregulated in individuals with HAND. First, the total
protein concentrations of the culture supernatants were
determined by a BCA assay (Thermo Fisher, #23225) toconfirm equivalent protein levels between treatment
conditions. The membrane arrays were incubated with
1X blocking buffer for 30 min at room temperature andthen with 1 mL of supernatant from the EV-treated cultures overnight. The membranes were incubated overnight with biotin-conjugated anti-cytokine antibodies
followed by incubation overnight with HRP-conjugatedstreptavidin. All the incubations were performed at 4 °C
with gentle shaking on a platform shaker. The membranes
were imaged with a GeneGnome XRQ chemiluminescence imaging system (Syngene). Densitometry data
were extracted from array images using ImageJ software(NIH). After background subtraction, each array was
normalized to its positive control signal, and the normalized signal intensities were compared between treatment
conditions to determine relative differences in cytokine
expression.
Western blot

Automated capillary western immunoblots were performed using the ProteinSimple Jess™ Simple Western
instrument (Bio-Techne) according to the manufacturer’s instructions as described previously [59]. Cells were
homogenized in NET buffer (1% NP-40 lysis buffer:
150 mM NaCl, 1% NP40, 50 mM Tris HCl at pH 8.0)
containing protease and phosphatase inhibitors (Thermo
Fisher). Cell lysates were centrifuged at 700g for 5 min.
Four microliters of cleared cell lysate (1 μg/μL protein)
were mixed with 1 μL of 5 × master mix containing fluorescent molecular weight markers and 200 mM dithiothreitol. For ABCA1 expression analysis, the mix was
incubated at 37 °C for 15 min. The 66–440-kDa Jess separation module (Bio-Techne, #SM-W008) was used forelectrophoresis. To prepare the assay plate for ABCA1
analysis, we loaded a molecular weight ladder, protein
samples, KPL blocking buffer (SeraCare Life Sciences,#5920-0004), total protein quantification reagent, rabbit
polyclonal anti-ABCA1 (1:40; Abcam, #ab63918), rabbit
polyclonal anti-GAPDH (1:100; Sigma, #G9545), secondary HRP conjugated anti-rabbit antibody (Bio-Techne,#042-206) and chemiluminescent substrate according
to the plate diagram. After plate loading, the separation,
electrophoresis, total protein quantification, and immunodetection steps were conducted in the fully automatedJess system. Digital image of chemiluminescence of thecapillary was captured with Compass Simple Western

Schenck et al. Journal of Neuroinflammation(2024) 21:127

software (version 5.1.0, Protein Simple) that automatically calculated heights (chemiluminescence intensity),
area, and signal/noise ratio. Results were visualized aselectropherograms representing the peak of chemiluminescence intensity and as lane view from the signal ofchemiluminescence detected in the capillary.
Data analysis

All the values are presented as the average ± SEM. Student’s t test, one-way ANOVA, or two-way ANOVA was
performed where appropriate using GraphPad Prism
software. p < 0.05 was considered to indicate statisticalsignificance.

Results
Nef EVs disrupt CNS myelin in vivo

To determine whether Nef EVs can contribute to theoligodendrocyte and white matter deficits observed inpatients with HAND, Nef-containing and control EVs(Nef EVs and Ctrl EVs, respectively), purified from empty
vector-transfected (Ctrl EVs) or from Nef-transfected(Nef EVs) HEK293T cells as previously described [60],were injected into the white matter of the mouse brain(corpus callosum of the right (ipsilateral) hemisphere)
via stereotactic surgery. Tissue was collected 3 days post
injection for analysis by immunohistochemistry and electron microscopy. Disruption of myelin in EV-injected
white matter was assessed by the expression of myelin
basic protein (MBP) within the corpus callosum at the
injection site (Fig. 1A). Compared to that in the Ctrl
group, the total MBP + immunofluorescence within the
Nef EV-injected white matter was significantly lower in
the ipsilateral corpus callosum (Fig. 1B), consistent with alocalized myelin lesion. This change was accompanied bya focal increase in the mean MBP + fluorescence intensity(Fig. 1C), a common finding within myelin lesions due
to increased antibody binding in the demyelinating tissue [61]. Electron microscopy (EM) examination of the
myelin ultrastructure at the injection site also revealed anincrease in the number of unmyelinated axons and perturbation of myelin within Nef EV-injected white matter, which was not observed in Ctrl EV-injected tissue(Fig. 1D). Compared to those in the control group, thewhite matter in the Nef EV-injected group displayed astatistically significant 2.4-fold increase in the number ofunmyelinated axons, which had a relatively normal ultrastructural appearance (Fig. 1E). Conversely, there was asignificant 31% decrease in myelinated axons (Fig. 1F).
Together, these findings suggest that Nef EVs directly disrupt CNS myelin sheaths in the absence of overt axonal
degeneration. This result resembles that reported for the
Plp1-transgenic mouse model of Pelizaeus-Merzbacher

Page 7 of 21

disease [60], and suggests that demyelination, at least in
the short term, does not lead to neuronal death.
Injection of Nef EVs was also associated with a significant local reduction (by 42%) in the density of CC1 + (amarker of mature oligodendrocytes) cells within theipsilateral corpus callosum after 3 days (Fig. 1G, H),
indicating that Nef EVs may cause the loss of mature oligodendrocytes in vivo. No change in the density of myelin or oligodendrocytes was detected in regions of the
corpus callosum contralateral to the site of EV injection(Fig. 1B, H), indicating that the effects of Nef EVs on oligodendrocytes and myelin are localized to sites of direct
Nef EV exposure.
Nef EVs disrupt astrocytes and microglia in vivo

We have previously shown that astrocytes are required
for maintaining myelin integrity in the adult CNS [55],and other studies have reported that Nef expression leads
to downregulation of the gene expression of the major
astrocytic protein glial fibrillary acidic protein (GFAP)[62]. Since disruptions to astrocytes may contribute to
Nef EV-mediated demyelination, we examined astrocytes
in the brains of EV-injected mice via immunohistochemistry and EM. Injection of Nef EVs into the right hemisphere corpus callosum resulted in a marked localized
loss of GFAP + cells at the injection site (Fig. 2A), with a
30% decrease in the number of GFAP + cells (Fig. 2B) anda 63% decrease in GFAP + immunofluorescence (Fig. 2C)
within the myelin lesion area compared with those in Ctrl
EV-injected animals. The loss of GFAP + cells was correlated with perturbations in the ultrastructural morphology of astrocytes in the lesion area, including swelling ofperivascular astrocyte processes (Fig. 2F). There was no
significant difference in GFAP + cell density or staining
in the contralateral corpus callosum, indicating that thiseffect on GFAP + expression in astrocytes was localized
to the area of the injection.
To further characterize the effect of Nef-derived EVs on
astrocytes, we analyzed the relationship between GFAP(a mature astrocyte marker) and vimentin (a panglial andimmature astrocyte marker implicated in reactive astrogliosis [63, 64]) in the lesion area of the ipsilateral corpus callosum via colocalization analysis. In all analyses,
Pearson’s correlation and Mander’s overlap coefficientswere estimated employing the ImageJ Fiji JaCoP plugin.
All colocalization coefficients were significantly greaterfor the Ctrl EV-injected white matter than for the Nef
EV-injected white matter (Fig. 2D, E). The mean Pearson’s correlation coefficient was approximately halved for
Nef EV- relative to that for Ctrl EV-injected white matter (0.91 to 0.50, a 45% decrease), denoting a shift froma strong to a moderately positive correlation between
GFAP and vimentin signals. Furthermore, Mander’sSchenck et al. Journal of Neuroinflammation(2024) 21:127

