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Published in final edited form as: Exp Neurol. 2019 Oct 31;323:113077. doi: 10.1016/j.expneurol.2019.113077

Increased matrix metalloproteinase levels and perineuronal net proteolysis in the HIV-infected brain; relevance to altered neuronal population dynamics

P Lorenzo Bozzelli 1,2, Adam Caccavano 1,2,3, Valeria Avdoshina 2, Italo Mocchetti 1,2, Jian-Young Wu 1,2, Katherine Conant 1,2
PMCID: PMC7919751  NIHMSID: NIHMS1542300  PMID: 31678140

Abstract

HIV-associated neurocognitive disorders (HAND) continue to persist despite effective control of viral replication. Although the mechanisms underlying HAND are poorly understood, recent attention has focused on altered neuronal population activity as a correlate of impaired cognition. However, while alterations in neuronal population activity in the gamma frequency range are noted in the setting of HAND, the underlying mechanisms for these changes is unclear. Perineuronal nets (PNNs) are a specialized extracellular matrix that surrounds a subset of inhibitory neurons important to the expression of neuronal oscillatory activity. In the present study, we observe that levels of PNN-degrading matrix metalloproteinases (MMPs) are elevated in HIV-infected post-mortem human brain tissue. Furthermore, analysis of two PNN components, aggrecan and brevican, reveals increased proteolysis in HIV-infected brains. In addition, local field potential recordings from ex vivo mouse hippocampal slices demonstrate that the power of carbachol-induced gamma activity is increased following PNN degradation. Together, these results provide a possible mechanism whereby increased MMP proteolysis of PNNs may stimulate altered neuronal oscillatory activity and contribute to HAND symptoms.

Keywords: HIV, matrix metalloproteinases, perineuronal net, gamma oscillations

INTRODUCTION

The advent of combination antiretroviral therapy (cART) has revolutionized HIV treatment by significantly prolonging the lifespan of infected individuals. Despite these life-saving drugs, infected individuals continue to experience cognitive impairments which are termed HIV-associated neurocognitive deficits (HAND). Up to 50% of infected individuals will develop HAND at some point in their lifetime (Heaton et al., 2010), and HAND symptoms often include deficits in working memory and attention (Mohamed et al., 2010). Although the underlying mechanisms of HAND remain poorly understood, recent attention has focused on altered neuronal population activity in HIV-infected individuals.

Alterations in overall neuronal excitability have been noted in the context of HIV. As such, HIV infected individuals are at an increased risk for developing new-onset seizures, with opportunistic infections being a major contributor (Chadha et al., 2000; Kellinghaus et al., 2008; Siddiqi et al., 2017). New-onset seizures have a high recurrence rate and are associated with high mortality (Elafros et al., 2018). Furthermore, non-epileptiform EEG abnormalities are also detected in infected individuals with new-onset seizures (Siddiqi et al., 2015).

Gamma frequency (25 – 85 Hz) oscillatory activity is thought to underlie various cognitive processes such as attention, memory, and perception (Bartos et al., 2007; Ray and Maunsell, 2015), which are altered in HAND (Woods et al., 2009). Gamma oscillations are closely associated with inhibitory interneuron activity (Bartos et al., 2007; Buzsáki and Wang, 2012), and specifically parvalbumin-positive (PV+) GABAergic neurons have been shown to critically influence gamma oscillations (Cardin et al., 2009). Of interest, PV+ neurons also represent the most common neuronal subtype that is ensheathed by a specialized extracellular matrix known as the perineuronal net (PNN) (Wen et al., 2018b).

PNNs are lattice-like structures that enhance the firing rate of fast-spiking neurons (Balmer, 2016), and as such, they are important in the maintenance of PV+ interneuron excitability. PNNs protect PV+ neurons from oxidative stress (Cabungcal et al., 2013), and PNN components, which include chondroitin sulfate proteoglycans (CSPGs), have been shown to be protective against glutamate excitotoxicity (Okamoto et al., 1994). Enzymatic digestion of PNNs in ex vivo brain slices results in a decrease in PV+ neuronal firing rate (Balmer, 2016), and genetic knock-out of the PNN CSPG, brevican, reduces synaptic input to PV+ cells (Favuzzi et al., 2017). Similarly, PNN digestion reduces glutamatergic input to PV+ interneurons (Hayani et al., 2018), providing evidence for a modulatory role in PV+ neuronal excitability. Since each PV+ interneuron makes contact with a variety of pyramidal neurons (Packer and Yuste, 2011), PNN disruption and consequent pyramidal cell disinhibition may substantially influence overall neuronal network activity. Consistent with this are studies linking PNN disruption with enhanced gamma power in the mouse visual cortex (Lensjø et al., 2017), as well as with an increase in the frequency of highly synchronous hippocampal sharp-wave ripple events (Sun et al., 2017).

