Proteomic Analysis of Chronic Binge Alcohol-Induced Hippocampal and Anterior Cingulate Cortex Neuroadaptations in Simian Immunodeficiency Virus (SIV)-Infected Female Rhesus Macaques

Abstract

Human immunodeficiency virus (HIV) infection produces neurological comorbidities including HIV-associated neurocognitive disorder (HAND) and chronic pain. HIV also increases the risk of developing an alcohol use disorder (AUD). With the rising prevalence of AUD in women and people with HIV (PWH), understanding the neurobiological impact of alcohol in these populations is important. We examined proteomic alterations in the hippocampus and anterior cingulate cortex (ACC), brain regions critical for cognition and affective pain, in a female rhesus macaque model of chronic binge alcohol administration and SIV infection. Adult female rhesus macaques received either chronic binge alcohol (CBA, 13–14 g/kg/week of alcohol) or water (VEH) via gastric catheter. All animals were inoculated with simian immunodeficiency virus (SIV_mac251) and treated with antiretroviral therapy (ART). Brain samples were processed for proteomic analysis, and quantitative discovery-based proteomics identified differentially expressed proteins in both brain regions comparing CBA treatment to VEH. Ingenuity Pathway Analysis (IPA) was also used to predict pathway activation. CBA significantly altered 147 proteins in the hippocampus and 176 proteins in the ACC. IPA revealed alterations in 39 canonical pathways in the hippocampus and 62 canonical pathways in the ACC. Fourteen common canonical pathways were enriched in both regions, including synaptogenesis and protein kinase A (PKA) signaling. These discoveries expand our understanding of how alcohol alters proteins of critical signaling pathways in vulnerable brain regions in the context of SIV/HIV infection and may lead to the development of new pharmacological treatment avenues for neurological dysfunction in women with HIV who use alcohol.

Introduction

Human immunodeficiency virus (HIV) infection leads to profound pathophysiological consequences at both peripheral and central nervous system levels, including increased neuroinflammation, impaired neurogenesis, and neurodegeneration (Kaul et al. 2005; Maxi et al. 2016). These neurological changes can manifest behaviorally in the forms of cognitive impairment, termed HIV-associated neurocognitive disorder (HAND), and enhanced pain symptoms, termed HIV-associated neuropathy (HIV-N) (Singer et al. 2010; Ghosh et al. 2012; Jazebi et al. 2021; Simon et al. 2021a). Furthermore, there is a high prevalence of psychiatric comorbidities in people with HIV (PWH), including alcohol use disorder (AUD), generalized anxiety disorder (GAD), major depressive disorder (MDD), and posttraumatic stress disorder (PTSD), also potentially driven by underlying neuroadaptations (Galvan et al. 2002; Neigh et al. 2016; Crane et al. 2017; Camara et al. 2020).

PWH have a greater likelihood of alcohol misuse compared to the general population, and approximately 30% of PWH meet criteria for an AUD, compared to 10% of the general population (Galvan et al. 2002; Park et al. 2016; Koch et al. 2019; Duko et al. 2019; Yen et al. 2022; SAMHSA 2022). Furthermore, AUD in PWH can result in aggravated HIV-related biomedical and psychological consequences and can exacerbate comorbidities including HAND and HIV-N (Molina et al. 2014; Fama et al. 2016). Indeed, alcohol consumption can exacerbate systemic and neuroinflammation and result in immune dysfunction, contributing to the severity of HIV-associated neuropsychiatric comorbidities, including HIV-associated neurocognitive disorder (Pandrea et al. 2010; Monnig 2017).

With antiretroviral therapy (ART) advances extending the longevity of PWH and the rising prevalence of AUD in women (Grant et al. 2017), understanding the impact of HIV and alcohol on the female nervous system is increasingly important.

Sex is a significant factor in the HIV/AIDS epidemic. In 2023, an estimated 20.5 million women were living with HIV globally, accounting for more than half of adult PWH (World Health Organization 2024). Women with HIV endure more mental health challenges than men with HIV, including a higher motivation to consume alcohol in response to negative emotions and pain (Cook et al. 2016). Higher alcohol consumption may exacerbate both cognitive decline and neuropathic pain (Pahng et al. 2017), putting women with comorbid HIV and AUD at a heightened risk for future declines in mental health. Importantly, women are more susceptible to brain damage due to chronic alcohol use, and women are at heightened risk for developing age-related cognitive decline and chronic pain in the context of AUD (Hommer 2003; Mann et al. 2005; Maki et al. 2009; Levine et al. 2021; Fitzpatrick-Schmidt and Edwards 2023). This elevated risk, coupled with the rising prevalence of AUD in women and longer life expectancy in PWH due to advances in ART, necessitates further investigation to better understand the mechanisms of cognitive decline and other brain-related disorders in this vulnerable patient population.

Dysregulation of hippocampal and anterior cingulate cortex (ACC) signaling may play a role in neuroplastic changes seen in both HIV and AUD. The hippocampus is a key component of the limbic system, plays a prominent role in learning and memory, and can undergo atrophy in the context of HAND due to impaired neural progenitor cell proliferation and differentiation (Venkatesan et al. 2007; Lee et al. 2013; Yonelinas 2013). The anterior cingulate cortex (ACC) is a frontal cortical region that integrates bidirectional connections with the hippocampus and other limbic system structures and has prominent roles in cognition, emotional regulation, and the affective processing of pain (Stevens et al. 2011; Pahng et al. 2017). Importantly, the hippocampus and ACC are particularly vulnerable to the effects of alcohol and may play a key role in the development of AUD due to their central role at the intersection of emotion and motivation (Edwards and Koob 2010; Egli et al. 2012; Kutlu and Gould 2016; Pahng et al. 2017; Mira et al. 2019). The two regions also functionally interact to regulate cognitive-affective processing (Wang et al. 2021). One recent study discovered that functional connectivity between the hippocampus and ACC is reduced in individuals suffering from both AUD and HIV infection, and that this deficit is correlated with lifetime alcohol consumption (Honnorat et al. 2022).

Our previous work revealed that alcohol promotes simian immunodeficiency virus (SIV)-induced cognitive deficits in male rhesus macaques (Winsauer et al. 2002). From a neurobiological perspective, we also discovered that chronic binge alcohol (CBA) administration in simian immunodeficiency virus (SIV)-infected male rhesus macaques upregulates hippocampal genes involved in inflammation and immune function and dysregulates genes involved in neurogenesis (Maxi et al. 2016). Additional work indicates that CBA in adolescent male rhesus macaques leads to a long-lasting impairment in hippocampal proliferation and neurogenesis and corresponding neurodegeneration (Taffe et al. 2010). Furthermore, a macaque model of binge alcohol self-administration has revealed a bidirectional relationship with heavy alcohol consumption and deficits in cognitive and behavioral flexibility (Shnitko et al. 2019, 2020). Unfortunately, few studies have examined the impact of both alcohol and HIV on the brain proteome, and only one study has utilized proteomic profiling in HIV + drinkers, although this was done on plasma exosomes (Kodidela et al. 2020).

To better understand the neurobiological impact of comorbid HIV and CBA use in women, the current study sought to investigate proteomic neuroadaptations in the hippocampus and ACC of SIV-infected female rhesus macaques exposed to CBA administration. The overarching goal of this experimental design is to determine how HIV and comorbid AUD risk (in this case, modeled using SIV infection in the context of longitudinal CBA administration) may lead to future cognitive and neuropsychiatric comorbidities by identifying proteins and pathways altered within this context. Our model approximates the definition of “extreme binge” drinking from the National Institute on Alcohol Abuse and Alcoholism (NIAAA), and such blood alcohol levels are commonly achieved in individuals either at-risk for or suffering from AUD. Based on our previous studies, we hypothesized that proteins associated with inflammation, immune signaling, and neurodevelopment would be altered by chronic binge alcohol in the context of SIV. The revelation of neuroadaptations and signaling networks within these key brain areas in highly valid preclinical models of comorbid HIV and AUD is critical for understanding neurological deficits and promoting novel therapeutic avenues to improve quality of life in women with HIV.