Page 8 of 21

Fig. 1 Effects of Nef EVs on myelination of axons in vivo. Mice were intracranially injected with either Ctrl or Nef EVs. At 3 days post-injection, brain
tissue was processed for analysis using immunohistochemistry (IHC) or scanning electron microscopy (SEM). Images depict representative fields
from the corpus collosum at the injection site. A Representative fluorescence images of myelin (MBP, red). B Quantification of the percentage
of MBP + immunofluorescence area within the ipsilateral (ipsi) and contralateral (contra) corpus callosum. C Quantification of MBP + fluorescenceintensity within the corpus callosum at the injection site. D Representative SEM images within the corpus callosum lesion. E Quantificationof the percentage of unmyelinated axons within the corpus callosum lesion by SEM. F Quantification of the percentage of myelinated axons
within the corpus callosum lesion by SEM. G Representative fluorescence images of mature oligodendrocytes (CC1, green) and nuclei (DAPI,
blue). H Quantification of CC1 + cells per µm2 within the ipsilateral and contralateral corpus callosum. The white arrow indicates the injection site
and the dotted line indicates the border of the corpus callosum. For IHC, n = 3–5 mice per group, scale bar is 100 µm. For SEM, n = 7–9 regionsof interest (ROIs) from 2 to 3 animals per group, scale bar is 5 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

M1 and M2 coefficients ranged between 0.7 and 1.0 in
the Ctrl EV-injected white matter, indicating the strong
overlap of GFAP fractions in the vimentin area (M1) andvimentin fractions in the GFAP area (M2); Mander’s M1
and M2 coefficients in the Nef EV-injected white matter ranged between 0 and 0.5, indicating only a weak tomoderate overlap of GFAP fractions in the vimentin area(M1) and vimentin fractions in the GFAP area (M2). The
overall correlation and co-occurrence coefficient results
suggested that GFAP and vimentin overlapped less in

Nef-EV-injected white matter than in control whitematter, indicating that Nef EVs induced a change in theexpression of astrocytic markers implicated in cell differentiation and activation in the setting of a lesion.
Next, we examined brain tissue from EV-injected micefor evidence of neuroinflammation, which generallyaccompanies HIV-mediated brain disease [65] and canpromote or reflect active demyelination. Previous studies showed that peripheral injection of Nef EVs in vivo
was associated with elevated plasma levels of IL-6 andSchenck et al. Journal of Neuroinflammation(2024) 21:127

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Fig. 2 Effects of Nef EVs on astrocytes in vivo. Mice were intracranially injected with either Ctrl or Nef EVs. At 3 days post injection, brain tissue
was processed for analysis using immunohistochemistry (IHC) or scanning electron microscopy (SEM). The images display representative
fields from the corpus collosum at the injection site. A Representative fluorescence images of vimentin (red), GFAP (green), and nuclei (DAPI).
Higher magnification insets illustrate the loss of GFAP within the lesion area. B Quantification of GFAP + cells per µm2 within the ipsilateral(ipsi) and contralateral (contra) corpus callosum at the lesion. C Quantification of the percentage of GFAP + immunofluorescence area
within the ipsilateral and contralateral corpus callosum at the lesion. D, E Analysis of colocalization of GFAP and vimentin within the ipsilateral
corpus callosum at the lesion. M1: fraction of GFAP in the vimentin area, and M2: fraction of vimentin in the GFAP area. F Representative SEM
images of perivascular astrocytes within the corpus callosum lesion. The white arrow indicates the injection site and the dotted line indicatesthe border of the corpus callosum. For IHC, n = 3–5 mice per group, scale bar is 100 µm. For SEM, scale bar is 5 µm. *p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001

Schenck et al. Journal of Neuroinflammation(2024) 21:127

TNFα [60], suggesting that Nef EVs can promote inflammation. To assess the inflammatory response to direct
CNS injection, the ipsilateral and contralateral corpus
callosum at the injection site were analyzed for microglial infiltration and activation (Fig. 3). Compared to Ctrl
EV-injected tissue, ipsilateral white matter from Nef EVinjected brains showed a 29% increase in DAPI positive
cells (Fig. 3A, B) accompanied by a 2.9-fold increase in
microglia/myeloid cell activation (extent of IBA1 + staining area) and a 2.4-fold increased density of activatedcells (Fig. 3C, D). Taken together, these data revealed
increased infiltration and activation of IBA1 + microglia

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within white matter injected with Nef EVs, indicatingthat an increased inflammatory response may contribute to Nef-mediated demyelination. There was no difference in the number of DAPI + cells, IBA1 + cell density,or IBA1 + activation in the contralateral corpus callosum,
indicating that this inflammatory response was localized
to the site of injection.
Nef EVs disrupt myelin in cerebellar slice cultures ex vivo

To assess the effects of Nef EVs on CNS myelin integrity in the absence of immune cell infiltration, cerebellar slice cultures from C57BL/6J mice at postnatal day

Fig. 3 Effects of Nef EVs on microglia in vivo. Mice were intracranially injected with Ctrl or Nef EVs. At 3 days post injection, brain tissue
was processed for analysis by immunohistochemistry. The images depict representative fields from the corpus collosum at the lesion site. A
Representative fluorescence images of microglia/myeloid cells (IBA1, green) and nuclei (DAPI, blue). B Quantification of DAPI + immunofluorescenceper µm2 within the ipsilateral (ipsi) and contralateral (contra) corpus callosum. C Quantification of IBA1 + cells per µm2 ­within2 the ipsilateraland contralateral corpus callosum. D Quantification of the percentage of IBA1 + immunofluorescence within the ipsilateral and contralateral
corpus callosum at the injection site. N = 5 mice per group. The white arrow indicates the injection site and the dotted line indicates the borderof the corpus callosum. For IHC, n = 5 mice per group, scale bar is 100 µm. *p < 0.05; **p < 0.01; ***p < 0.001

Schenck et al. Journal of Neuroinflammation(2024) 21:127

10 were grown on tissue culture inserts for 4 days andtreated once daily with Nef EVs or Ctrl EVs for 2 or
4 days. The concentration of EV-associated Nef added
to the cultures was 241.4 pg/mL, approximately tenfold
less than the concentration of Nef found in the blood of
more than 50% of ART-treated HIV-infected individuals with undetectable viral load [43]. After 2 days, thelength of the unmyelinated axons in the Nef EV-treatedslices significantly increased (twofold relative to that in
the untreated slices and 2.2-fold relative to that in the
Ctrl EV-treated slices) (Fig. 4A, B). After 4 days, sections from Nef EV-treated slices had significantly less
MBP expression than their counterparts (61% decrease
relative to untreated, and 54%—relative to Ctrl EVtreated) with visibly disrupted myelin sheaths (Fig. 4C,
D). These results suggest that less total myelin waspresent in cerebellar tissue exposed to Nef EVs. Taken
together, these findings indicate that Nef EVs inducechanges in myelin structure and integrity in the absenceof an infiltrating inflammatory cell response and axonal
degradation and that these effects increase over timewith continued exposure to Nef EVs.

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Nef EVs disrupt oligodendrocytes in mixed primary mousebrain cultures

Oligodendrocyte-specific expression of recombinant Nef
affects proper differentiation of oligodendrocytes in vitro[28]. To determine the effects of extracellular Nef EVs
on oligodendrocytes in vitro, mixed brain cultures wereobtained from neonatal C57BL/6J mice at postnatal day
3, grown in media promoting oligodendrocyte development for 5 days in vitro, and then were treated once daily
with Nef EVs or Ctrl EVs for 2 days. Oligodendrocytes
were detected using a monoclonal antibody against O4
since O4 expression begins shortly after lineage commitment and is associated with the duration of oligodendrocyte differentiation [28, 66]. In the presence of Nef EVs,
O4 + cells were visibly disrupted, and their morphology was altered (Fig. 5A, B); this disruption included a
decreased number and disorder of the branching process. We quantified the number of oligodendrocyteswith normal branching and found that Nef EV treatment resulted in a decrease in the number of branching O4 + oligodendrocytes (62% relative to that in theuntreated group and 68% relative to that in the Ctrl EVtreated group) (Fig. 5C). The total number of O4 + cells