Degradation of the PNN occurs through enzymatic cleavage. One such class of endogenous enzymes that are capable of degrading PNNs are matrix metalloproteinases (MMPs) (Fosang et al., 1996; Nakamura et al., 2000; Struglics and Hansson, 2012). Of interest, various MMPs have been shown to be pathologically elevated in cerebrospinal fluid, plasma, and serum of HIV-infected patients (Conant et al., 1999; Li et al., 2013; Liuzzi et al., 2000; Xing et al., 2017), and levels of MMPs are correlated with brain atrophy and cognitive impairment (Conant et al., 1999; Ragin et al., 2011, 2009).

Interestingly, HIV-infected individuals exhibit stronger gamma range spontaneous activity in the somatosensory cortex as detected by magnetoencephalography (MEG) (Spooner et al., 2018). Spontaneous gamma activity is also elevated in the visual cortex of HIV-infected individuals regardless of HAND diagnosis (Wiesman et al., 2018). Furthermore, increases in other oscillatory frequencies have recently been noted in various cortical brain regions of HIV-infected participants, and these different frequency bands have been associated with cognitive processes such as selective attention (Lew et al., 2018).

Given that gamma power is elevated in HAND, and that reductions in PNN integrity can increase gamma power (Alaiyed et al., 2019; Lensjø et al., 2017), in this study we tested the possibility that PNN-degrading proteases remain elevated in the era of cART. We also examined cleavage of critical PNN components in post-mortem brain tissues from HIV-infected patients who received cART. Lastly, we examined the effect of PNN degradation on gamma power in murine hippocampal slices. While the hippocampus is an area that is difficult to examine in human studies of gamma frequency, this brain region is thought to be important to cognitive impairments in HIV (Castelo et al., 2006; Maki et al., 2009).

MATERIALS AND METHODS

In vitro digest

Recombinant MMPs and PNN proteins aggrecan and brevican were incubated in vitro to assess MMP-dependent cleavage of PNN substrate. Recombinant human MMP-3 (Millipore Calbiochem, catalog # 444217) was used at a concentration of 5 μg/mL and recombinant human MMP-13 (Enzo Life Sciences, catalog # BML-SE246-0010) was used at a concentration of 17.5 μg/mL. Recombinant human aggrecan (R&D, catalog # 1220-PG-025) was used at a concentration of 62.5 μg/mL and recombinant human brevican (R&D, catalog # 5800-NC-050) was used at a concentration of 50 μg/mL. The broad-spectrum MMP-inhibitor GM6001 (Tocris, catalog # 2983) was used at a concentration of 10 μM. Enzyme and substrate were incubated together for two hours at 37° C. Following incubation, an equal volume of 2X Laemmli buffer containing 2-mercaptoethanol was added, heated for 5 minutes at 95° C, resolved using SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were then stained with Coomassie Blue (BioRad, R-250) for two hours. Excess staining was removed using a de-stain solution containing 50% distilled water, 40% ethanol, and 10% glacial acetic acid. Membranes were placed into a total of three fresh de-stain solution containers for 20 minutes each. After the last de-stain, the membranes were allowed to air-dry and then scanned for image acquisition.