Materials and Methods

Animal Study Design

Experiments were approved by the Institutional Animal Care and Use Committee at Louisiana State University Health Sciences Center in New Orleans, LA (IACUC #5128, LSUHSC-NO Comprehensive Alcohol-HIV/AIDS Research Center, Expiration Date: June 20, 2025). The study adhered to the National Institute of Health (NIH) guidelines for the care and use of experimental animals. The experimental design has been described in detail in previous publications in both male (Molina et al. 2014; Maxi et al. 2016) and female (Poret et al. 2021; Simon et al. 2021b) rhesus macaques, and an overview of the timeline is shown in Fig. 1. In brief, adult female rhesus macaques (Macaca mulatta) ages 6–9 years were randomly assigned to a group receiving either chronic binge alcohol (CBA) or isovolumetric water (VEH) administration. Gastric catheter implantation surgeries were performed to administer 13–14 g/kg/week of alcohol (30% ethanol in water, w/v). A single 30-min infusion of 30% w/v alcohol was delivered via intragastric catheter in the mornings 5 days a week. Blood alcohol levels peaked at 50–60 mM (~ 230 mg/dL) at approximately two hours after initiation of alcohol infusion. The alcohol dosing regimen models extreme binge alcohol administration that places humans at risk for developing an AUD and has been historically used in our alcohol center and non-human primate model of alcohol-induced organ pathophysiology and cognitive impairments (Winsauer et al. 2002). CBA-treated animals do not lose consciousness and are intoxicated for no more than 2–3 h. In order to determine the effects of alcohol on SIV disease progression in a controlled setting, the same dose of alcohol and SIV virus were provided to all animals. Following three months of CBA or VEH administration, all animals were inoculated intravaginally with SIV_mac251. After 2.5 months, animals achieved viral set-point and were started on daily subcutaneous injections of antiretroviral therapy (ART) consisting of 30 mg/kg of emtricitabine (FTC) and 20 mg/kg of Tenofovir [9-(2-phosphonomethoxypropyl) adenine, PMPA] (Gilead Sciences Inc., Foster City, CA). In total, two treatment groups were assigned randomly: VEH (n = 6) and CBA (n = 6). Animals received CBA or VEH for a total of 14.5 months, were infected with SIV for 11.5 months, and were on ART for 9 months. At the end of the treatment regimens, all animals were euthanized according to the American Veterinary Medical Associations guidelines. Data for SIV viral loads, T-cell counts, and markers of immune activation for these animals have been published previously (McTernan et al. 2022; Gallegos et al. 2024).

Fig. 1.

A schematic showing the timeline for the nonhuman primate study. Adult female rhesus macaques were randomly assigned to chronic binge alcohol (CBA) or isovolumetric water (VEH) groups. Alcohol was administered at 13–14 g/kg/week (30% ethanol in water, wt/vol; infusions 30 min/day, 5d/wk). peaked at 50–60 mM (230 mg%) at approximately two hours after initiation of alcohol infusion. Following three months of CBA or VEH administration, all animals were inoculated intravaginally with SIV_mac251. After 2.5 months, animals achieved viral set-point and were started on daily subcutaneous injections of antiretroviral therapy consisting of 30 mg/kg of emtricitabine (FTC) and 20 mg/kg of Tenofovir [9-(2-phosphonomethoxypropyl) adenine, PMPA] (Gilead Sciences Inc., Foster City, CA). Finally, all animals were euthanized humanely at the study end-point, brains were snap-frozen, and the anterior cingulate cortex and hippocampus were isolated and prepared for proteomic analysis. Animals received CBA or VEH for a total of 14.5 months, were infected with SIV for 11.5 months, and were on ART for 9 months

Tissue Preparation and Protein Assay

Whole brains were hemisected and snap frozen in −30 °C isopentane at necropsy and the right hemisphere was stored at −80°C until dissection. Due to the nature of this longitudinal study (2015–2021), samples were stored at −80 for a period of years. Cohorts of animals were balanced in terms of treatment groups to mitigate effects related to differential time of tissue storage. Regional brain dissections were taken from 6 mm coronal brain slices and guided by the Paxinos Rhesus Macaque Brain Atlas, Second Edition (Paxinos et al. 2009). Regions collected for this analysis include the anterior cingulate cortex (Brodmann area 24, + 12.0 mm to + 6.0 mm from Bregma) and whole hippocampus (−10 mm to −16 mm from Bregma) from the right hemisphere only.

Protein Extraction and Digestion

To the weighted tissue (20 mg minimum) from regional brain samples, the same volume (in μL) of ABC Buffer (100 mM Ammonium Bicarbonate, pH 8) and 8 volumes of 100% methanol (MetOH) were added (i.e. for 20 mg tissue, add 20 μL buffer and 160 μL MetOH). Samples were then homogenized using Bead Ruptor 96 (OMNI International, USA) and 6 large beads. Suspensions (180 μL) were transferred into a new Eppendorf tube, centrifuged at 14,000 × g for 5 min and the supernatant was discarded. Pellets were washed twice with cold acetone and dried in a SpeedVac concentrator. For digestion, protein pellets were solubilized in 20 μL of Denaturation Buffer (25 mM ammonium bicarbonate, pH8.0; 10 mM TCEP; 5% SDC (sodium deoxycholate)), incubated for 10 min at 60°C, and alkylated using 5 μL Alkylation Buffer (100 mM Iodoacetamide in water, prepared fresh). The incubation continued for an additional 60 min at room temperature in the dark. Following alkylation, samples were diluted with 175 μL of dilution buffer (25 mM ammonium bicarbonate, pH8.0), and 2 μL of Trypsin solution (1 μg/μL) were added. Samples were subsequently incubated overnight at 37°C. To stop the reaction and remove the SDC, 10 μL of 10% trifluoroacetic acid (TFA) was added, and the mixture was incubated for 30 min at room temperature and centrifuged at 15,000 × g (SDC precipitates at low pH). Supernatants were then transferred into the new tubes and directly used for liquid chromatographymass spectrometry (LC–MS) analysis.

Liquid Chromatography-Mass Spectrometry (LC–MS) Analysis

All analyses were carried out using a Bruker nanoElute2 System coupled to a timsTOF fleX 2 mass spectrometer. The mobile phases were composed of Solvent A (0.1% formic acid in 3% ACN) and Solvent B (0.1% formic acid in ACN). The injection volume was 2 μL. Following injection, the peptides were separated on C-18 reversed phase PepSep column (0.150 × 250 mm; 1.5 nm particle; Bruker) coupled with the CaptiveSpray ionization source of the mass spectrometer. The flow rate and temperature of the column chamber were set to 800 nL/min and 50 °C. Separation of peptides was achieved at the following gradient: T = 0 min: 0% B; T = 40 min: 26% B; T = 40.5 min: 95% B; T = 41.5 min: 95% B; T = 42 min: 0% B; T = 45 min: 0% B (column re-equilibration). Mass spectrometry data was collected in positive, Data Dependent Acquisition (DDA) – PASEF mode under the following conditions: a capillary voltage of 1,500 V; source temperature of 180 °C; dry gas flow at 3 L/min; and an acquisition range of 100 – 1,700 m/z. The time settings were as follows: 1/K0 Start: 0.60 Vs/cm²; 1/K0 End: 1.60 Vs/cm²; Ramp Time: 100 ms; Accumulation Time: 100 ms; Duty Cycle: 100%; and Ramp Rate: 9.42 Hz.

Proteomic Data Analysis

Proteomic data were initially processed using MaxQuant, leveraging the UniProt Macaca mulatta database to identify proteins with the label-free quantification (LFQ) method. LFQ intensities were processed and prepared using an in-house R script. Proteins detected in at least five out of six replicates of samples in any given group were classified as detected proteins for that group. The median gap-filling method addressed potential gaps in protein intensity measurements. Statistical analyses were conducted using moderated Student’s t-test with Empirical Bayes statistics of the R limma package (limma_3.58.1) to identify differentially expressed proteins, with the significance level alpha set at 0.05 and absolute log2-transformed fold change (|log2FC|) > 0.322 (corresponding to a linear FC of 1.25) (Smyth et al. 2005). Using the R stat package (stat_4.4.1), principal component analysis (PCA) was performed on centered protein expression levels to visualize the variation among groups for each region separately. For further analysis, Ingenuity Pathway Analysis (IPA) and GraphPad Prism 9 for macOS (San Diego, CA, USA) were employed to elucidate pathway interactions and perform statistical analyses.

Results

Proteomic analysis of the hippocampus and anterior cingulate cortex (ACC) detected a total of 3,756 proteins in the hippocampus and 3,805 proteins in the ACC (Fig. 2). Of the proteins detected in the hippocampus, 3,534 (94.1%) were detected in both the CBA and VEH groups, while 127 proteins (3.4%) were detected only in the CBA group and 95 proteins (2.5%) were detected only in the VEH group (Fig. 2a). In the ACC, 3,594 proteins (94.5%) were detected in both groups, while 92 proteins (2.4%) were detected only in the CBA group and 119 proteins (3.1%) were detected only in the VEH group (Fig. 2b). Pairwise analysis revealed CBA significantly altered levels of 147 proteins in the hippocampus (Fig. 3a) and 176 proteins in the ACC (Fig. 3b). In the hippocampus, 79 proteins were upregulated, and 68 proteins were downregulated, while in the ACC 96 proteins were upregulated and 80 proteins were downregulated.