Fig. 4 Effects of Nef EVs on myelin in cerebellar slice cultures. Cerebellar slice cultures derived from C57BL/6J mice at postnatal day 10 were
cultured for 4 days and subsequently treated with either Ctrl or Nef EVs once daily for 2 or 4 days. A Representative fluorescence images depict
myelin (MBP, red) and axons (NF, green) 2 days after treatment. B Quantification of unmyelinated axon length 2 days post-treatment, measured
as MBP-/NF + axon length in µm. Axons were measured on a single slice each for untreated (n = 33 axons), Ctrl EV-treated (n = 23 axons), and Nef
EV-treated (n = 31 axons) groups. C Representative fluorescence images display myelin (MBP, red) 4 days after treatment. D Quantificationof the percentage of MBP + immunofluorescence per field. N = 2–3 sections per treatment condition. Scale bar is 100 µm. *p < 0.05; **p < 0.01;***p < 0.001; ****p < 0.0001

Schenck et al. Journal of Neuroinflammation(2024) 21:127

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Fig. 5 Effects of Nef EVs on oligodendrocytes in primary brain cultures. Mouse primary brain cells were collected at postnatal day 3 and grownfor 5 days in media to promote oligodendrocyte growth. Subsequently, the cells were treated with either Ctrl or Nef EVs for 48 h. A, B Untreated(Unt) cultures served as negative controls. Representative fluorescence images illustrate oligodendrocytes (O4, green) and nuclei (DAPI, blue).
Higher magnification images displaying branching oligodendrocyte morphology are presented in (B). C Quantification of O4 + oligodendrocyteswith clear immunostaining and normal branching as a proportion of total DAPI + cells. D Representative fluorescence images of the TUNEL assay. E
Quantification of the TUNEL assay. Scale bar is 50 µm. N = 6, ****p < 0.0001

was not significantly different in the presence of Nef EVs,
and there was no significant change in TUNEL immunoreactivity between treatment conditions (Fig. 5D, E),suggesting that Nef EVs were not immediately toxic tooligodendrocytes but rather compromised their ability togenerate or maintain normal cellular architecture in thepresence of other neural cells.
Nef‑EVs disrupt oligodendrocytes in A2B5 + ‑enriched
cultures

To assess the effects of Nef EV treatment on oligodendrocyte lineage cells, A2B5 + oligodendrocyte progenitor cells were isolated from C57BL/6J mice at postnatal

day 5 and differentiated into O4 + oligodendrocytes for
2 or 6 days before treatment with Ctrl or Nef EVs once
daily for 2 days (Supplemental Fig. S3A, E). After the cells
were cultured for 2 days in oligodendrocyte differentiation medium prior to EV treatment, there was no significant change in the percentage of O4 + oligodendrocytes
between the treatment groups (Supplemental Fig. S3B).
However, Nef EV treatment resulted in a significant
increase in the number of chromatin fragments with adiameter less than 5 µm. Notably, this effect was observed
throughout the culture (Supplemental Fig. S3C) but wasmore pronounced within cell clusters (Supplemental
Figs. S3A, C). Chromatin condensation and degradation

Schenck et al. Journal of Neuroinflammation(2024) 21:127

of genomic DNA into fragments are common morphological markers of apoptosis [58]; accordingly, phase andfluorescence imaging of A2B5 + -enriched oligodendrocyte cultures revealed that DAPI + fragments overlayed
injured cells (via phase contrast microscopy) and disrupted oligodendrocytes (via O4 + immunofluorescence)(Supplemental Fig. S2).
To determine the effect of Nef EVs on more matureoligodendrocytes, EVs were added to A2B5 + OPCs after
6 days of differentiation (Supplemental Fig. S3E). In addition to an increase in DAPI + fragments, we observed asignificant decrease in the percentage of O4 + cells in Nef
EV-treated cultures, with significant disruptions to oligodendrocyte morphology (Supplemental Fig. S3E-G).
Taken together, these results suggest that in the absenceof other neural cells, Nef EVs directly damage matureoligodendrocytes, and the lack of effect on the total cellnumber in younger cultures may reflect continued generation of O4 + cells from the original A2B5 progenitors.
Nef EVs alter microglia and astrocytes in mixed primarymouse brain cultures

Since Nef-mediated demyelination and oligodendrocyte
damage in vivo are associated with changes in astrocytes
and microglia, we also examined the effects of Nef EVs on
these cell types in primary mouse brain cultures. Treating mixed brain cultures for 48 h with Nef EVs resulted
in a shift in the morphology of IBA1 + microglia/myeloid
cells toward a highly ramified morphology (25% increase
relative to that of the untreated group; 42% increase relative to that of the Ctrl EV-treated group; Supplemental
Fig. S4A-B), with no change in the number of IBA1 + cells
across all treatment conditions (Supplemental Fig. S4C).
This shift in microglial morphology may indicate achange in phenotype toward an activated state. To further determine whether Nef EVs potentiate a proinflammatory response in CNS cells that could contribute tooligodendrocyte damage, we analyzed the supernatant ofprimary brain cultures treated with Ctrl or Nef EVs forthe presence of 22 cytokines. A cytokine array did notreveal significant changes in protein expression between
Ctrl and Nef EV-treated cultures (Supplemental Fig. S5),
indicating that Nef EVs alone do not induce an inflammatory response in culture and that oligodendrocyte damage is not dependent on the expression of the cytokines
assayed. These results are in line with our previous studies on monocyte-derived macrophages in vitro, whichindicated that Nef alone is unable to induce a cytokine
response [60].
As in vivo (Fig. 2), astrocytes demonstrated a marked
shift in morphology in mixed cultures treated with Nef
EVs compared to controls (Fig. 6A–D): There was a significant decrease in the proportion of fibrous GFAP + cells

Page 13 of 21(25% decrease relative to untreated, 24% decrease relative to Ctrl EV-treated; Fig. 6B) that was accompanied
by significant increase in rod/bipolar (207% relative tountreated, 192% relative to Ctrl EV-treated; Fig. 6C) andmultipolar/stellated cells (649% relative to untreated,
415% relative to Ctrl EV-treated; Fig. 6D). There was nochange in the number of GFAP + cells across treatment
conditions (Fig. 6E), indicating no loss of astrocytes. This
shift in astrocyte morphology may reflect phenotypicchanges that could impact astrocyte function.
Since Nef EVs had a pronounced effect on astrocytes
both in vivo and in mixed cultures and because astrocytes
are critical for maintaining oligodendrocyte survival
and myelin maintenance [55], we examined the effectof Nef EVs on purified astrocytes (Fig. 6F–H). Treating GFAP + -enriched astrocyte cultures with Nef EVs
resulted in a decrease in the total number of DAPI + cells
and an increase in the GFAP + staining area per cell
(Figs. 6G, H), indicating a decrease in GFAP + astrocytes
but increased activation of the remaining astrocytes. Nef
EV treatment further resulted in changes in GFAP + cellorganization: whereas GFAP + cells in control samples
were scattered diffusely with no discernable orientation of astrocyte processes, Nef EV-treated GFAP + cells
were aggregated in clusters with processes extending radially around the center point (Fig. 6F). Taken
together, these data indicate that Nef EVs directly affect
GFAP + astrocytes.
Nef‑induced oligodendrocyte damage is prevented
by agents that inhibit the effect of Nef on ABCA1

Previously, we demonstrated that Nef EVs decreased
ABCA1 protein abundance in peripheral myeloid cells,
impairing cholesterol efflux and promoting inflammatory
responses [60, 67]. Analysis of ABCA1 in primary mousebrain cultures treated with Nef EVs demonstrated specific downmodulation of ABCA1 (Supplemental Fig. S6).
We have shown that the liver X receptor (LXR) agonist
TO-901317 restores ABCA1-mediated cholesterol efflux
in Nef-expressing HIV-infected macrophages [68, 69].
We also identified the small-molecule compound AMS55, which potently reversed the negative effects of Nef
on ABCA1 abundance by inhibiting the binding of Nef
to calnexin, an endoplasmic reticulum chaperone protein integral for ABCA1 maturation [70–72]. To determine the role of Nef EV-mediated disruption of ABCA1
in oligodendrocyte damage, we tested whether this damage could be prevented by treatment with TO-901317
or AMS-55. Mixed brain cultures obtained from neonatal C57BL/6J mice at postnatal day 3 were cultured
for 7 days and then pretreated once daily for 3 days with
1 µM TO-901317 or AMS-55. Cultures were then treatedonce daily with EVs for 2 days before immunostaining