Western blot

Cortical tissue was dissected from post-mortem human brains (Table 1). Tissue was homogenized in a standard RIPA lysis buffer containing protease/phosphatase inhibitors. Samples were sonicated, spun down, and the soluble supernatant was separated. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (ThermoFisher, catalog # 23225). Samples were mixed with 2X Laemmli buffer containing 2-mercaptoethanol, and heated for 5 minutes at 95° C. Forty μg of total protein was loaded per lane and resolved in a 4% - 20% gradient polyacrylamide tris glycine gel (BioRad). Following electrophoresis, proteins were transferred to a nitrocellulose membrane in a Trans-Blot Turbo Transfer System (BioRad) set to mixed molecular weight. Membranes were blocked with 5% milk in TBST for 1 hour prior to overnight incubation with primary antibodies. The dilution of the primary antibodies were: MMP-3 (Abcam, catalog # ab52915, 1:1000), MMP-13 (ThermoFisher, catalog #MA5-14238, 1:220), aggrecan (ThermoFisher, catalog # MA3-16888, 1:2000), brevican (ThermoFisher, catalog # PA5-47563, 1:2000), GAPDH (Millipore, Catalog # MAB374, 1:5000). Membranes were washed multiple times the next day and incubated at room temperature with HRP-conjugated secondary antibodies, and followed by additional washes. Signal was detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, catalog # 34580). Bands were visualized using an Amersham Imager 600 (GE Healthcare Life Sciences). Band densitometry was conducted using ImageJ (NIH). Band density for all proteins of interest were normalized to GAPDH. A biological mixture was used as a standard control that was run on every gel in order to compare band density across different blots. When reprobing membranes for proteins of similar molecular weight, membranes were stripped with the Restore Western Blot Stripping Buffer (ThermoFisher, catalog # 21059) according to manufacturer’s directions. Stripping efficiency was assessed each time by incubating membranes with ECL and exposing for an extended duration. After successful stripping, membranes were blocked again and probed for proteins as mentioned above.

Table 1.

Post-mortem human brain tissue demographics and clinical characteristics

Controls (n=15) HIV+ (n=26)
Age 51.1 +/− 15.1 (100%) 46.2 +/− 7.1 (69%)
Gender 9 male, 6 female (100 %) 11 male, 5 female (62%)
CD4 cell count N/A 112.8 +/− 123.5 (62%) range: 2–471
Plasma VL (copies/ml) N/A 22975.3 +/− 50312.5 (38%, 4/10 und.) range: 0 (und.)122642
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(% reported from total n); und. = undetectable

Slice preparation and incubation

21 – 32-day old male and female C57BL6/J mice were used to prepare paired hippocampal hemi-slices in accordance with the National Institutes of Health guidelines and a protocol that had been approved by the Institutional Animal Care and Use Committee at Georgetown University Medical Center. Following deep isoflurane anesthesia, mice were decapitated, and the brain was dissected in ice-cold sucrose-based cutting artificial cerebrospinal fluid (aCSF) containing (in mM) 252 sucrose, 3 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 10 dextrose and bubbled with 95% O2/5% CO2. Horizontal hippocampal slices were cut at 490 μm thickness using a vibratome (Leica, VT1000s). Slices were bisected to obtain paired hemislices for subsequent incubation in either control aCSF or aCSF containing hyaluronidase. The aCSF contained (in mM): 132 NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO2, 10 dextrose and bubbled with 95% O2/5% CO2 at 26° C. Slices were incubated for at least four hours before being moved to the recording chamber. Lyophilized hyaluronidase (Sigma, catalog # H4272) was reconstituted in aCSF prior to brain slice incubation. The final concentration of hyaluronidase in the incubation solution was 0.25 g/mL which was based on previously published work (Bikbaev et al., 2015; Sun et al., 2017).

Local field potential recordings

Low-resistance glass microelectrodes (50–150 KΩ) filled with aCSF were used for recording local field potentials in hippocampal slices. The recordings were conducted in a submerged chamber and slices were perfused on both sides at a high flow rate (20 mL/min). The recording electrodes were placed in CA1 stratum radiatum. Comparisons between control and hyaluronidase-treated slices were performed with paired hemispheres. After baseline recording, gamma oscillations were induced by adding the cholinergic-agonist carbachol (carbamoylcholine chloride, Sigma-Aldrich, catalog #: C4382) at a concentration of 40 μM into the recording chamber. Analysis of the LFP signal was performed using a custom MATLAB algorithm. A Gaussian FIR band-pass filter with corrected phase delay was applied between 1 – 1000 Hz, after which additional band-pass filters were applied for theta (4 – 12 Hz), low gamma (25 – 55 Hz), and high gamma (65 – 85 Hz) ranges. The power was computed by integrating the entire recorded block band-pass filtered signals. Traces in figures were additionally notch filtered at 60 Hz to correct for line noise. Spectrograms of LFP traces were calculated in MATLAB with a short-time Fourier transform, in 1 Hz frequency bins from 1 – 100 Hz, and in 30ms time windows. Presented results display the Z-scored power to show temporal deviations from baseline. The average and standard deviation (SD) were computed for each frequency bin from a baseline recording before carbachol administration, and the carbachol-induced deviations for each frequency bin were computed as [P(t) – Average(Pbaseline)] / SD(Pbaseline).