Fig. 2.

Venn diagram depicting proteins detected in the CBA, VEH, or both groups in the hippocampus (a) and anterior cingulate cortex (ACC) (b). a. 3,756 proteins were detected in the hippocampus, with 3,534 of these proteins identified in both groups. b. 3,805 proteins were detected in the ACC, with 3,594 of these proteins identified in both groups

Fig. 3.

Volcano plot depicting proteins significantly altered by chronic binge alcohol (CBA) in the hippocampus and anterior cingulate cortex (ACC). a. In the hippocampus, CBA resulted in upregulation of 79 proteins and downregulation of 68 proteins. b. In the ACC, CBA resulted in upregulation of 96 proteins and downregulation of 80 proteins

Of the significantly regulated proteins, 14 were detected in both the ACC and hippocampus (Fig. 4). Five proteins were upregulated in both brain regions, including adhesion G protein-coupled receptor B2 (ADGRB2), neuron-specific calcium-binding protein hippocalcin (HPCA), synemin (SYNM), thiol methyltransferase 1A (TMT1A), and transthyretin (TTR). Seven proteins were downregulated in both regions, including aldehyde dehydrogenase 3 family member B1 (ALDH3B1), Rac/Cdc42 guanine nucleotide exchange factor 6 (ARHGEF6), carboxylic ester hydrolase (CES1), citramalyl Co-A lyase (CLYBL), 5-oxoprolinase (ATP-hydrolysing; OPLAH), synapsin III (SYN3), and zinc finger CCCH domain-containing protein 15 (ZC3H15). The remaining two proteins were differentially regulated with both proteins upregulated in the hippocampus and downregulated in the ACC (alanyl-tRNA synthetase domain containing 1 (AARSD1) and glycogen debranching enzyme (AGL)). Table 1 shows these 14 proteins and their log2-fold change (log2FC) for each brain region.

Fig. 4.

Venn diagram (A) and heat map (B) showing proteins altered by CBA in the hippocampus and anterior cingulate cortex (ACC). 14 proteins were altered by CBA in both the hippocampus and ACC

Table 1.

Chronic binge alcohol significantly altered 14 proteins in both the hippocampus and anterior cingulate cortex of SIV-infected female macaques

			Hippocampus			Anterior cingulate cortex
UniProt	Gene name	Description	log2FC	P-Value	Expression	log2FC	P-Value	Expression
F6PQH0	AARSD1	Alanyl-tRNA synthetase domain containing 1	0.54063771	0.03173684	Up-regulated	−0.386603	0.0172036	Down-regulated
A0A5F8A4G7	ADGRB2	Adhesion G protein-coupled receptor B2	0.56426081	0.01017682	Up-regulated	0.45588502	0.01384375	Up-regulated
F7EAR8	AGL	Glycogen debranching enzyme	0.33182155	0.0366304	Up-regulated	−0.4206269	0.01371138	Down-regulated
A0A1D5QTX0	ALDH3B1	Aldehyde dehydrogenase 3 Family Member B1	−0.4680561	0.02047928	Down-regulated	−0.575973	0.0263026	Down-regulated
F6Q0Q9	ARHGEF6	Rac/Cdc42 guanine nucleotide exchange factor 6	−0.4172266	0.00474548	Down-regulated	−0.4440599	0.01484991	Down-regulated
F7AI42	CES1	Carboxylic ester hydrolase	−0.4499579	0.01313481	Down-regulated	−0.5073875	0.0248774	Down-regulated
F7ETT5	CLYBL	Citramalyl-CoA lyase	−1.0751604	0.00519463	Down-regulated	−14.486856	1.82E-11	Down-regulated
F7HSC9	HPCA	Neuron-specific calcium-binding protein hippocalcin	0.64673274	0.00967482	Up-regulated	0.47923652	0.00684953	Up-regulated
A0A5F8ARZ5	OPLAH	5-oxoprolinase, ATP-hydrolysing	−0.4194829	0.04640646	Down-regulated	−0.4572641	0.00826011	Down-regulated
F7HFD1	SYN3	Synapsin III	−0.4407369	0.01491037	Down-regulated	−0.4689005	0.00429813	Down-regulated
F7C3M5	SYNM	Synemin	0.69988633	1.78E-05	Up-regulated	0.60734439	0.0199949	Up-regulated
A0A5F8AU93	TMT1A	Thiol methyltrans-ferase 1A	0.64149998	0.00479771	Up-regulated	0.46004815	0.02060176	Up-regulated
A0A1D5QXZ0	TTR	Transthyretin	1.25076596	0.04387351	Up-regulated	1.19095385	0.01180823	Up-regulated
F7H9Q2	ZC3H15	Zinc finger CCCH domain-containing protein 15	−0.5009104	0.01454721	Down-regulated	−14.202414	1.57E-21	Down-regulated

Several proteins were detected in only one group in both brain regions. In the hippocampus, 20 proteins were detected only in VEH animals, while 17 proteins were detected only in CBA animals. Table 2 shows these hippocampal proteins and their respective p-values for each group. In the ACC, 16 proteins were detected only in the VEH animals and 11 proteins were detected only in the CBA animals. Table 3 shows these ACC proteins and their respective p-values for each group.

Table 2.

In the hippocampus, 20 proteins were detected only in the vehicle animals, while 17 proteins were only detected in the chronic binge alcohol animals

UniProt	Gene name		Description	P-value
Only detected in VEH
F6QST9	CACNG2	Voltage-dependent calcium channel gamma-2 subunit		1.28E-17
F6R9U7	SLC38A3	Sodium-coupled neutral amino acid transporter 3		1.34E-20
F7DKJ0	CLVS1	Clavesin-1		1.56E-25
G7NFS5	SLC27A4	long-chain-fatty-acid–CoA ligase		9.16E-19
A0A5F8AVF2	MFSD6	Major facilitator superfamily domain containing 6		1.44E-22
F6ZS77	CHD4	DNA helicase		4.87E-18
F7EQ66	PDS5B	PDS5 cohesin associated factor B		4.88E-25
G7NCH2	PPP2R5B	Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit		3.16E-20
F6S0I2	XKR4	XK-related protein		2.65E-19
F6WRA1	SPECC1L	Cytospin-A		3.04E-23
F7CB80	SESTD1	SEC14 domain and spectrin repeat-containing protein 1		1.26E-24
F6V197	KCNJ10	ATP-sensitive inward rectifier potassium channel 10		7.11E-16
Q5TM62	MRPS18B	Small ribosomal subunit protein mS40		5.57E-19
F6S7U8	LTN1	E3 ubiquitin-protein ligase listerin		8.25E-23
F6PQ65	HSPB6	Heat shock protein beta-6		2.97E-17
F7G2I9	ERMP1	Endoplasmic reticulum metallopeptidase 1		4.03E-19
H9H3P6	CFH	Complement factor H		2.40E-17
F7GDT2	SNX30	Sorting nexin-30		3.37E-13
F7GYQ8	RPAP1	RNA polymerase II associated protein 1		6.54E-19
F7AG75	RGS6	Regulator of G protein signaling 6		2.37E-14
Only detected in CBA
F7B8J7	NSMF	NMDA receptor synaptonuclear signaling and neuronal migration factor		1.10E-22
A0A5F8AHI7	GSTA2	Glutathione S-transferase		4.67E-15
G7MYN0	MESD	LRP chaperone MESD		1.12E-24
A0A1D5QYD1	STRADA	STE20-related kinase adapter protein alpha		3.16E-16
A0A1D5QYW7	FXN	Frataxin, mitochondrial		2.38E-22
I0FM31	FGFR1OP2	FGFR1 onco partner 2		6.37E-23
H9Z9T3	RWDD1	RWD domain containing 1		1.32E-23
F7DUH0	CPSF7	Cleavage and polyadenylation specific factor 7		1.01E-21
F7HNU4	SHROOM2	Shroom family member 2		4.21E-21
I2CTJ0	RHOT2	Mitochondrial Rho GTPase		6.18E-23
F7GJI8	DPY30	Protein dpy-30 homolog		2.84E-22
F6QI72	KCNIP2	Kv channel-interacting protein 2 isoform 2q		1.81E-21
F6ZWH1	PTDSS1	Phosphatidylserine synthase		9.96E-24
A0A5F7ZTS4	RGS10	Regulator of G protein signaling 10		3.08E-23
F7F4F6	UFL1	E3 UFM1-protein ligase 1		2.14E-20
A0A5F8A8L3	F13A1	Coagulation factor XIII A chain		5.13E-21
F7A6I9	ZNF638	Zinc finger protein 638		1.68E-24

Table 3.