Schenck et al. Journal of Neuroinflammation(2024) 21:127

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Fig. 6 Effects of Nef EVs on astrocyte morphology in vitro. Mouse primary brain cells were collected at postnatal day 3. After 5 days of growth
in media to promote oligodendrocyte development, cells were treated with either Ctrl or Nef EVs for 48 h, with untreated (Unt) cultures served
as negative controls. A. Representative fluorescence images depict the three primary types of astrocyte morphology observed, including (i)fibrous, (ii) bipolar, and (iii) multipolar. GFAP (red) and DAPI (blue) label astrocytes and nuclei, respectively. B–D Quantification of fibrous, bipolar,
and multipolar GFAP + cells as a proportion of the total GFAP + cell population. N = 5 images/group. E Quantification of GFAP + cells as a proportionof DAPI + cells. F A2B5 + cells were purified via MACS from dissociated mouse brains, cultured for 3 days in media to promote astrocyte
differentiation, and then treated with either Ctrl or Nef EVs for 48 h. GFAP (red) and DAPI (blue) label astrocytes and nuclei, respectively. Scalebar is 50 µm. G Quantification of DAPI + cells. H Quantification of GFAP + cells. N = 5, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Schenck et al. Journal of Neuroinflammation(2024) 21:127

for O4. Consistent with the results shown in Fig. 5, treatment with Nef EVs resulted in stark morphological disruption of O4 + cells (Fig. 7A), accompanied by a 50%
reduction in total O4 + cell density (Fig. 7B) and a 66%
reduction in O4 + cell immunofluorescence (Fig. 7C).
These effects of Nef EVs are consistent with our findings in purified oligodendrocyte cultures and support
our conclusion that Nef EVs preferentially affect matureoligodendrocytes. Pretreatment with TO-901317 prior
to treatment with Nef EVs restored O4 + cell counts to

Fig. 7 Treatment of brain cells with agents targeting Nef-mediateddownmodulation of ABCA1. Mouse primary brain cells werecollected at postnatal day 3 and allowed to grow for 7 days in media
conducive to oligodendrocyte development. Subsequently, they
were pre-treated with the LXR agonist TO-901317 (TO, 1 µM)
or the inhibitor of the Nef-calnexin interaction, AMS-55 (AMS, 1 µM),
for 3 days, followed by treatment with either Ctrl or Nef EVs for 48 h.
A—Representative fluorescence images display oligodendrocytes(O4, green) and nuclei (DAPI, blue). B Quantification of total
O4 + oligodendrocytes as a proportion of total DAPI + cells. C
Quantification of the percentage of O4 immunofluorescence. Scalebar is 50 µm. N = 4. *p < 0.05; **p < 0.01; ****p < 0.0001

Page 15 of 21

the levels observed in Ctrl EV-treated cultures but only
partially restored O4 + immunofluorescence. Conversely,
AMS-55 only partially restored O4 + cell density, but
O4 + immunofluorescence was more than twice that in
Ctrl EV-treated cultures. Taken together, these data suggest that TO-901317 and AMS-55 partially prevent Nefmediated oligodendrocyte damage and implicate ABCA1
in the mechanism of oligodendrocyte damage caused by
Nef EVs.

Discussion
Previous reports have linked Nef to HAND pathogenesis[33–35, 59], but no studies have investigated the role of
Nef in demyelination. In this study, we discovered that
Nef-containing EVs impair oligodendrocytes and disrupt
CNS myelin. Our findings align with those of Radja et al.
who observed a significant reduction in O4 + immunofluorescence and alterations in oligodendrocyte morphology induced by Nef in a transgenic model expressing
Nef under the oligodendrocyte-specific MBP promoter[28]. Since HIV-1 does not infect oligodendrocytes dueto their lack of CD4 and CCR5 receptors, our study
offers a biologically significant context by examining theeffects of exogenously produced EV-associated Nef onoligodendrocytes.
Other studies have provided evidence of the toxic
effects of both Nef and Nef-containing EVs on neurons[36, 37, 73]. Considering that disruptions in myelin canlead to severe axonal degeneration and impair neuronal
function [74], we aimed to investigate the impact of Nef
EVs on myelin sheaths. We employed various experimental methods to explore Nef-mediated myelin perturbation and oligodendrocyte damage. Initially, we utilized
electron microscopy (EM) analysis after administering
Nef EVs in vivo and observed the depletion of myelin
sheaths around intact axons. Subsequently, ex vivo treatment of cerebellar slice cultures with Nef EVs revealed
an increase in the number of MBP-negative axons,indicating a reduction in axon myelination without significant neuronal death. Finally, in vitro experiments
demonstrated that Nef EVs directly inhibited the enrichment of A2B5 + cells in culture, indicating an impact onoligodendrocytes. Taken together, these findings suggest that Nef EVs exert direct effects on myelin and oligodendrocytes, potentially contributing to subsequentneurodegeneration.
Oligodendrocyte maturation is typically classified
into four stages based on morphology and the temporal
expression of cell surface protein markers (as reviewed
in [75, 76]): bipolar oligodendrocyte precursor cells(OPCs), branching premyelinating oligodendrocytes(preOLs), differentiated immature oligodendrocytes, andmature myelinating oligodendrocytes. The sensitivity of

Schenck et al. Journal of Neuroinflammation(2024) 21:127

oligodendrocytes to programmed cell death varies withmaturation: while OPCs express potent mitogens andsurvival factors, such as PDGF-A [77], preOLs are highly
susceptible to apoptosis induced by various stressors[78, 79]. Indeed, the majority of oligodendrocytes arelost during the preOL stage of development and during
adult oligodendrogenesis [80–82]. In both mixed primary and enriched A2B5 + oligodendrocyte cultures,
we observed more pronounced morphological disruptions in O4 + cells in older cultures. This could be attributed to the heightened susceptibility of oligodendrocytes
to stress during the preOL stage, when cells experience
increased metabolic demands associated with myelin
synthesis pathways.
We demonstrate that agents targeting the effect of
Nef on ABCA1 prevent Nef-induced oligodendrocyte
damage in vitro. The LXR agonist TO-901317, known
for its potent induction of ABCA1, has previously beenshown to enhance oligodendrocyte proliferation, differentiation, and maturation, and to alleviate ischemia andlysolecithin-induced demyelination [83, 84]. Although
TO-901317 treatment resulted in an overall increase inO4 + cells, we observed two distinct oligodendrocyte
morphologies in cultures treated with TO-901317: moremature oligodendrocytes with highly complex branching
and less mature oligodendrocytes with fewer branchingprocesses. While TO-901317 prevented O4 + cell loss,
its effect on immunofluorescence was less pronounced,
suggesting that the ABCA1 pathway might not be thesole target of Nef-mediated oligodendrocyte toxicity.
Additionally, the interaction of AMS-55, an inhibitor of
the Nef-calnexin complex necessary for Nef-mediated
ABCA1 downregulation [70, 72], partially prevented
oligodendrocyte damage. AMS-55 did not fully restore
the O4 + cell number but did significantly increase theO4 + staining area to a level greater than that observedin Ctrl EV-treated cultures. These findings suggested
that while inhibiting the Nef-calnexin interaction (thusrestoring ABCA1 abundance) was not sufficient to completely prevent Nef-mediated oligodendrocyte damage,it did protect a certain population of cells. Furthermore,cultures treated with Nef + AMS-55 displayed a sheathing morphology reflective of more mature oligodendrocytes. Overall, these results suggest that oligodendrocyte
damage caused by Nef EVs is at least partially mediatedby ABCA1. Notably, LXRs are critical regulators of cholesterol homeostasis in the CNS and induce the expression of several genes involved in cholesterol transport
in oligodendrocytes [85]. LXRs directly regulate reverse
cholesterol transport through the transcription of apoEand cholesterol transporters, including ABCA1, ABCG1,
and ABCG4 [86]. LXRs also regulate the uptake of cholesterol via low-density lipoprotein receptors (LDLRs)

