Accelerated neurodegeneration of basal forebrain cholinergic neurons in HIV-1 gp120 transgenic mice: Critical role of the p75 neurotrophin receptor.

Human Immunodeficiency Virus-1 (HIV) infection of the brain induces HIV-associated neurocognitive disorders (HAND). The set of molecular events employed by HIV to drive cognitive impairments in people living with HIV are diverse and remain not completely understood. We have shown that the HIV envelope protein gp120 promotes loss of synapses and decreases performance on cognitive tasks through the p75 neurotrophin receptor (p75NTR). This receptor is abundant on cholinergic neurons of the basal forebrain and contributes to cognitive impairment in various neurological disorders. In this study, we examined cholinergic neurons of gp120 transgenic (gp120tg) mice for signs of degeneration. We observed that the number of choline acetyltransferase-expressing cells is decreased in old (12-14-month-old) gp120tg mice when compared to age matched wild type. In the same animals, we observed an increase in the levels of pro-nerve growth factor, a ligand of p75NTR, as well as a disruption of consolidation of extinction of conditioned fear, a behavior regulated by cholinergic neurons of the basal forebrain. Both biochemical and behavioral outcomes of gp120tg mice were rescued by the deletion of the p75NTR gene, strongly supporting the role that this receptor plays in the neurotoxic effects of gp120. These data indicate that future p75NTR-directed pharmacotherapies could provide an adjunct therapy against synaptic simplification caused by HIV.

Mice deficient for G-protein-coupled receptor 75 display altered presynaptic structural protein expression and disrupted fear conditioning recall.

There are a number of G-protein-coupled receptors (GPCRs) that are considered "orphan receptors" because the information on their known ligands is incomplete. Yet, these receptors are important targets to characterize, as the discovery of their ligands may lead to potential new therapies. GPR75 was recently deorphanized because at least two ligands appear to bind to it, the chemokine CCL5 and the eicosanoid 20-Hydroxyeicosatetraenoic acid. Recent reports suggest that GPR75 may play a role in regulating insulin secretion and obesity. However, little is known about the function of this receptor in the brain. To study the function of GPR75, we have generated a knockout (KO) mouse model of this receptor and we evaluated the role that this receptor plays in the adult hippocampus by an array of histological, proteomic, and behavioral endpoints. Using RNAscope® technology, we identified GPR75 puncta in several Rbfox3-/NeuN-positive cells in the hippocampus, suggesting that this receptor has a neuronal expression. Proteomic analysis of the hippocampus in 3-month-old GPR75 KO animals revealed that several markers of synapses, including synapsin I and II are downregulated compared with wild type (WT). To examine the functional consequence of this down-regulation, WT and GPR75 KO mice were tested on a hippocampal-dependent behavioral task. Both contextual memory and anxiety-like behaviors were significantly altered in GPR75 KO, suggesting that GPR75 plays a role in hippocampal activity.

Recent Advances in the Molecular and Cellular Mechanisms of gp120-Mediated Neurotoxicity.

Axonal degeneration and loss of synapses are often seen in different brain areas of people living with human immunodeficiency virus (HIV). Nevertheless, the underlying causes of the pathological alterations observed in these individuals are poorly comprehended, considering that HIV does not infect neurons. Experimental data have shown that viral proteins, including the envelope protein gp120, cause synaptic pathology followed by neuronal cell death. These neurotoxic effects on synapses could be the result of a variety of mechanisms that decrease synaptic plasticity. In this paper, we will briefly present new emerging concepts connected with the ability of gp120 to promote the degeneration of synapses by either directly damaging the axonal cytoskeleton and/or the indirect activation of the p75 neurotrophin receptor death domain in dendrites.

Race-Dependent Association of Single-Nucleotide Polymorphisms in TrkB Receptor in People Living with HIV and Depression.