In the anterior cingulate cortex, 16 proteins were detected only in the vehicle animals, while 12 proteins were only detected in the chronic binge alcohol animals

UniProt	Gene name	Description	P-value
Onlv detected in VEH
A0A5F7ZB67	PKLR	Pyruvate kinase	1.05E-11
G7NGN4	LOC706710	Large ribosomal subunit protein mL62	6.10E-21
F7GFS1	GGACT	Gamma-glutamylaminecyclotransferase	8.53E-23
A0A5F8A0J9	COQ3	Ubiquinone biosynthesis O-methyltransferase, mitochondrial	1.01E-14
F7AHG4	CCDC47	PAT complex subunit CCDC47	2.04E-22
A0A5F7ZPN9	SNRPE	Small nuclear ribonucleoprotein E	6.42E-19
A0A1D5Q9D5	SKIC2	SKI2 subunit of superkiller complex	1.47E-21
F7HS52	GAB1	GRB2 associated binding protein 1	8.99E-19
F7AVV4	RNF141	RING finger protein 141	5.10E-16
F7G9W1	NRP2	Neurolipin	2.91E-13
F6T885	ADHFE1	Hydroxyacid-oxoacid transhydrogenase, mitochondrial	9.59E-13
F6YW03	STARD10	PCTP-like protein	8.87E-13
F7BX36	VARS2	Valine-tRNA ligase, mitochondrial	2.08E-21
F6QI47	POLR2B	DNA-directed RNA polymerase subunit beta	1.31E-21
F7ETT5	CLYBL	Citramalyl-CoA lyase	1.82E-11
F7H9Q2	ZC3H15	Zinc finger CCCH domain-containing protein 15	1.57E-21
Only detected in CBA
F6Q743	CNN2	Calponin	1.69E-18
A0A1D5QQF1	FTL	Ferritin light chain	3.81E-18
A0A5F8A311	BZW1	Basic leucine zipper and W2 domains 1	4.34E-23
I0FLZ7	NHP2	H/ACA ribonucleoprotein complex subunit 2	2.36E-20
F7B3S2	MRPL14	Large ribosomal subunit protein uL14m	1.22E-18
F6UV16	TEX2	Testis expressed 2	1.70E-23
F7GMS1	MTMR7	Phosphatidylinositol-3-phosphatase	2.54E-17
F7BAI3	RBKS	Ribokinase	3.41E-18
Q5PXZ9	TIMP3	Metalloproteinase inhibitor 3	5.27E-12
H9EQ69	RMND5A	RING-type E3 ubiquitin transferase	2.19E-18
A0A1D5QYR9	SNAP23	Synaptosomal-associated protein	2.67E-16
F7GUL5	MMAA	Metabolism of cobalamin associated A	8.66E-14

To better understand how CBA alters the hippocampal and ACC proteomes, we utilized principal component analysis (PCA) to compare the proteomic profiles from the two treatment groups in each brain region. PCA analysis revealed that the first two principal components (PCs) account for about 90% of the variability in our data in each region (Fig. 5, Supplementary Fig. 1). In the hippocampus, PC1 accounted for 89.0% of the variability, while PC2 accounted for 3.04%. In the ACC, PC1 accounted for 82.9%, while PC2 accounted for 6.5%. The percentage of variances explained by individual components for Hippocampus and ACC regions are provided in the supplementary bar plot Supplementary Fig. 1. Supplementary Table 1 and 2 contain the loading values for the top 50 proteins in the hippocampus and the ACC, respectively.

Fig. 5.

Principal component analysis (PCA) score plot of proteomics data for the hippocampus (A) and anterior cingulate cortex (B)

Ingenuity pathway analyses (IPA) was performed to identify canonical pathways altered by CBA in the hippocampus and ACC of SIV-infected female macaques. Canonical pathways are defined as well-characterized, essential metabolic and cell signaling pathways that are generally conserved across species. In the hippocampus, 39 canonical pathways were significantly enriched by CBA, including SNARE signaling, glutaminergic receptor signaling, and opioid signaling (Table 4). In the ACC, 62 canonical pathways were significantly enriched by CBA, including actin cytoskeleton signaling, extracellular matrix organization, and post-translational protein phosphorylation (Table 5).

Table 4.

Ingenuity pathway analysis revealed 39 hippocampal pathways were significantly enriched by chronic binge alcohol administration (p < 0.05)

Ingenuity canonical pathway	Molecules	Z-score	P-value
Acetylcholine Receptor Signaling Pathway	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GUCY1A1	−2.449	0.001
Adrenergic Receptor Signaling Pathway (Enhanced)	ADCY5, CACNA2D2, CACNG2, GUCY1A1	−2	0.028
Autism Signaling Pathway	ALDH3B1, CACNA2D2, CACNG2, CAMK2D, CAMK2G	−0.447	0.032
Calcium Signaling	CACNA2D2, CACNG2, CAMK2D, CAMK2G, MYL1	−2	0.009
cAMP-mediated signaling	ADCY5, AKAP12, CAMK2D, CAMK2G, GUCY1A1, RGS10	−2	0.002
Cardiac conduction	CACNA2D2, CAMK2D, CAMK2G, KCNIP2, SCN3B	0	0.001
Corticotropin Releasing Hormone Signaling	ADCY5, CACNA2D2, CACNG2, GUCY1A1	0	0.011
CREB Signaling in Neurons	ADCY5, ADGRB2, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GRIK5, GUCY1A1	−1.134	0.024
Dilated Cardiomyopathy Signaling Pathway	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GUCY1A1, MYL1	−2	0.000
GABAergic Receptor Signaling Pathway (Enhanced)	ADCY5, CACNA2D2, CACNG2, GUCY1A1, SLC38A3	1.342	0.001
Glutaminergic Receptor Signaling Pathway (Enhanced)	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GRIK5, GUCY1A1, SCN3B, SLC38A3	−1.667	0.000
Gustation Pathway	ADCY5, CACNA2D2, CACNG2, GUCY1A1, SCN3B	−1	0.006
ILK Signaling	ARHGEF6, MYL1, PPP2R5B, RHOT2	2	0.029
Mitochondrial Dysfunction	CACNA2D2, CACNG2, CAMK2D, CAMK2G, RHOT2	−0.447	0.048
Necroptosis Signaling Pathway	CAMK2D, CAMK2G, CYLD, PYGL	−2	0.012
Netrin Signaling	CACNA2D2, CACNG2, CAMK2D, CAMK2G, DCC, ENAH	−0.816	0.000
Neurovascular Coupling Signaling Pathway	CACNA2D2, CACNG2, GUCY1A1, KCNJ10	0	0.045
Neutrophil degranulation	AGL, ALDH3B1, ANXA2, ATP8A1, PYGL, TMEM30A, TMT1A, TTR	0	0.006
Opioid Signaling Pathway	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GUCY1A1, RGS10, RGS6	−1.134	0.000
Orexin Signaling Pathway	ADCY5, CACNA2D2, CACNG2, GUCY1A1	−2	0.048
Parkinson’s Signaling Pathway	ALDH3B1, CACNA2D2, CACNG2, CAMK2D, CAMK2G	−2.236	0.032
Protein Kinase A Signaling	ADCY5, AKAP12, CAMK2D, CAMK2G, DCC, GUCY1A1, MYL1, PYGL	−0.447	0.003
RAF/MAP kinase cascade	CAMK2D, CAMK2G, KIT, KSR1, PPP2R5B	−0.447	0.018
RAR Activation	ADCY5, CAMK2D, CAMK2G, GUCY1A1, KIT, PRMT2, RHOT2	−1.134	0.012
Response to elevated platelet cytosolic Ca2 +	ENDOD1, F13A1, HABP4, SCG3	1	0.007
RHO GTPase cycle	ABL2, AKAP12, ARHGEF6, BRK1, CIT, CPSF7, ELMO2	1.134	0.015
Role of NFAT in Cardiac Hypertrophy	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, GUCY1A1	−2.236	0.002
Serotonin Receptor Signaling	ADCY5, CACNA2D2, CACNG2, CAMK2D, CAMK2G, F13A1, GUCY1A1, RHOT2	−1.414	0.006
Signaling by Rho Family GTPases	ARHGEF6, CIT, MYL1, RHOT2, SEPTIN6	0.447	0.019
Signaling by ROBO receptors	ABL2, DCC, ENAH, GPC1, NRP1	2.236	0.001
Signaling by VEGF	BRK1, ELMO1, ELMO2, NRP1	1	0.003
Sleep NREM Signaling Pathway	ADCY5, CAMK2D, CAMK2G, GUCY1A1	0	0.004
SNARE Signaling Pathway	ADCY5, CACNA2D2, CAMK2D, CAMK2G, GUCY1A1, MYL1, SYN3	−1.134	0.000
Synaptic Long Term Depression	CACNA2D2, CACNG2, GUCY1A1, PPP2R5B	−1	0.028
Synaptogenesis Signaling Pathway	ADCY5, CACNA2D2, CAMK2D, CAMK2G, GUCY1A1, SYN3	−2	0.010
Thrombin Signaling	ADCY5, ARHGEF6, CAMK2D, CAMK2G, GUCY1A1, MYL1, RHOT2	0.447	0.000
White Adipose Tissue Browning Pathway	ADCY5, CACNA2D2, CACNG2, GUCY1A1	−2	0.008
WNT/SHH Axonal Guidance Signaling Pathway	ADCY5, CAMK2D, CAMK2G, GPC1, GUCY1A1	0.447	0.002
Xenobiotic Metabolism PXR Signaling Pathway	ALDH3B1, CAMK2D, CAMK2G, GSTA3	1	0.026

Table 5.