Page 16 of 21[87–89]. These findings suggested that the effects of Nef
EVs on oligodendrocytes may be due to interactions withseveral aspects of cholesterol homeostasis regulated by
LXRs. Future studies will further examine the effects of
Nef EVs on cholesterol metabolism in the CNS.
Microglia and astrocytes are involved in myelin maintenance and repair; alterations to these cells can contribute to demyelination and disrupt the process ofremyelination [39], and both cell types have been implicated in various mechanisms of neuronal damage thatlikely contribute to HAND [90, 91]. Both cell types arelikely infected by HIV and are capable of producing
Nef [92], but the effect of exogenous Nef on these cells
in relation to the subsequent effect on myelin has notbeen previously reported. Our finding that Nef EVs disrupt oligodendrocytes in enriched cultures implies that
Nef-containing EVs directly damage oligodendrocytes
in vitro, but does not preclude indirect effects from othercell types, including microglia or astrocytes. In fact, ourresults suggest that in the context of mixed cultures, oligodendrocytes are less susceptible to the cytotoxic effects
of Nef EVs. We report that Nef EV injection induces anincrease in IBA1 + microglia/myeloid cell infiltration atmyelin lesions. However, our ex vivo and in vitro data
indicate that systemic inflammation is not essential for
Nef EV-mediated disruption of myelin and oligodendrocytes. For example, altered myelination of NF + axons wasobserved in cerebellar slice cultures, an ex vivo system
ideal for studying myelin in the absence of an infiltrating immune response. Additionally, we did not observechanges in cytokine protein expression in mixed primarybrain cultures containing oligodendrocytes, astrocytes,
and microglia, and observed oligodendrocyte damage inenriched cultures derived from A2B5 + oligodendrocyte
precursor cells. While our results show that inflammation is not necessary for oligodendrocyte damage, it likely
contributes to the persistent demyelinating pathology
observed in individuals with HAND.
Our finding that Nef EVs do not stimulate inflammatory cytokine release by mixed brain cultures appears
to contradict the findings of previous reports that
Nef exosomes stimulate the release of cytokines andchemokines from CHME-5 microglia [93]. A possible
explanation is that IBA1 + microglia/macrophages makeup only 10% of the cells in our primary brain cultures,
which may be insufficient to elicit a notable inflammatory response. In addition, there may be differences in EV
preparation and contamination with proinflammatory
agents (e.g., LPS) between studies. Indeed, we have previously shown that Nef alone is unable to elicit a cytokine
response in macrophages in vitro, but Nef EVs reorganize lipid rafts and potentiate proinflammatory responses
in bystander cells to inflammatory stimuli [60]. This maySchenck et al. Journal of Neuroinflammation(2024) 21:127

be particularly relevant to in vivo experimental models,
where low-level inflammation is difficult to avoid. Therefore, Nef EV-exposed astrocytes and microglia likely
indirectly contribute to oligodendrocyte damage via thepotentiation of inflammation.
Disruptions to GFAP + astrocytes were observed after
Nef EV treatment, including a decrease in cell density
and altered co-expression of the astrocytic maturation
status and reactivity markers GFAP and vimentin in vivo,
and a shift in cell morphology and orientation in vitro.
Conflicting reports on the effects of HIV and Nef on
astrocytic expression of GFAP have been published: one
study reported increased expression of GFAP, indicativeof a reactive astrocyte phenotype [94], while the othershowed that Nef downregulates GFAP expression inastrocytes in vitro [62]. These conflicting findings may be
due to the heterogeneity of CNS astrocytes. In our studies, we consistently observed a disruption in GFAP + cells
in Nef EV-injected white matter and in purified astrocyte
cultures. This result was unexpected given the astrogliosis commonly observed in the brains of HIV-positive
individuals [95]. The principal driver of astrogliosis in
HIV-infected brain is likely the viral protein Tat, known

Page 17 of 21

to elevate GFAP expression [96]. In the context of HIV
brain infection, particularly in the era of ART, the collective effect on astrocytes could vary based on the local
concentrations of Tat and Nef, alongside other potential
factors. The reduction in GFAP expression observed inour study may signify a specific influence of Nef EVs on
astrocytic function, potentially contributing to astrocyte
dysfunction during astrogliosis. Suppression of GFAP
expression in astrocytes by Nef EVs does not contradict
their pro-inflammatory effect and may represent the consequences of abnormal activation. These results suggest apotential shift in phenotype or cellular dysfunction that
could affect astrocytic homeostatic mechanisms. Since
astrocytes are critical for myelin maintenance [55], Nef
EV-mediated astrocyte damage likely contributes to oligodendrocyte damage and myelin impairment.
We propose a mechanism whereby Nef EVs directly
and indirectly damage oligodendrocytes and disrupt
myelin in the CNS (Fig. 8). Nef EVs are released fromHIV-infected cells and taken up by bystander cells in the
CNS. Oligodendrocytes are directly damaged by Nef EVs,
which compromises the survival and/or metabolism ofthese cells and impairs myelin integrity. Additionally, Nef

Fig. 8 Schematic for Nef EV-mediated oligodendrocyte damage and myelin impairment. Nef is released from extracellular vesicles (EVs)and is internalized by bystander cells in the brain. Nef EVs directly damage oligodendrocytes, compromising their survival or metabolism
and impairing myelin integrity. This damage is partially mediated by the downregulation of ABCA1. Additionally, Nef may indirectly harmoligodendrocytes by disrupting ABCA1-mediated cholesterol efflux in astrocytes and microglia. This disruption leads to increased intracellularcholesterol levels, lipid raft formation, activation of inflammasomes, and the secretion of pro-inflammatory cytokines. Inflammation is further
exacerbated in microglia during recycling and clearance of myelin debris. In astrocytes, degradation of ABCA1 may hinder the transport of criticalcholesterol to oligodendrocytes, further disrupting myelin homeostasis. Image created with BioRenderSchenck et al. Journal of Neuroinflammation(2024) 21:127

EVs may indirectly damage oligodendrocytes by disrupting cholesterol homeostasis in astrocytes and microglia.
Nef reduces the amount of ABCA1, the primary cholesterol efflux transporter in these cells [50, 97], throughpreviously described mechanisms [60]. Disrupting
ABCA1-mediated cholesterol efflux leads to increased
intracellular cholesterol levels, lipid raft formation,
inflammasome activation, and proinflammatory cytokine
secretion stimulation induced by an inflammatory stimulus, all of which contribute to demyelinating pathology.
In astrocytes, degradation of ABCA1 may also preventthe transport of critical cholesterol to oligodendrocytes,
which further damages oligodendrocytes and disrupts
myelin integrity. The inflammatory process is further
exacerbated in phagocytic microglia during the recycling
and clearance of cholesterol-rich myelin debris, leading
to a persistent inflammatory state.
The relevance of our findings to the pathogenesis of
HAND is underscored by the demonstrated expression of
Nef and Nef-containing EVs in the brains of HIV-infectedindividuals, including those undergoing ART treatment[36, 43, 59, 98–100]. Notably, Nef EVs can originate fromHIV-infected brain cells such as microglial cells, perivascular macrophages, and astrocytes, which constitute viralreservoirs in the brain. These reservoirs are establishedshortly after initial infection and persist despite the initiation of ART [101]. Furthermore, circulating Nef EVs[43] represent another potential source of Nef in thebrain, as these vesicles possess the capability to traverse
the blood–brain barrier [102].