Statistical analyses

All statistical analyses were performed using GraphPad Prism 8.0 (Graphpad Software). Data were subjected to unpaired t-tests. Pearson correlation coefficients were calculated for relationships between protein levels in human brain tissue. All data are reported as mean +/− SEM and significance was set at p ≤ 0.05. Some human samples were omitted from the correlational analyses given that they did not have a value for at least one of the four probed proteins. Furthermore, an outlier analysis was conducted for each probed protein and identified outliers were omitted from subsequent analyses.

RESULTS

MMPs cleave the PNN proteins aggrecan and brevican

MMPs often demonstrate restricted substrate specificity. We focused on MMP-3 and MMP-13 given recent observations of their colocalization with PNN proteolysis following status epilepticus (Dubey et al., 2017; Rankin-Gee et al., 2015). Furthermore, these MMPs are thought to be upstream activators of other MMPs, given that both MMP-3 (Johnson, Jason L. et al., 2011; Ogata et al., 1992; Ramos-DeSimone et al., 1999) and MMP-13 (Dreier et al., 2004; Knäuper et al., 1997) are able to activate the latent form of MMP-9. We conducted an in vitro digest to assess the ability of these MMPs to cleave two main PNN proteins: aggrecan and brevican. We found that MMP-3 and MMP-13 were able to cleave both PNN substrates, and that this cleavage event was prevented by the broad-spectrum MMP inhibitor GM6001 (Figure 1).

Figure 1.

Figure 1.

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MMPs cleave the perineuronal net proteins aggrecan and brevican. Active recombinant matrix metalloproteinases were incubated with recombinant aggrecan and brevican. In vitro digests revealed that A. aggrecan (Acan) and B. brevican (Bcan) are both cleaved by MMP-3 and MMP-13, as demonstrated by the appearance of the indicated cleavage fragments. Enzymatic cleavage is prevented by addition of the broad-spectrum MMP inhibitor GM6001.

HIV infection increases MMP-3 and MMP-13 in post-mortem brain tissue

Recently, analyses of post-mortem human brain revealed MMP-13 mRNA levels as being particularly altered in HIV-infection (Sanna et al., 2017). To our knowledge, however, protein levels of MMP-3 and MMP-13 have not previously been assessed in infected brain tissue. We probed for both MMPs in HIV- and HIV+ brain samples, and found that HIV-infected samples exhibited significantly higher levels of both MMP-3 (unpaired t test, df = 34, t = 4.535, p < 0.0001, n = 36, Figure 2A) and MMP-13 (unpaired t test, df = 37, t = 2.902, p = 0.0062, n = 38, Figure 2B). We also assessed MMP levels within HIV+ samples in order to assess whether MMP levels were dependent on HAND diagnosis severity, however, no differences within HIV+ samples were observed, and this may be due to the relatively smaller sample size per each of the different HAND diagnoses (data not shown).

Figure 2.

Figure 2.

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MMP-3 and MMP-13 are elevated in HIV-infected human brain tissue. MMP-3 and MMP-13 were detected by Western blot in post-mortem human cortical lysates. Representative bands from infected and non-infected are shown. A. MMP-3 (53 kDa) levels were elevated in HIV-infected tissue (unpaired t test, df = 34, t = 4.534, p < 0.0001, n = 36). B. Catalytic MMP-13 (26 kDa) levels were also elevated in HIV-infected tissue (unpaired t test, df = 37, t = 2.902, p = 0.0062, n = 38). ** p < 0.01, **** p <0.0001

PNN proteins demonstrate increased proteolysis in the HIV-infected brain

Various brain disease and injury models have demonstrated damage to PNNs (Andrews et al., 2012; Fowke et al., 2018; Hobohm et al., 2005). Here, we sought to measure protein levels of aggrecan and brevican. Specifically, aggrecan has been identified as particularly important in protecting neurons from oxidative stress (Suttkus et al., 2014), and brevican has been shown to be important to PV neuron-mediated inhibition of pyramidal cells (Favuzzi et al., 2017). Using an antibody that detects aggrecan cleavage fragments, HIV-infected samples were found to exhibit significantly higher expression of the cleavage fragment (unpaired t test, df = 36, t = 2.086, p = 0.0441, n = 38, Figure 3A). Also, using an antibody that detects full-length (intact) brevican, HIV-infected brain tissue expressed significantly less full-length brevican, suggesting increased proteolysis of the full-length protein (unpaired t test, df = 36, t = 5.985, p < 0.0001, n = 38, Figure 3B). These results are consistent with increased PNN proteolysis in the brains of HIV-infected individuals.