Ingenuity pathway analysis revealed 62 anterior cingulate cortex pathways were significantly enriched by chronic binge alcohol administration (p < 0.05)

Ingenuity canonical pathway	Molecules	Z-score	P-value
ABRA Signaling Pathway	ACTA2, ACTB, ACTC1, CALD1, GSN, MYH11, MYL9, TAGLN, TPM1, TPM2, VCL	2.111	0.000
Actin Cytoskeleton Signaling	ACTA2, ACTB, ACTC1, ACTN4, APC, ARHGEF6, FGF12, FLNA, FN1, GSN, MYH11, MYL12B, MYL9, RRAS, TLN1, VCL	2.138	0.000
Acute Phase Response Signaling	ALB, APOA1, APOA2, C1QB, CRABP1, FN1, FTL, RRAS, SERPINA3, TTR, VWF	0.816	0.000
Agrin Interactions at Neuromuscular Junction	ACTA2, ACTB, ACTC1, ARHGEF6, PKLR, RRAS	1.342	0.000
Amyloid fiber formation	APOA1, GSN, MFGE8, TTR	2	0.002
Atherosclerosis Signaling	ALB, APOA1, APOA2, APOD, COL18A1	2.236	0.002
Autism Signaling Pathway	ALDH18A1, ALDH3B1, ALDH4A1, APC, FMR1, RRAS	−0.816	0.019
Binding and Uptake of Ligands by Scavenger Receptors	ALB, APOA1, COL4A1, COL4A2, FTL, HBB	2.449	0.000
Caveolar-mediated Endocytosis Signaling	ACTA2, ACTB, ACTC1, ALB, CAVIN1, FLNA	2	0.000
Cell junction organization	ACTB, ARHGEF6, FLNA, NECTIN3, RSU1	1.342	0.000
Deubiquitination	ACTB, APC, PSME1, PSME2, RNF123, WDR20	0	0.009
DHCR24 Signaling Pathway	ALB, APOA1, APOA2, APOD, RRAS, TTR	2.449	0.000
Dilated Cardiomyopathy Signaling Pathway	ACTA2, ACTB, ACTC1, CAMK1, CAMK4, CNN1, DES, GAB1, MYH11, MYL9, TPM1	−1.667	0.000
EPH-Ephrin signaling	ACTB, MYH11, MYL12B, MYL9	2	0.004
Epithelial Adherens Junction Signaling	MYL12B, NECTIN3, RRAS, VCL	1	0.022
Extracellular matrix organization	BGN, COL18A1, COL4A1, COL4A2, FN1, LUM, TTR	2.646	0.000
Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes	ACTA2, ACTB, ACTC1, TLN1	2	0.004
Gap Junction Signaling	ACTA2, ACTB, ACTC1, CSNK1D, PLCD3, RRAS	0.816	0.023
Glutaminergic Receptor Signaling Pathway (Enhanced)	CAMK4, DGKE, FMR1, PLCD3, SCN4B, VAMP2	−0.816	0.023
Glycerophospholipid biosynthesis	CHKB, GPCPD1, SLC44A1, STARD10	−2	0.005
GP6 Signaling Pathway	COL18A1, COL4A1, COL4A2, TLN1	2	0.011
Hepatic Fibrosis Signaling Pathway	ACTA2, APC, CSNK1D, FTL, MYL12B, MYL9, RRAS	2.236	0.023
ILK Signaling	ACTA2, ACTB, ACTC1, ACTN4, ARHGEF6, FLNA, FN1, MYH11, MYL9, RSU1, TGFB1I1, VCL	1.155	0.000
Integrin cell surface interactions	COL18A1, COL4A1, COL4A2, FBN1, FN1, LUM, VWF	2.646	0.000
Integrin Signaling	ACTA2, ACTB, ACTC1, ACTN4, GSN, MYL12B, MYL9, RRAS, TLN1, VCL	3	0.000
L1CAM interactions	ACTB, ANK3, LYPLA2, NRP2, SCN4B, SPTBN4	−1.633	0.000
Leukocyte Extravasation Signaling	ACTA2, ACTB, ACTC1, ACTN4, TIMP3, VCL	1.342	0.002
LXR/RXR Activation	ALB, APOA1, APOA2, APOD, TTR	2.236	0.002
Neutrophil degranulation	AGL, ALDH3B1, CNN2, FTL, GSN, HBB, S100A11, SER-PINA3, SNAP23, TMT1A, TTR, VCL	2.309	0.000
Nuclear Cytoskeleton Signaling Pathway	ACTA2, ACTB, ACTC1, DES, FN1	2.236	0.017
PAK Signaling	ARHGEF6, MYL12B, MYL9, RRAS	1	0.008
Parkinson’s Signaling Pathway	ACTA2, ACTB, ACTC1, ALDH18A1, ALDH3B1, ALDH4A1	−2.449	0.018
Paxillin Signaling	ACTA2, ACTB, ACTC1, ACTN4, ARHGEF6, RRAS, TLN1, VCL	2.121	0.000
Post-translational protein phosphorylation	ALB, APOA1, APOA2, FBN1, FN1, MFGE8, VWA1	2.646	0.000
Production of Nitric Oxide and Reactive Oxygen Species in Macrophages	ALB, APOA1, APOA2, APOD	2	0.041
Protein Kinase A Signaling	FLNA, MTMR7, MYL12B, MYL9, PDE4B, PLCD3, PYGM	−1	0.022
Pulmonary Fibrosis Idiopathic Signaling Pathway	ACTA2, ACTB, ACTC1, COL18A1, COL4A1, COL4A2, FN1, RRAS	2.449	0.002
RAF/MAP kinase cascade	ACTB, FN1, NEFL, PSME1, PSME2, SPTBN4, TLN1, VCL, VWF	1.667	0.000
Regulation of Actin-based Motility by Rho	ACTA2, ACTB, ACTC1, GSN, MYL12B, MYL9	2.236	0.000
Regulation of Insulin-like Growth Factor (IGF) transport and uptake by IGFBPs	ALB, APOA1, APOA2, FBN1, FN1, MFGE8, VWA1	2.646	0.000
Response to elevated platelet cytosolic Ca2 +	ACTN4, ALB, APOA1, FLNA, FN1, SERPINA3, TIMP3, TLN1, VCL, VWF	3.162	0.000
RHO GTPase cycle	ACTB, ACTC1, ARHGAP39, ARHGEF6, CAVIN1, CDC42SE2, CHN1, SNAP23, TEX2, WIPF3	0.632	0.001
RHO GTPases activate PAKs	FLNA, MYH11, MYL12B, MYL9	2	0.000
RHOA Signaling	ACTA2, ACTB, ACTC1, MYL12B, MYL9, NRP2	2.236	0.000
RHOGDI Signaling	ACTA2, ACTB, ACTC1, ARHGEF6, MYH11, MYL12B, MYL9	−1.633	0.001
RNA Polymerase II Transcription	POLR2B, RNMT, SNRPE, SRSF6	−2	0.019
Role of Osteoclasts in Rheumatoid Arthritis Signaling Pathway	CAMK4, COL18A1, COL4A1, COL4A2, GSN, RRAS	1.633	0.019
Semaphorin interactions	MYH11, MYL12B, MYL9, RRAS, TLN1	2.236	0.000
Semaphorin Neuronal Repulsive Signaling Pathway	MYL12B, MYL9, NRP2, PDE4B, RRAS	−0.447	0.004
Sertoli Cell-Germ Cell Junction Signaling Pathway (Enhanced)	ACTA2, ACTB, ACTC1, NECTIN3, RRAS	−1.342	0.022
Sertoli Cell-Sertoli Cell Junction Signaling	ACTA2, ACTB, ACTC1, ACTN4, NECTIN3, RRAS, VCL	2.646	0.001
Signaling by Rho Family GTPases	ACTA2, ACTB, ACTC1, ARHGEF6, DES, MYL12B, MYL9	1.89	0.002
Smooth Muscle Contraction	ACTA2, CALD1, MYH11, MYL12B, MYL9, TLN1, TPM1, TPM2, VCL	3	0.000
SNARE Signaling Pathway	MYH11, MYL9, SNAP23, SYN3, VAMP2	2.236	0.002
Striated Muscle Contraction	ACTC1, DES, TPM1, TPM2	2	0.000
Synaptogenesis Signaling Pathway	CHN1, NECTIN3, RRAS, SYN3, TLN1, VAMP2	0.816	0.020
Thrombin Signaling	ARHGEF6, CAMK1, CAMK4, MYL12B, MYL9, PLCD3, RRAS	−0.447	0.001
VEGF Signaling	ACTA2, ACTB, ACTC1, ACTN4, RRAS, VCL	1.633	0.000
Virus Entry via Endocytic Pathways	ACTA2, ACTB, ACTC1, FLNA, RRAS	2.236	0.001
Wound Healing Signaling Pathway	ACTA2, COL18A1, COL4A1, COL4A2, FN1, RRAS	2.449	0.007
Xenobiotic Metabolism AHR Signaling Pathway	ALDH18A1, ALDH3B1, ALDH4A1, NQO2	−2	0.003
Xenobiotic Metabolism CAR Signaling Pathway	ALDH18A1, ALDH3B1, ALDH4A1, SOD3	−1	0.043