Limitations
It is important to note that in this study, EVs were derived
from HEK293T cells rather than from primary or immortalized CNS cells. Given that EVs facilitate the transfer
of proteins, lipids, and nucleic acids (including noncoding RNAs and microRNAs) [103], brain-derived EVs(BDEVs) may contain specific materials that further contribute to Nef-mediated pathology in the CNS. However,the characterization of BDEVs and the methodology for
their production are still evolving scientific approaches.
In contrast, HEK293T cells are widely recognized as amodel system for EV production. In fact, EVs derived
from HEK293T cells are extensively characterized andconsidered reliable, to the extent that they can be commercially purchased for therapeutic delivery (System
Biosciences, #EXOP-110A-1) and are commonly utilized
in preclinical studies involving EV-based therapeutics
to treat both neurological and systemic diseases [104].
Furthermore, our laboratory has extensively characterized the effects of HEK293T-derived Nef EVs on cholesterol metabolism [42, 60, 105], suggesting that these EVs
are well-suited for foundational studies of white matter

Page 18 of 21

damage caused by Nef EVs. Future studies will aim toinvestigate the effects of astrocyte- and microglia-derived
Nef EVs on myelin and oligodendrocytes in the CNS.

Conclusions
In conclusion, our study presents the initial characterization of the effects of Nef EVs on myelin loss within the
CNS. We observed that Nef EVs induce direct damage
to oligodendrocytes and potentially exert indirect effects
by perturbing microglia and astrocytes. Demyelination
is likely mediated, at least in part, by the impairment ofcholesterol homeostasis resulting from Nef EV-mediateddownmodulation of ABCA1. Previously reported agents
capable of blocking or reversing ABCA1 downregulation by Nef have shown promising results in protecting
oligodendrocytes from damage. Further elucidation of
the mechanisms underlying Nef EV-mediated oligodendrocyte damage, particularly its impact on cholesterol
metabolism, holds potential for identifying therapeutics
aimed at preventing or treating HAND. Our study sheds
light on the intricate interplay between Nef, glial cells,
and myelin integrity, thereby contributing to a deeper
understanding of HAND pathogenesis. Additionally, ourfindings may pave the way for the identification of potential targets for therapeutic intervention aimed at alleviating neurocognitive deficits in individuals living with HIV.
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03124-5.
Supplementary Material 1.
Acknowledgements
The following reagent was obtained through the NIH HIV Reagent Program,
Division of AIDS, NIAID, NIH: Plasmid pcDNA3.1 Expressing Human CodonOptimized HIV-1 96ZM651.8 Nef Expression Vector, ARP-8677, contributed byDrs. Yingying Li, Feng Gao and Beatrice H. Hahn. AMS-55 was kindly provided
by Dr. Amol Kulkarni at Howard University College of Pharmacy in Washington,
DC. We are indebted to Dr. Christopher Vanpouille (NIH, NICHD) for testing oursamples in the Nef ELISA.
Author contributions
JKS performed mouse experiments, performed histological examinationof the brain, analyzed the data, and was a major contributor in writing themanuscript. MTK supervised animal experiments and performed section
preparation for EM. CCP performed EM imaging and analysis. AB performed EV
analysis. TP performed Western blot analysis. BB prepared EVs. RHM analyzed
and interpreted brain tissue results and contributed to manuscript writing.
MIB designed the study and was a major contributor in writing the manu‑
script. All authors read and approved the final manuscript.
Funding
This study was supported by AHA Grant 20PRE35080036 (J.K.S.) and NIH
Grants to M.I.B. R01 HL140977, R01 HL158305, R01 NS124477, and P30
AI117970.

Schenck et al. Journal of Neuroinflammation(2024) 21:127

Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information files.

Declarations
Declarations
Ethics approval and consent to participate
All the experimental procedures were approved by the George Washington
University Institutional Animal Care and Use Committee (protocol A2020-40,
approved on December 15, 2020).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 28 January 2024 Accepted: 4 May 2024

References
1. Cysique LA, et al. HIV-associated neurocognitive disorder in Australia: acase of a high-functioning and optimally treated cohort and implica‑
tions for international neuroHIV research. J Neurovirol. 2014;20:258–68.
2. Heaton RK, et al. HIV-associated neurocognitive disorders persist in
the era of potent antiretroviral therapy: CHARTER Study. Neurology.
2010;75:2087–96.
3. Robertson K, et al. Screening for neurocognitive impairment, depres‑sion, and anxiety in HIV-infected patients in Western Europe andCanada. AIDS Care. 2014;26:1555–61.
4. Robertson KR, et al. The prevalence and incidence of neurocognitive
impairment in the HAART era. AIDS. 2007;21:1915–21.
5. Sacktor N, et al. Prevalence of HIV-associated neurocognitive disorders
in the Multicenter AIDS Cohort Study. Neurology. 2016;86:334–40.
6. Tozzi V, et al. Prevalence and risk factors for human immunodeficiency
virus–associated neurocognitive impairment, 1996 to 2002: results froman urban observational cohort. J Neurovirol. 2005;11:265–73.
7. Bloch M, et al. A screening strategy for HIV-associated neurocognitive
disorders that accurately identifies patients requiring neurologicalreview. Clin Infect Dis. 2016;63:687–93.
8. Crum-Cianflone NF, et al. Low prevalence of neurocognitive impair‑ment in early diagnosed and managed HIV-infected persons. Neurol‑ogy. 2013;80:371–9.
9. De Francesco D, et al. Defining cognitive impairment in people-livingwith-HIV: the POPPY study. BMC Infect Dis. 2016;16:617.
10. McDonnell J, et al. Minimal cognitive impairment in UK HIV-positivemen who have sex with men: effect of case definitions and comparison
with the general population and HIV-negative men. J Acquir Immune
Defic Syndr. 2014;67:120–7.
11. Wright E, et al. Factors associated with neurocognitive test performanceat baseline: a substudy of the INSIGHT Strategic Timing of AntiRetroviral
Treatment (START) trial. HIV Med. 2015;16:97–108.
12. Simioni S, et al. Cognitive dysfunction in HIV patients despite longstanding suppression of viremia. AIDS. 2009;24:1.
13. Doyle KL, et al. Real-world impact of neurocognitive deficits in acuteand early HIV infection. J Neurovirol. 2013;19:565–73.
14. Saylor D, et al. HIV-associated neurocognitive disorder—pathogenesis
and prospects for treatment. Nat Rev Neurol. 2016;12:234.
15. Su T, et al. White matter hyperintensities in relation to cognition in
HIV-infected men with sustained suppressed viral load on combination
antiretroviral therapy. AIDS. 2016;30:2329–39.
16. Hoare J, et al. White matter correlates of apathy in HIV-positive subjects:
a diffusion tensor imaging study. J Neuropsychiatry Clin Neurosci.
2010;22:313–20.