Figure 3.

Figure 3.

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Perineuronal net protein levels are altered in HIV-infected human brain tissue. Aggrecan and brevican were detected by Western blot using post-mortem human cortical lysates. Representative bands from infected and non-infected shown. A. Levels of the aggrecan (Acan) cleavage fragment (27 kDa) were elevated in HIV-infected samples (unpaired t test, df = 36, t = 2.086, p = 0.0441). B. Levels of intact full-length brevican (Bcan) (160 kDa) were decreased in HIV-infected samples (unpaired t test, df = 36, t = 5.985, p < 0.0001). C. Correlations between MMPs and PNN protein expression were assessed. MMP-3 levels were positively correlated with levels of aggrecan cleavage fragments (Pearson correlation; r = 0.4023, R2 = 0.1618, p = 0.0150, n = 36), and D. negatively correlated with full-length brevican (Pearson correlation; r = −0.4450, R2 = 0.1980, p = 0.0065, n = 36). E. There was a significant positive correlation between levels of MMP-13 and the aggrecan cleavage fragment (Pearson correlation; r = 0.9301, r2 = 0.8651, p < 0.0001, n = 36), and F. there was a trending negative correlation between levels of MMP-13 and brevican (Pearson correlation; r = −0.2928, r2 = 0.08575, p = 0.0831, n = 36). HIV-negative (black arrows) n = 15, HIV-positive (purple circles) n = 21; for all correlations the best fit line and 95% confidence bands were generated using linear regression. * p < 0.05, **** p < 0.0001.

In order to determine a relationship between MMP expression and PNN proteolysis, correlational analyses were conducted. Given that the antibodies used for the two PNN proteins detect either a cleavage fragment (aggrecan) or intact full-length (brevican) bands, correlations with the detected forms is expected to be in the opposite direction. As such, MMP-3 was positively correlated with levels of the aggrecan cleavage fragment (Pearson correlation; r = 0.4023, r2 = 0.1618, p = 0.0150, n = 36, Figure 3C), and negatively correlated with full-length brevican (Pearson correlation; r = −0.4450, r2 = 0.1980, p = 0.0065, n = 36, Figure 3D). Levels of MMP-13 and the aggrecan fragment were significantly correlated (Pearson correlation; r = 0.9301, r2 = 0.8651, p < 0.0001, n = 36, Figure 3E), and there was a negative trend between levels of MMP-13 and full-length brevican (Pearson correlation; r = −0.2928, r2 = 0.08575, p = 0.0831, n = 36, Figure 3F).

Removal of PNNs in the hippocampus increases gamma frequency

Previous work has found an increase in in vivo gamma frequency following digestion of PNNs in mouse visual cortex (Lensjø et al., 2017). We sought to evaluate the effect in the hippocampus, an HIV-relevant region of the brain, which has been implicated in episodic and working memory impairments in HIV-infected individuals (Castelo et al., 2006; Maki et al., 2009). Using ex vivo mouse brain slices, we sought to evaluate the role of PNNs in hippocampal gamma frequency by comparing control and hyaluronidase (Hyal) treated slices. Hyaluronidase targets the hyaluronan backbone of PNNs which maintains the binding of CSPGs such as aggrecan and brevican (Deepa et al., 2006). In order to detect gamma oscillations ex vivo, carbachol was added to the recording chamber solution. Hyaluronidase digestion of PNNs resulted in no difference in theta frequency (unpaired t test, df = 8, t = 0.8983, p = 0.3953, n = 5 mice, 1–2 hemislices/condition, Figure 4A); however, hyaluronidase treatment resulted in a trending increase in low gamma (unpaired t test, df = 8, t = 1.975, p = 0.0837, n = 5 mice, 1–2 hemislices/condition, Figure 4B), and a significant increase in high gamma (unpaired t test, df = 8, t = 4.260, p = 0.0028, n = 5 mice, 1–2 hemislices/condition, Figure 4C). These results provide evidence for PNN modulation of hippocampal neuronal population activity, and specifically modulation of gamma oscillations.

Figure 4.

Figure 4.