IPA analysis provides z-score values representing potential pathway activation (positive z-score) or potential pathway inhibition (negative z-score). In the hippocampus, CBA inhibited 29 pathways and activated 10 pathways (Fig. 6). The hippocampal pathways with the lowest z-scores (i.e., greatest inhibition) included acetylcholine receptor signaling, Parkinson’s signaling, and synaptogenesis signaling. Among the most activated hippocampal pathways (highest z-scores) were signaling by ROBO receptors and ILK signaling. In the ACC, CBA activated 49 canonical pathways and inhibited 13 pathways (Fig. 7). The most activated pathways in the ACC included response to elevated platelet cytosolic Ca²⁺, integrin signaling, smooth muscle contraction, post-translational protein phosphorylation, and extracellular matrix organization, while the most inhibited pathways included Parkinson’s signaling, xenobiotic metabolism AHR signaling, glycerophospholipid biosynthesis, and RNA polymerase II transcription.

Fig. 6.

Chronic binge alcohol significantly altered 39 canonical pathways in the hippocampus

Fig. 7.

Chronic binge alcohol significantly altered 62 canonical pathways in the anterior cingulate cortex

Several pathways were altered by CBA in both the hippocampus and ACC (Fig. 8). Five canonical pathways were inhibited in both brain regions, including Parkinson’s signaling, dilated cardiomyopathy signaling, protein kinase A signaling, autism signaling, and glutaminergic receptor signaling pathways. Four canonical pathways were activated in both regions, including RHO GTPase cycle, ILK signaling, signaling by RHO Family GTPases, and response to elevated platelet cytosolic Ca²⁺. Interestingly, five pathways were differentially altered in the two brain regions, with one pathway (thrombin signaling) activated in the hippocampus and inhibited in the ACC and three pathways (synaptogenesis signaling, RAF/MAP kinase cascade, and SNARE signaling) inhibited in the hippocampus and activated in the ACC. One pathway (neutrophil degranulation) was activated in the ACC but had a z-score of 0 in the hippocampus, indicating that, although individual proteins in the pathway were significantly altered, overall pathway activity was not altered.

Fig. 8.

Chronic binge alcohol significantly enriched 14 canonical pathways in both the hippocampus and anterior cingulate cortex

Discussion

Extending our previous work describing the pathophysiological effects of alcohol in a preclinical rhesus macaque animal model of chronic SIV infection and ART treatment (Molina et al. 2015), the current study aimed to examine the impact of longitudinal chronic binge alcohol (CBA) exposure on the hippocampal and ACC proteome in female macaques. Our quantitative proteomic approach discovered that CBA results in differential protein expression profiles in the hippocampus and ACC of female macaques. Our analyses also identified numerous proteins and several pathways significantly enriched by CBA across the two brain regions. This study builds on previous work from our group, which investigated CBA-induced alterations in the hippocampal and frontal cortex transcriptome in SIV-infected male macaques. Interestingly, and in contrast to this previous study and our initial hypothesis, we did not observe CBA-induced enrichment of proteins involved in inflammatory and immune signaling in female macaques. Other recent CNS proteomic analyses have focused on the impact of HIV or alcohol on the brain proteome (Doke et al. 2021; Teng et al. 2023; Enculescu et al. 2019; Grantham and Farris 2019); but to our knowledge, this is the first study to examine the dual neuroproteomic impact of chronic binge alcohol in the context of SIV infection. Below, we summarize our overall findings for each region, highlight the most significantly differentially expressed proteins, and also discuss select systems related to HIV, AUD, and cognition that could serve as viable targets for pharmacotherapy and future mechanistic investigation.

Effects of CBA in the Hippocampus

In the hippocampus, 147 proteins were dysregulated by CBA, with about half of these proteins up-regulated and half down-regulated (Fig. 3A). Of hippocampal proteins expressed in both CBA and VEH animals, the largest expression increase observed was transthyretin (Table 6). Transthyretin (TTR) is a carrier protein responsible for transportation of thyroxine and retinol (Sousa et al. 2007; Ueda 2022). Importantly, TTR can also bind to amyloid beta peptide, a peptide whose accumulation is a key pathological feature of Alzheimer’s disease (AD; Sousa et al. 2007). Amyloid beta has also been shown to accumulate in the frontal cortex of HIV-positive individuals and act synergistically with Tat, one of the HIV viral proteins, to synergistically increase neurotoxicity (Achim et al. 2009; Hategan et al. 2017). TTR levels have been shown to be altered both in patients with AD and in rodent models of AD (Riisøen 1988; Serot et al. 1997; Li et al. 2011). One clinical study found that participants with higher blood TTR levels were at heightened risk of mild cognitive impairment (Nakamura et al. 2023). Another study showed serum TTR concentrations are elevated in association with higher daily alcohol consumption (Jono et al. 2016); however, to our knowledge, no studies have investigated TTR in the context of CBA exposure or AUD. Future studies should explore neuronal TTR as a potential mechanism of alcohol-related cognitive decline. In addition, neuronal pentraxin-2 (NPTX2) was also upregulated in the hippocampus of CBA animals. NPTX2 is a protein involved in excitatory synapse formation involved in clustering AMPA glutamate receptors at synapses (Chapman et al. 2019). Previous studies have shown that NPTX2 may contribute to brain atrophy and memory decline in AD (Swanson et al. 2016). Furthermore, there is evidence that NPTX2 is also upregulated in the context of Parkinson’s disease and epilepsy (Chapman et al. 2019). Previous work using an alcohol self-administration model in cynomolgus macaques found chronic ethanol altered ionotropic glutamate receptor complexes in the prefrontal cortex, and a proteomic study performed in post-mortem human brain tissue revealed changes in the glutamate transporter GLAST/EAAT1 (Acosta et al. 2010; Kashem et al. 2021). Thus, NPTX2 and glutamatergic signaling may also play a role in SIV- and CBA-induced neurological deficits.

Table 6.