Page 19 of 21

17. Tate DF, et al. Regional areas and widths of the midsagittal corpus cal‑losum among HIV-infected patients on stable antiretroviral therapies. J
Neurovirol. 2011;17:368.
18. Leite SCB, et al. Diffusion tensor MRI evaluation of the corona radiata,
cingulate gyri, and corpus callosum in HIV patients. J Magn Reson
Imaging. 2013;38:1488–93.
19. Borjabad A, et al. Significant effects of antiretroviral therapy on global
gene expression in brain tissues of patients with HIV-1-associatedneurocognitive disorders. PLoS Pathog. 2011;7:1002213.
20. Solomon IH, et al. White matter abnormalities linked to interferon,stress response, andenergy metabolism gene expression changes inolder HIV-positive patients onantiretroviral therapy. Mol Neurobiol.
2020;57:1115.
21. Lackner P, et al. Antibodies to myelin oligodendrocyte glycoprotein
in HIV-1 associated neurocognitive disorder: a cross-sectional cohort
study. J Neuroinflammation. 2010;7:79.
22. Corrêa DG, et al. Diffusion tensor MR imaging of white matter integ‑
rity in HIV-positive patients with planning deficit. Neuroradiology.
2015;57:475–82.
23. Gongvatana A, et al. White matter tract injury and cognitive impairment
in human immunodeficiency virus–infected individuals. J Neurovirol.
2009;15:187–95.
24. Tate DF, et al. Quantitative diffusion tensor imaging tractography
metrics are associated with cognitive performance among HIV-infectedpatients. Brain Imaging Behav. 2010;4:68–79.
25. Müller-Oehring EM, Schulte T, Rosenbloom MJ, Pfefferbaum A, Sullivan
EV. Callosal degradation in HIV-1 infection predicts hierarchical percep‑
tion: a DTI study. Neuropsychologia. 2010;48:1133–43.
26. Zou S, et al. Oligodendrocytes are targets of HIV-1 tat: NMDA and AMPA
receptor-mediated effects on survival and development. J Neurosci.
2015;35:11384–98.
27. Hauser KF, et al. HIV-1 Tat and morphine have interactive effects on
oligodendrocyte survival and morphology. Glia. 2009;57:194–206.
28. Radja F, Kay DG, Albrecht S, Jolicoeur P. Oligodendrocyte-specificexpression of human immunodeficiency virus type 1 Nef in transgenic
mice leads to vacuolar myelopathy and alters oligodendrocyte pheno‑
type in vitro. J Virol. 2003. https://​doi.​org/​10.​1128/​jvi.​77.​21.​11745-​11753.​
2003.
29. Li G-H, Henderson L, Nath A. Astrocytes as an HIV reservoir: mechanismof HIV infection. Curr HIV Res. 2016;14:373–81.
30. Minagar A, et al. The role of macrophage/microglia and astrocytes
in the pathogenesis of three neurologic disorders: HIV-associateddementia, Alzheimer disease, and multiple sclerosis. J Neurol Sci.
2002;202:13–23.
31. Nath A. Eradication of human immunodeficiency virus from brain
reservoirs. J Neurovirol. 2015;21:227–34.
32. Sviridov D, Mukhamedova N, Makarov AA, Adzhubei A, Bukrinsky M.
Comorbidities of HIV infection: role of Nef-induced impairment ofcholesterol metabolism and lipid raft functionality. AIDS. 2020;34:1–13.
33. Khan MB, et al. Nef exosomes isolated from the plasma of individuals
with HIV-associated dementia (HAD) can induce Aβ(1–42) secretion in
SH-SY5Y neural cells. J Neurovirol. 2016;22:179–90.
34. Saribas AS, Khalili K, Sariyer IK. Dysregulation of autophagy by HIV-1 Nef
in human astrocytes. Cell Cycle. 2015;14:2899–904.
35. Acharjee S, et al. HIV-1 Nef expression in microglia disrupts dopaminer‑
gic and immune functions with associated mania-like behaviors. Brain
Behav Immun. 2014;40:74–84.
36. Chompre G, et al. Astrocytic expression of HIV-1 Nef impairs spatial andrecognition memory. Neurobiol Dis. 2013;49:128.
37. Rivera J, et al. Infusion of HIV-1 Nef-expressing astrocytes into therat hippocampus induces enteropathy and interstitial pneumonitis
and increases blood–brain-barrier permeability. PLoS ONE. 2019;14:e0225760.
38. de Waard DM, Bugiani M. Astrocyte–oligodendrocyte–microglia cross‑talk in astrocytopathies. Front Cell Neurosci. 2020;14:608073.
39. Domingues HS, Portugal CC, Socodato R, Relvas JB. Oligodendrocyte,
astrocyte, and microglia crosstalk in myelin development, damage, andrepair. Front Cell Dev Biol. 2016;4:1–16.
40. Siegel J, Darwish C, Popratiloff A, Bukrinsky M, Brichacek B. Live cell
imaging of ABCA1 downregulation by HIV-1 Nef in an experimental
model of HeLa ABCA1-GFP. AIDS Res Hum Retrovir. 2016;32:872–3.

Schenck et al. Journal of Neuroinflammation(2024) 21:127

41. Cui HL, et al. HIV protein Nef causes dyslipidemia and formation of foamcells in mouse models of atherosclerosis. FASEB J. 2014;28:2828–39.
42. Asztalos BFF, et al. Circulating Nef induces dyslipidemia in simian
immunodeficiency virus-infected macaques by suppressing cholesterol
efflux. J Infect Dis. 2010;202:614–23.
43. Ferdin J, et al. Viral protein Nef is detected in plasma of half of HIVinfected adults with undetectable plasma HIV RNA. PLoS ONE. 2018;13:
e0191613.
44. Caobi A, et al. Protein cargo of Nef-containing exosomal extracellular
vesicles may predict HIV-associated Neurocognitive Impairment status.
Res Sq. 2023. https://​doi.​org/​10.​21203/​RS.3.​RS-​27401​35/​V2.
45. Jurevics H, Morell P. Cholesterol for synthesis of myelin is made locally,
not imported into brain. J Neurochem. 1995;64:895–901.
46. Corraliza-Gomez M, Sanchez D, Ganfornina MD. Lipid-binding proteinsin brain health and disease. Front Neurol. 2019;10:1152.
47. Nave K-A, Werner HB. Myelination of the nervous system: mechanisms
and functions. Annu Rev Cell Dev Biol. 2014;30:503–33.
48. Nieweg K, Schaller H, Pfrieger FW. Marked differences in cholesterolsynthesis between neurons and glial cells from postnatal rats. J Neuro‑
chem. 2009;109:125–34.
49. Camargo N, et al. Oligodendroglial myelination requires astrocytederived lipids. PLOS Biol. 2017;15: e1002605.
50. Kim WS, Guillemin GJ, Glaros EN, Lim CK, Garner B. Quantitation of ATPbinding cassette subfamily-A transporter gene expression in primary
human brain cells. NeuroReport. 2006;17:891–6.
51. Dubrovsky L, et al. Inhibition of HIV replication by apolipoprotein A–I
binding protein targeting the lipid rafts. MBio. 2020;11:10–128.
52. Livshts MA, et al. Isolation of exosomes by differential centrifugation:
theoretical analysis of a commonly used protocol. Sci Rep. 2015;5:1–4.
53. Gelibter S, et al. The impact of storage on extracellular vesicles: a sys‑
tematic study. J Extracell Vesicles. 2022;11: e12162.
54. Russell AE, et al. Biological membranes in EV biogenesis, stability,
uptake, and cargo transfer: an ISEV position paper arising from the ISEV
membranes and EVs workshop. J Extracell Vesicles. 2019;8:1684862.
55. Tognatta R, et al. Astrocytes are required for oligodendrocyte survival
and maintenance of myelin compaction and integrity. Front Cell Neuro‑sci. 2020;14:74.
56. Najm FJ, et al. Drug-based modulation of endogenous stem cells
promotes functional remyelination in vivo. Nature. 2015;522:216.
57. Gogolla N, Galimberti I, DePaola V, Caroni P. Staining protocol fororganotypic hippocampal slice cultures. Nat Protoc. 2006;1:2452–6.
58. Dobrucki J, Darzynkiewicz Z. Chromatin condensation and sensitivity of
DNA in situ to denaturation during cell cycle and apoptosis—a confo‑
cal microscopy study. Micron. 2001;32:645–52.
59. Pushkarsky T, et al. Abundance of Nef and p-Tau217 in brains ofindividuals diagnosed with HIV-associated neurocognitive disorders
correlate with disease severance. Mol Neurobiol. 2022;59:1088–97.
60. Mukhamedova N, et al. Exosomes containing HIV protein Nef reorgan‑
ize lipid rafts potentiating inflammatory response in bystander cells.
PLoS Pathog. 2019;15: e1007907.
61. Edgar JM, et al. Demyelination and axonal preservation in a transgenic
mouse model of Pelizaeus-Merzbacher disease. EMBO Mol Med.
2010;2:42.
62. Wilson KM, He JJ. HIV Nef expression down-modulated GFAP expression
and altered glutamate uptake and release and proliferation in astro‑cytes. Aging Dis. 2023;14:152.
63. Jurga AM, Paleczna M, Kadluczka J, Kuter KZ. Beyond the GFAP-astro‑cyte protein markers in the brain. Biomolecules. 2021;11:1361.
64. Bramanti V, Tomassoni D, Avitabile M, Amenta F, Avola R. Biomarkers
of glial cell proliferation and differentiation in culture. Front Biosci.
2010;2:558–70.
65. Scaravilli F, Bazille C, Gray F. Neuropathologic contributions tounderstanding AIDS and the central nervous system. Brain Pathol.
2007;17:197.
66. Pfeiffer SE, Warrington AE, Bansal R. The oligodendrocyte and its manycellular processes. Trends Cell Biol. 1993;3:191–7.
67. Ditiatkovski M, et al. Modification of lipid rafts by extracellular vesicles
carrying HIV-1 protein Nef induces redistribution of amyloid precur‑sor protein and Tau, causing neuronal dysfunction. J Biol Chem.
2020;295:13377–92.