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Perineuronal net digestion leads to increased hippocampal gamma frequency. Representative local field potential recordings shown from A. control and B. hyaluronidase (Hyal)-treated slices following carbachol (40 μM) induction of gamma oscillations in the CA1 region of the hippocampus. The LFP was filtered in the theta (4 – 12 Hz), low gamma (25 – 55 Hz), and high gamma (65 – 85 Hz) ranges. The Z-scored spectrogram was computed with a short-time Fourier transform, normalizing the power in each frequency bin to the average and SD of a baseline recording prior to carbachol administration. C. There was no significant difference in theta frequency (unpaired t test, df = 8, t = 0.8983, p = 0.3953). D. There was, however, a trend for an increase in low gamma (unpaired t test, df = 8, t = 1.975, p = 0.0837), and E. the power of high gamma activity was significantly increased following hyaluronidase digestion of PNNs (unpaired t test, df = 8, t = 4.260, p = 0.0028). N = 5 mice, 1 – 2 hemisected slices/condition; values were averaged when recorded from 2 slices/condition. Each hemisected pair of recordings were done using different mice on separate days. # p < 0.1, ** p < .01.

DISCUSSION

Analyses of post-mortem human cortical samples reveals that despite cART, HIV infection is associated with increased levels of MMP-3 and MMP-13. In addition, observed changes in the MMP-3 and MMP-13 substrates aggrecan and brevican suggest that increased MMP activity and PNN remodeling may occur in the brains of HIV infected individuals. Moreover, the observation that ex vivo digestion of mouse PNNs results in a significant increase in hippocampal gamma power suggests that PNN remodeling may affect neuronal population dynamics in region critical for learning and memory. Together, these data provide potential mechanistic insight into the observation that spontaneous gamma power is increased in the background of HAND (Spooner et al., 2018; Wiesman et al., 2018).

In select situations, enhanced gamma power may be expected to increase attention and working memory. For example, gamma frequency sensory stimulation improves spatial learning and memory in a mouse model of Alzheimer’s disease (Adaikkan et al., 2019). In addition, a high definition transcranial alternating current protocol that enhances theta-gamma phase amplitude coupling improves working memory in older adult humans (Reinhart and Nguyen, 2019). Moreover, in conditions of excessive PNN deposition, certain therapeutics such as monoamine reuptake inhibitors may increase MMP expression proximal to sites of monoamine release, to potentially restore physiologically appropriate PNN levels, enhance gamma power (Alaiyed et al., 2019), and improve learning and memory (Riga et al., 2017). In other settings, however, this relationship between enhanced gamma power and cognition is not as straightforward. For example, in schizophrenia, spontaneous gamma power is elevated (Hirano et al., 2015), and this is thought to be a reflection of disrupted excitatory/inhibitory balance in brain circuits that in turn interferes with salient information processing (McNally and McCarley, 2016). Similarly, the dissociative anesthetic ketamine can enhance gamma power through predominant inhibition of NMDA receptors localized to PV+ neurons (Cunningham et al., 2006).

In HIV infection and other inflammatory disorders, leukocytes may release PNN-degrading MMPs in a non-uniform manner, lacking in neuronal circuit specificity, which could lead to poorly localized or otherwise aberrant increases in local gamma power. Gamma increases in specific frequency ranges may be especially beneficial (Adaikkan et al., 2019; Singer et al., 2018), and HIV infection might instead increase gamma power in a broader frequency range. Aberrant increases in gamma power could also influence processes such as theta-gamma cross frequency coupling, which play a role in information routing (Scheffer-Teixeira and Tort, 2016). Consistent with this, widespread and circuit non-specific knockout of the obligatory NMDA receptor subunit NR1 on PV+ interneurons increases gamma power, but reduces theta-gamma phase locking, and impairs spatial working memory (Korotkova et al., 2010). Relevant to the disconnect between increased gamma power and cognition in HIV infection, recent work has shown that while HIV-infected individuals had stronger spontaneous gamma, the somatosensory evoked gamma responses were dampened (Spooner et al., 2018).

Interestingly, previous immunohistochemical analysis revealed that the HIV-encephalitis brain is largely devoid of PNNs (Belichenko et al., 1997). Also, in a simian immunodeficiency virus model, PNNs were found to be drastically depleted, and especially so in encephalitic cases (Medina-Flores et al., 2004). These previous reports both provided initial evidence that retroviral infection leads to a loss of PNNs. Our data with decreased levels of full-length brevican and increased levels of an aggrecan cleavage product suggest that PNN alterations persist in the cART era.