Literature review of top differentially expressed proteins in the"hippocampus

UniProt ID	Gene name	Description	Relevant literature
Top Upregulated Proteins (Expressed in Both Groups)
A0A1D5QXZ0	TTR	Transthyretin	Preclinical Mice who consumed alcohol for 30 days and were treated with naltrexone had increased transthyretin in the prefrontal cortex (Yu et al. 2011) Clinical Daily alcohol consumption is associated with increased serum transthyretin (Jono et al. 2016) People with higher levels of transthyretin are at increased risk of mild cognitive impairment (Nakamura et al. 2023) Women with HIV had lower serum transthyretin than seronegative women (Kiefer et al. 2018)
F7FQM7	APOA1	Apolipoprotein A1	Clinical People with HIV have increased anti-apolipoprotein A1 auto-antibodies (Frias et al. 2024)
A0A5F8AMA1	CA1	Carbonic anhydrase 1	Preclinical Alcohol inhibits erythrocyte carbonic anhydrase in rats (Coban et al. 2008) Review that discusses potential of carbonic anhydrases as targets to protect against neurovascular dysfunction in Alzheimer’s disease (Lemon et al. 2021)
Top Downregulated Proteins (Expressed in Both Groups)
A0A5F8A6B8	MPZ	Myelin protein P0	Preclinical Chronic alcohol exposure in mice inhibits myelinogenesis in the prefrontal cortex and corpus callosum (Guo et al. 2021) Clinical Two patients with HIV encephalopathy had increased cerebrospinal fluid myelin basic protein, a marker for central nervous system demyelination (Pfister et al. 1989) MPZ mutations are associated with peripheral neuropathies, including Charcot-Marie-Tooth disease (Bienfait et al. 2006; Raasakka et al. 2019)
A0A1D5RDW1	PMP2	Myelin P2 protein	Preclinical PMP2 plays a role in myelin thickening and ATP production during remyelination following nerve crush injury (Hong et al. 2024)
F6XQH6	PRX	Periaxin	Periaxin mutations are associated with peripheral neuropathies including Charcot-Marie Tooth and Dejerine-Sottas neuropathy (Datta et al. 2019; Boerkoel et al. 2001; Peddareddygari 2012)

The most downregulated hippocampal protein detected in both CBA and VEH animals was myelin protein zero (MPZ; Table 6). MPZ is the main structural component of myelin sheath in the peripheral nervous system, and disruption of MPZ is associated with the development of peripheral neuropathies (Raasakka et al. 2019). Chronic alcohol consumption can lead to the development of alcoholic neuropathy, a polyneuropathy characterized by damage to sensory, motor, and autonomic nerve fibers (Dudek et al. 2020). Although the exact mechanism of alcoholic neuropathy is not well characterized, it is thought that disruption of myelin in the peripheral nerves contributes, as in other peripheral neuropathies. The downregulation of MPZ thus provides evidence of myelin disruption in a macaque model of CBA, which has been shown in previous transcriptomic studies examining the prefrontal cortex in mice and rhesus macaques exposed to chronic alcohol (Bogenpohl et al. 2019). Several proteins were only detected in the hippocampus of CBA animals, including the mitochondrial proteins glutathione S-transferase and frataxin and the ER protein phosphatidylserine synthase. Indeed, previous work has reported alterations in nucleus accumbens glutathione signaling pathways in alcohol-drinking macaques (Womersley et al. 2024). Alteration of these mitochondrial proteins, including glutathione S-transferase, suggests disruptions due to CBA are impacting the mitochondria of hippocampal neurons, which could suggest dysregulation of hippocampal bioenergetics that would be expected to be critical for cognitive function.

Ingenuity pathway analyses revealed that CBA significantly altered 39 hippocampal pathways, including inhibition of acetylcholine receptor signaling, orexin signaling, and serotonin receptor signaling, each representing potential pharmacotherapeutic targets. Acetylcholine is a neurotransmitter critically involved in memory, attention, and motivation and normally supports cognition and anti-inflammatory processes (Hasselmo 2006; Han et al. 2017). Reductions in acetylcholine signaling may reflect cognitive deficits and heightened inflammation in the hippocampus of CBA animals. Importantly, hippocampal acetylcholine deficiency is one of the hallmarks of AD, a progressive neurodegenerative disorder and the most common cause of dementia, and cholinesterase inhibitors such as donepezil and rivastigmine are prescribed for mild and moderate AD to improve cognitive and behavioral symptoms (Chen et al. 2022). Interestingly, acetylcholine receptor antagonists may impair cognition in monkeys with a history of chronic heavy drinking, while acetylcholine receptor agonists improved cognitive deficits in socially subordinate, but not socially dominant, monkeys (Galbo-Thomma et al. 2024).

Deficits in hippocampal serotonin signaling have been shown in several monkey models involving early life stress and alcohol consumption. Early maternal separation in male rhesus macaques results in lower serotonin transporter binding potential in several brain regions, including the hippocampus and anterior cingulate gyrus (Ichise et al. 2006). Another study similarly showed early maternal separation reduces serotonin metabolites and serotonin turnover in the hippocampus and increases ethanol consumption in male rhesus macaques (Huggins et al. 2012). Furthermore, low baseline serotonin levels in the cerebrospinal fluid were associated with greater alcohol intake in male and female nonhuman primates (Wood et al. 2022). These studies, together with the present study, provide evidence that there may be a link between serotonergic dysfunction and alcohol dependence. To further explore the potential contribution of these and other neurotransmitter signaling pathways in cognition and negative affect, recently developed behavioral paradigms can be applied to functionally investigate future cohorts of SIV-infected and CBA-treated macaques (Shnitko et al. 2017).

A previous study conducted a proteomic analysis of multiple brain regions, including the hippocampus and prefrontal cortex, on the brains of adult males with and without AUD (Teng et al. 2023). Interestingly, only two hippocampal pathways were associated with differentially expressed in the AUD brains compared to control, and these pathways did not overlap with the pathways we observed in the present analysis. The majority of the AUD-altered pathways they observed were in the amygdala, including the enrichment of several pathways we saw altered by CBA in the hippocampus (adrenergic, opioid, and GABA receptor signaling).

Effects of CBA in the Anterior Cingulate Cortex

In the anterior cingulate cortex (ACC), 176 proteins were significantly altered by CBA (Fig. 3B). Of ACC proteins expressed in both CBA and VEH animals, the largest increases observed were actin alpha-2 and calponin-1, both of which are involved in smooth muscle contraction (Yuan 2015; Liu and Jin 2016; Table 7). The most inhibited ACC protein detected in both groups was voltage-gated sodium channel subunit β4, a protein that promotes excitability by slowing sodium channel inactivation and may be involved in alcohol consumption based on evidence from rodent models and humans with alcohol use disorder (Blednov et al. 2019; Table 7). Other significantly upregulated proteins in the ACC included apolipoproteins A1, A2, and D. Apolipoproteins are proteins that bind to lipids to form lipoproteins, or soluble complexes that transport lipids (Elliott et al. 2010). While apolipoprotein E is largely recognized as a major genetic risk factor for AD, recent work suggests other apolipoproteins, including apolipoprotein A1 and apolipoprotein D, may also play a role in neurodegenerative and neuropsychiatric disorders, including AD, Parkinson’s disease, and schizophrenia (Elliott et al. 2010; Qiang et al. 2013; Dassati et al. 2014; Tong et al. 2022). Furthermore, apolipoprotein A2 was found to be elevated in the serum in a nonhuman primate model of ethanol self-administration, suggesting its potential as a peripheral biomarker of ethanol consumption (Freeman et al. 2006). Interestingly, a proteomic study examining exosomal proteins derived from plasma of HIV-infected alcohol drinkers have also reported a downregulation of apolipoprotein A2 (Kodidela et al. 2020).

Table 7.

Literature review of top differentially expressed proteins in the"anterior cingulate cortex

UniProt ID	Gene name	Description	Relevant literature
Top Upregulated Proteins (Expressed in Both Groups)
F7HJJ8	ACTA2	Alpha-2 actin	Preclinical Chronic alcohol consumption in mice alters hippocampal actin cytoskeleton (Gao et al. 2024) A review that discusses the role of the actin cortex in the initiation of HIV infection (Spear et al. 2012)
A0A1D5QTD2	CNN1	Calponin 1	Preclinical CNN1 expression reduced with alcohol consumption in oligodendrocytes of mice (Bazzi et al. 2023)
A0A1D5QLJ5	MFGE8	Milk fat globule epidermal growth factor 8	Preclinical MFGE8 improves reduces apoptosis and attenuates organ injury in alcohol intoxicated rats (Wu et al. 2010; Chaung et al. 2019) Pre-treatment with MFGE8 reduces apoptosis and inflammation after surgical brain injury and traumatic brain injury in rats (Xiao et al. 2018; Gao et al. 2018)
Top Downregulated Proteins (Expressed in Both Groups)
A0A5F8A459	SCN4B	Sodium voltage-gated channel beta subunit 4	Preclinical Scn4b knockout in mice alters hypnotic effects of alcohol but does not alter alcohol consumption (Blednov et al. 2019) Scn4b gene expression altered in alcohol-preferring mice and rats (Mulligan et al. 2006; Tabakoff et al. 2008) Clinical Scn4b gene expression altered in humans with alcohol use disorder (Farris et al. 2015)
F7DWC1	MAGI3	Membrane associated guanylyl kinase, WW, and PDZ domain containing 3	—
F7ATN1	MOXD1	Monooxygenase DBH-like 1	Preclinical MOXD1 is predictive of alcoholic liver disease in a mouse model (Pan et al. 2024)