Human immunodeficiency virus (HIV)-associated cognitive disorders (HAND) is characterized by impaired motor and intellectual functions, as well as mood disorders. Brain-derived neurotrophic factor and its receptor TrkB (or NTRK2) mediate the efficacy of antidepressant drugs. Genomic studies of BDNF/TrkB have implicated common single-nucleotide polymorphisms in the pathology of depression. In the current study, we investigated whether single-nucleotide polymorphisms (SNPs) (rs1212171, rs1439050, rs1187352, rs1778933, rs1443445, rs3780645, rs2378672, and rs11140800) in the NTRK2 has a functional impact on depression in HIV-positive subjects. We have utilized the Central Nervous System (CNS) HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. Our methods explored the univariate associations of these SNPs with clinical (current and lifetime) diagnosis of depression via chi-square. The distribution of alleles was significantly different for African-Americans and Caucasians (non-Hispanic) for several SNPs, so our regression analyses included both "statistical controls" for race group and models for each group separately. Finally, we applied a method of simultaneous analysis of associations, estimating the mutually shared information across a system of variables, separately by race group. Our results indicate that there is no significant association between clinical diagnosis of major depression and these SNPs for either race group in any analysis. However, we identified that the SNP associations varied by race group and sex.

Extracellular Vesicles and HIV-Associated Neurocognitive Disorders: Implications in Neuropathogenesis and Disease Diagnosis.

Extracellular vesicles are heterogeneous cell-derived membranous structures of nanometer size that carry diverse cargoes including nucleic acids, proteins, and lipids. Their secretion into the extracellular space and delivery of their cargo to recipient cells can alter cellular function and intracellular communication. In this review, we summarize the role of extracellular vesicles in the disease pathogenesis of HIV-associated neurocognitive disorder (HAND) by focusing on their role in viral entry, neuroinflammation, and neuronal degeneration. We also discuss the potential role of extracellular vesicles as biomarkers of HAND. Together, this review aims to convey the importance of extracellular vesicles in the pathogenesis of HAND and foster interest in their role in neuroinflammatory diseases.

Neuronal apoptosis induced by morphine withdrawal is mediated by the p75 neurotrophin receptor.

Morphine withdrawal evokes neuronal apoptosis through mechanisms that are still under investigation. We have previously shown that morphine withdrawal increases the levels of pro-brain-derived neurotrophic factor (BDNF), a proneurotrophin that promotes neuronal apoptosis through the binding and activation of the pan-neurotrophin receptor p75 (p75NTR). In this work, we sought to examine whether morphine withdrawal increases p75NTR-driven signaling events. We employed a repeated morphine treatment-withdrawal paradigm in order to investigate biochemical and histological indicators of p75NTR-mediated neuronal apoptosis in mice. We found that repeated cycles of spontaneous morphine withdrawal promote an accumulation of p75NTR in hippocampal synapses. At the same time, TrkB, the receptor that is crucial for BDNF-mediated synaptic plasticity in the hippocampus, was decreased, suggesting that withdrawal alters the neurotrophin receptor environment to favor synaptic remodeling and apoptosis. Indeed, we observed evidence of neuronal apoptosis in the hippocampus, including activation of c-Jun N-terminal kinase (JNK) and increased active caspase-3. These effects were not seen in saline or morphine-treated mice which had not undergone withdrawal. To determine whether p75NTR was necessary in promoting these outcomes, we repeated these experiments in p75NTR heterozygous mice. The lack of one p75NTR allele was sufficient to prevent the increases in phosphorylated JNK and active caspase-3. Our results suggest that p75NTR participates in the neurotoxic and proinflammatory state evoked by morphine withdrawal. Because p75NTR activation negatively influences synaptic repair and promotes cell death, preventing opioid withdrawal is crucial for reducing neurotoxic mechanisms accompanying opioid use disorders.

Up-regulation of the p75 neurotrophin receptor is an essential mechanism for HIV-gp120 mediated synaptic loss in the striatum.