Our experimental ex vivo slice paradigm using hyaluronidase results in a nearly complete digestion of PNNs as detected by the lectin wisteria floribunda (Sun et al., 2017). However, in non-encephalitic cases of brain infection, complete degradation of PNNs is unlikely and PNN disruption may instead occur in a non-uniform/irregular distribution. The enzyme hyaluronidase was chosen to degrade PNNs given experimental limitations of using exogenous MMPs which include low enzymatic activity and longer incubation periods (Tewari et al., 2018), as well as a potential lack of tissue penetration and insufficient enzyme concentrations in the slice recording chamber. Recent work has, however, shown that even partial digestion of PNNs at the exposed slice surface is sufficient in altering fast-spiking neuron firing rate (Tewari et al., 2018). Since each PV+ interneuron contacts numerous pyramidal neurons (Packer and Yuste, 2011), these irregular/patchy disruptions in PNN integrity have the potential to influence population-wide dynamics.

We focused our attention on two specific MMPs given recent evidence implicating their involvement in proteolysis of PNNs following aberrant neuronal activity (Dubey et al., 2017; Tewari et al., 2018). Moreover, the Dubey et al. study found that levels of another PNN-degrading class of enzymes, those belonging to the ADAMTS family (also known as aggrecanases), were unchanged in a rodent model of status epilepticus (Dubey et al., 2017). MMP-3 and MMP-13 have additional substrates that may increase pyramidal cell activity through direct mechanisms including astrocytic protease-activated receptor 1 (PAR-1)-dependent glutamate release and subsequent NMDA-receptor activation (Lee et al., 2007). However, due to relatively abundant PNN expression as compared to other MMP substrates, and the high density of PV+ cell connections onto pyramidal cells (Packer and Yuste, 2011), we posit that PNN disruption makes a particularly strong contribution to altered gamma power in HIV-infected patients. While we and others have previously examined MMP-9 as an effector of PNN proteolysis (Alaiyed et al., 2019; Murase et al., 2017; Wen et al., 2018a), MMP-9 levels have also been shown to be elevated in HIV-infected CNS (Conant et al., 1999). Moreover, both MMP-3 and MMP-13 are able to activate MMP-9 (Dreier et al., 2004; Johnson, Jason L. et al., 2011; Knäuper et al., 1997; Ogata et al., 1992; Ramos-DeSimone et al., 1999), which highlights the importance of examining upstream activators of MMP-9.

In conclusion, we have established a correlation between increased MMPs and PNN proteolysis in post-mortem HIV-infected brain despite cART treatment. We have also demonstrated that attenuation of hippocampal PNNs leads to alterations in gamma power, an alteration that has previously been observed in HIV-infected individuals (Wiesman et al., 2018). In the future, it would be of interest to assess MMP levels in cerebrospinal fluid and to determine whether they are correlated with altered oscillatory activity in patients. It is also of interest to determine whether targeting MMPs released from infiltrating or activated monocyte-derived cells, which play a role in HAND-related cognitive injury (Lyons et al., 2011; Shikuma et al., 2012; Shiramizu et al., 2009), could reduce cognitive deficits and disordered neuronal population activation in the context of HIV-infection. Further work is warranted examining MMPs as potential therapeutic candidates in HIV-associated neurocognitive deficits.

Highlights.

  • Altered brain oscillations have been previously reported in HIV+ patients

  • Increased MMP expression and PNN proteolysis is observed in HIV+ brain tissue

  • PNN degradation in murine hippocampal slices results in increased gamma power

  • Aberrant PNN proteolysis may underlie altered brain oscillations in HIV+ patients

ACKNOWLEDGEMENTS

This study was funded by the National Institutes of Health (NIH) R01 NS108810 (KC), NIH R01 NS079172 (IM), NIH T32 NS041218 (PLB), the Cosmos Club Foundation of Washington, D.C. (PLB), and NIH/NCATS TL1TR001431 (AC). HIV+ human brain samples were provided by the National NeuroAIDS Tissues Consortium (NNTC) and made possible through NIH funding U24MH100931, U24MH100928, and U24MH100925 to the Manhattan HIV Brain Bank, California NeuroAIDS Tissue Network, and Data Coordinating Center, respectively. Control human brain samples were kindly provided by Craig Stockmeier, PhD and Gouri Mahajan, PhD from the University of Mississippi.

Footnotes

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