Several ACC proteins were detected only in CBA animals, including ferritin light chain and metabolism of cobalamin associated A. Ferritin is an intracellular protein that stores iron and is comprised of a light chain and a heavy chain (Kotla et al. 2022). Elevated ferritin is associated with iron overload and heavy alcohol consumption and can result in ferroptosis, or cell death due to iron accumulation and lipid peroxidation (Kotla et al. 2022). Previous work has shown chronic intermittent alcohol increases iron-positive cells in the hippocampus and prefrontal cortex and is associated with anxiety and depression (Xu et al. 2022). Furthermore, inhibiting ferroptosis prevents alcohol-induced neuronal damage (Xu et al. 2022). Interestingly, higher ferritin heavy chain in the cerebrospinal fluid may be neuroprotective in PWH (Kaur et al. 2021). Future work with our nonhuman primate model will follow up on this finding with ex vivo assessments (e.g., Seahorse-based assays) to better understand the role of ferroptosis in CBA-induced deficits. Metabolism of cobalamin associated A is a protein involved in translocation of cobalamin (vitamin B12) into the mitochondria. Importantly, cobalamin is one of the vitamins that can become deficient in patients with chronic AUD, resulting in megaloblastic anemia and demyelination of peripheral nerves (Green et al. 2017; Madebo et al. 2022). Thus, it is interesting that the metabolism and translocation of this vitamin is upregulated in our CBA animals. This may be due to a compensatory upregulation of cobalamin-associated enzymes due to a CBA-induced vitamin deficiency.

Canonical pathways that were most significantly activated by CBA in the ACC included Parkinson’s signaling, xenobiotic metabolism aryl hydrocarbon receptor (AhR) signaling, and glycerophospholipid biosynthesis. CBA also inhibited several canonical pathways in the ACC, including response to elevated platelet cytosolic Ca2+, post-translational protein phosphorylation, and extracellular matrix organization.

Commonalities Between the Hippocampus and Anterior Cingulate Cortex

Overall, 14 common proteins (Fig. 4, Table 1) and 14 common canonical pathways (Fig. 8) were altered by CBA in both the hippocampus and ACC, suggesting some similarities in the effects of alcohol on distinct brain regions. As described above, the negative effects of lifetime alcohol consumption on executive function were found to be mediated by functional connectivity between the hippocampus and ACC in humans (Honnorat et al. 2022), suggesting that circuit-based mechanisms linking these two crucial regions may be particularly vulnerable to excessive alcohol exposure. Many of the individual protein alterations were in the same direction (i.e., upregulation or downregulation in both brain regions). For example, transthyretin was upregulated in both regions, while citramaloyl-CoA lyase was downregulated in both regions. Synapsin III was also downregulated in both regions, potentially indicating reductions in neurogenesis and neuronal plasticity based on its role in these processes (Porton et al. 2011). Indeed, previous work has shown CBA in nonhuman primates impairs hippocampal neurogenesis (Taffe et al. 2010).

Several canonical pathways were altered in both brain regions. Interestingly, synaptogenesis and SNARE signaling were significantly enriched by CBA in each brain region. In the hippocampus, synaptogenesis and SNARE signaling were inhibited, suggesting chronic alcohol reduces the formation of new synapses and neurotransmitter release in the hippocampus. In contrast, synaptogenesis and SNARE signaling pathways were activated in the ACC, suggesting CBA increases synapse formation and neurotransmitter release in this region. Thus, chronic alcohol may differentially alter synaptic signaling across these two regions, which may be due to functionally distinct roles for the two areas. For example, the hippocampus is heavily involved in spatial learning and long-term memory, while the ACC plays a more specific role in working memory and affective pain processing. These unique roles may explain the differential effects of CBA on regional physiology and how alcohol impacts these two sets of functional memory domains.

Other pathways affected by CBA were predicted to be altered in the same direction (i.e., both activated or both inhibited). Parkinson’s signaling was inhibited in both regions, which may indicate altered dopaminergic signaling in response to CBA in these dopaminoceptive regions, while integrin-linked kinase (ILK) signaling was activated in both regions, which may play influence hippocampal neurogenesis (Xu et al. 2015). Protein kinase A (PKA) signaling was also inhibited across both regions, suggesting a decrease in PKA-driven phosphorylation events and intracellular signaling cascades, a function that regulates diverse physiological processes including synaptic plasticity, memory, and metabolism that may be dysregulated across several neurological and psychiatric disorders (Glebov-McCloud et al. 2024). Interestingly, alcohol acutely activates PKA signaling, and PKA signaling may be critically involved in regulation of voluntary alcohol consumption (Thiele et al. 2000; Ron and Messing 2013). Particularly notable is the previously described role of reduced PKA activity in the nucleus accumbens in alcohol drinking (Misra and Pandey 2006) and reduced PKA signaling in the central amygdala in alcohol withdrawal-related anxiety (Pandey et al. 2005). Chronic intermittent alcohol exposure also dysregulates PKA-regulated genes in the medial prefrontal cortex, nucleus accumbens, and amygdala (Repunte-Canonigo et al. 2007). These deficits may extend to other areas including the hippocampus and ACC to mediate additional deleterious alcohol-related behaviors. Numerous studies have described interventions targeting an inhibition of cAMP phosphodiesterases (PDEs) to boost cAMP/PKA activity and reduce alcohol drinking (Logrip et al. 2014; Logrip 2015), and a recent NIH study recommended that PET imaging of PDE4B in individuals with alcohol use disorder (AUD) should be considered in conjunction with ongoing trials of PDE4 inhibitors to treat alcohol withdrawal and reduce alcohol consumption (Tang et al. 2024). A previous study examining proteomic alterations in postmortem brains from patients with AUD found metabolic changes in proteins associated with glycolysis (phosphofructokinase, glyceraldehyde-3-phosphate) and oxidative phosphorylation (NADH dehydrogenase iron-sulfur protein 8, cytoplasmic aspartate aminotransferase) in the prefrontal cortex (Enculescu et al. 2019); however, we did not see similar alterations in the ACC.

Limitations and Future Directions

While the analysis of female macaque brain represents a major strength of the current study, there are some limitations of bioinformatics analyses on rhesus macaque systems, as the monkey proteomic and IPA databases are less well characterized compared to those for humans and rodents. Another limitation is the exclusion of male animals in the present cohort, although ongoing studies are currently being conducted in male macaques. Future studies are necessary to extend these findings at the individual protein level and functional studies will be vital to confirm the role of specific pathways in corresponding behavioral tests. Another limitation of this study is the lack of SIV-negative control animals. Female macaques were particularly dimcult to source during the study period to include this control group. Ongoing macaque cohorts incorporate this factor, and we recognize that the results from the present analyses must be limited to interpretation of alterations in the context of SIV. Additionally, all animals in this experiment were treated with ART, which can itself have significant neurotoxic effects (Robertson et al. 2012). Thus, we are unable to tease out the effects of CBA without exposure to SIV and ART, and future studies should examine this to better understand how ART modulates the relationship between SIV infection and CBA exposure. Furthermore, the ART regimen we use here was the current standard at the time when the experiment was conducted (2015–2021); current cohorts of animals have an updated ART regimen that includes bictegravir. Finally, in comparison to our fixed alcohol administration model, Grant and colleagues have developed a model of alcohol self-administration in monkeys that allows for the study of the impact of voluntary chronic alcohol consumption and its comorbidities (Baker et al. 2023), including effects on cognitive behaviors (Shnitko et al. 2020; Grant et al. 2021). Nevertheless, while there are many advantages of the voluntary self-administration model, the ability to control the amount of alcohol consumed in our animal model is a strength of our present study.

Summary

Together, the results from this study suggest that CBA administration in SIV-infected female macaques produces widespread alterations in the hippocampal and ACC proteomes. Our discoveries indicate that certain proteins and pathways are similarly altered across both brain regions, including upregulation of transthyretin and inhibition of Parkinson’s and protein kinase A signaling pathways. Importantly, CBA may also differentially impact these regions, suggesting CBA-induced deficits may differ by brain region and may be specific to the functional role of that region. Ongoing studies seek to expand this work to males to better understand the role of sex on the impact of CBA on brain regional proteomics, especially the differential impact on specific brain areas including the hippocampus and ACC. Overall, the hippocampus and ACC represent important areas at the intersection of alcohol- and HIV-induced cognitive decline and for the discovery of potential therapeutic targets for treatment of brain-related disorders in women with HIV.

Data Availability

No datasets were generated or analysed during the current study.