Page 20 of 21

68. Morrow MP, et al. Stimulation of the liver X receptor pathway inhibits
HIV-1 replication via induction of ATP-binding cassette transporter A1.
Mol Pharmacol. 2010;78:215.
69. Ramezani A, et al. Stimulation of liver X receptor has potent anti-HIV
effects in a humanized mouse model of HIV infection. J Pharmacol Exp
Ther. 2015;354:376–83.
70. Adzhubei AA, et al. Modelling interaction between HIV-1 Nef andcalnexin. AIDS. 2018;32:2103–11.
71. Hunegnaw R, et al. Interaction between HIV-1 Nef and calnexin: frommodeling to small molecule inhibitors reversing HIV-induced lipid
accumulation. Arterioscler Thromb Vasc Biol. 2016;36:1758–71.
72. Adzhubei AA, et al. Direct interaction between ABCA1 and HIV-1 Nef:molecular modeling and virtual screening for inhibitors. Comput
Struct Biotechnol J. 2021;19:3876.
73. Sami Saribas A, et al. HIV-1 Nef is released in extracellular vesicles
derived from astrocytes: evidence for Nef-mediated neurotoxicity.
Cell Death Dis. 2017;8: e2542.
74. Mu J, et al. Myelin damage in diffuse axonal injury. Front Neurosci.
2019;13:435840.
75. Hughes EG, Stockton ME. Premyelinating oligodendrocytes: mecha‑
nisms underlying cell survival and integration. Front Cell Dev Biol.
2021;9:714169.
76. Butts BD, Houde C, Mehmet H. Maturation-dependent sensitivity ofoligodendrocyte lineage cells to apoptosis: implications for normal
development and disease. Cell Death Differ. 2008;157(15):1178–86.
77. Frost EE, Nielsen JA, Le TQ, Armstrong RC. PDGF and FGF2 regulateoligodendrocyte progenitor responses to demyelination. J Neurobiol.
2003;54:457–72.
78. Sun LO, et al. Spatiotemporal control of CNS myelination by oligo‑
dendrocyte programmed cell death through the TFEB-PUMA axis.
Cell. 2018;175:1811-1826.e21.
79. Cai Q, Ma T, Tian Y, Li C, Li H. Catalpol inhibits ischemia-induced
premyelinating oligodendrocyte damage through regulation ofintercellular calcium homeostasis via N­ a+/Ca2+ exchanger 3. Int J Mol
Sci. 2018;19:1925.
80. Trapp BD, Nishiyama A, Cheng D, Macklin W. Differentiation anddeath of premyelinating oligodendrocytes in developing rodent
brain. J Cell Biol. 1997;137:459–68.
81. Barres BA, et al. Cell death and control of cell survival in the oligoden‑
drocyte lineage. Cell. 1992;70:31–46.
82. Hughes EG, Orthmann-Murphy JL, Langseth AJ, Bergles DE. Myelin
remodeling through experience-dependent oligodendrogenesis in
the adult somatosensory cortex. Nat Neurosci. 2018;21:696–706.
83. Gao T, et al. T0901317, a liver X receptor agonist, ameliorates perina‑
tal white matter injury induced by ischemia and hypoxia in neonatal
rats. Neurosci Lett. 2023;793:136994.
84. Meffre D, et al. Liver X receptors alpha and beta promote myelina‑
tion and remyelination in the cerebellum. Proc Natl Acad Sci USA.
2015;112:7587–92.
85. Nelissen K, et al. Liver X receptors regulate cholesterol homeostasis inoligodendrocytes. J Neurosci Res. 2012;90:60–71.
86. Jasmin SB, et al. Differential regulation of ABCA1 and ABCG1 gene
expressions in the remodeling mouse hippocampus after entorhi‑
nal cortex lesion and liver-X receptor agonist treatment. Brain Res.
2014;1562:39–51.
87. Courtney R, Landreth GE. LXR regulation of brain cholesterol: fromdevelopment to disease. Trends Endocrinol Metab. 2016;27:404–14.
88. Ishimoto K, et al. Identification of human low-density lipoprotein
receptor as a novel target gene regulated by liver X receptor alpha.
FEBS Lett. 2006;580:4929–33.
89. Zelcer N, Hong C, Boyadjian R, Tontonoz P. LXR regulates cholesterol
uptake through idol-dependent ubiquitination of the LDL receptor.
Science. 2009;325:100–4.
90. Lee KM, et al. Form follows function: astrocyte morphology andimmune dysfunction in SIV neuroAIDS. J Neurovirol. 2014;20:474.
91. Borrajo A, Spuch C, Penedo MA, Olivares JM, Agís-Balboa RC. Impor‑tant role of microglia in HIV-1 associated neurocognitive disorders
and the molecular pathways implicated in its pathogenesis. Ann
Med. 2021;53:43.
92. Trillo-Pazos G, et al. Detection of HIV-1 DNA in microglia/mac‑
rophages, astrocytes and neurons isolated from brain tissue with

Schenck et al. Journal of Neuroinflammation

93.
94.
95.
96.

97.
98.
99.
100.
101.
102.
103.
104.
105.

(2024) 21:127

HIV-1 encephalitis by laser capture microdissection. Brain Pathol.
2003;13:144–54.
Raymond AD, et al. Microglia-derived HIV Nef+ exosome impair‑ment of the blood-brain barrier is treatable by nanomedicine-based
delivery of Nef peptides. J Neurovirol. 2016;22:129–39.
Kohleisen B, Hutzler P, Shumay E, Ovod V, Erfle V. HIV-1 Nef co-localizes
with the astrocyte-specific cytoskeleton protein GFAP in persistently
Nef-expressing human astrocytes. J Neurovirol. 2001;7:52–5.
Sabri F, Titanji K, De Milito A, Chiodi F. Astrocyte activation and apop‑
tosis: their roles in the neuropathology of HIV infection. Brain Pathol.
2003;13:84.
Joshi CR, Stacy S, Sumien N, Ghorpade A, Borgmann K. Astrocyte HIV-1
tat differentially modulates behavior and brain MMP/TIMP balance
during short and prolonged induction in transgenic mice. Front Neurol.
2020;11:593188.
Karasinska JM, et al. ABCA1 influences neuroinflammation and neuronal
death. Neurobiol Dis. 2013;54:445–55.
Yarandi SS, et al. Characterization of Nef expression in different brain
regions of SIV-infected macaques. PLoS ONE. 2020;15: e0241667.
Clauss M, Chelvanambi S, Cook C, Elmergawy R, Dhillon N. Viral bad
news sent by EVAIL. Viruses. 2021;13:1168.
Raymond AD, et al. HIV Type 1 Nef is released from infected cells in
CD45(+) microvesicles and is present in the plasma of HIV-infectedindividuals. AIDS Res Hum Retrovir. 2011;27:167–78.
Tang Y, et al. Brain microglia serve as a persistent HIV reservoir despite
durable antiretroviral therapy. J Clin Invest. 2023;133: e167417.
Banks WA, et al. Transport of extracellular vesicles across the blood–
brain barrier: brain pharmacokinetics and effects of inflammation. Int J
Mol Sci. 2020;21:1–21.
Zhang N, et al. Role of exosomes in brain diseases. Front Cell Neurosci.
2021;15:743353.
Gupta D, Zickler AM, El Andaloussi S. Dosing extracellular vesicles. Adv
Drug Deliv Rev. 2021;178:113961.
Dubrovsky L, et al. Extracellular vesicles carrying HIV-1 Nef induce longterm hyperreactivity of myeloid cells. Cell Rep. 2022;41:111674.

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