Reduced synaptodendritic complexity appears to be a key feature in human immunodeficiency virus (HIV)-associated neurological disorder (HAND). Viral proteins, and in particular the envelope protein gp120, play a role in the pathology of synapses. Gp120 has been shown to increase both in vitro and in vivo the proneurotrophin brain-derived neurotrophic factor, which promotes synaptic simplification through the activation of the p75 neurotrophin receptor (p75NTR). To provide evidence that p75NTR plays a role in gp120-mediated loss of synapses in vivo, we intercrossed gp120tg mice with p75NTR null mice and used molecular, histological and behavioral analyses to establish a link between p75NTR and gp120-mediated synaptic simplification. Synaptosomes obtained from the striatum of gp120tg mice exhibited a significant increase in p75NTR levels concomitantly to a decrease in synaptic markers such as TrkB and PSD95. Analysis of striatal dendritic spines by Golgi staining revealed that gp120tg mice display a reduced proportion of mushroom-type spines in addition to fewer spines overall, when compared to wild type or gp120tg lacking one or two p75NTR alleles. Moreover, removal of one p75NTR allele in gp120 transgenic mice abolished the gp120-driven impairment on a task of striatal-dependent motor learning. These data indicate that p75NTR could be a key player in HIV-mediated synaptic simplification in the striatum.

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

OBJECTIVE: Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. DESIGN: Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. METHODS: First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. RESULTS: PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform. CONCLUSION: Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.

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

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.

Histone deacetylase 6 inhibition rescues axonal transport impairments and prevents the neurotoxicity of HIV-1 envelope protein gp120.

Despite successful antiretroviral drug therapy, a subset of human immunodeficiency virus-1 (HIV)-positive individuals still display synaptodendritic simplifications and functional cognitive impairments referred to as HIV-associated neurocognitive disorders (HANDs). The neurological damage observed in HAND subjects can be experimentally reproduced by the HIV envelope protein gp120. However, the complete mechanism of gp120-mediated neurotoxicity is not entirely understood. Gp120 binds to neuronal microtubules and decreases the level of tubulin acetylation, suggesting that it may impair axonal transport. In this study, we utilized molecular and pharmacological approaches, in addition to microscopy, to examine the relationship between gp120-mediated tubulin deacetylation, axonal transport, and neuronal loss. Using primary rat cortical neurons, we show that gp120 decreases acetylation of tubulin and increases histone deacetylase 6 (HDAC6), a cytoplasmic enzyme that regulates tubulin deacetylation. We also demonstrate that the selective HDAC6 inhibitors tubacin and ACY-1215, which prevented gp120-mediated deacetylation of tubulin, inhibited the ability of gp120 to promote neurite shortening and cell death. We further observed by co-immunoprecipitation and confirmed with mass spectroscopy that exposure of neurons to gp120 decreases the association between tubulin and motor proteins, a well-established consequence of tubulin deacetylation. To assess the physiological consequences of this effect, we examined the axonal transport of brain-derived neurotrophic factor (BDNF). We report that gp120 decreases the velocity of BDNF transport, which was restored to baseline levels when neurons were exposed to HDAC6 inhibitors. Overall, our data suggest that gp120-mediated tubulin deacetylation causes impairment of axonal transport through alterations to the microtubule cytoskeleton.

Reversal of Cognitive Impairment in gp120 Transgenic Mice by the Removal of the p75 Neurotrophin Receptor.

Activation of the p75 neurotrophin receptor (p75NTR), by the proneurotrophin brain-derived neurotrophic factor (proBDNF), triggers loss of synapses and promotes neuronal death. These pathological features are also caused by the human immunodeficiency virus-1 (HIV) envelope protein gp120, which increases the levels of proBDNF. To establish whether p75NTR plays a role in gp120-mediated neurite pruning, we exposed primary cultures of cortical neurons from p75NTR -/- mice to gp120. We found that the lack of p75NTR expression significantly reduced gp120-mediated neuronal cell death. To determine whether knocking down p75NTR is neuroprotective in vivo, we intercrossed gp120 transgenic (tg) mice with p75NTR heterozygous mice to obtain gp120tg mice lacking one or two p75NTR alleles. The removal of p75NTR alleles inhibited gp120-mediated decrease of excitatory synapses in the hippocampus, as measured by the levels of PSD95 and subunits of the N-methyl-D-Aspartate receptor in synaptosomes. Moreover, the deletion of only one copy of the p75NTR gene was sufficient to restore the cognitive impairment observed in gp120tg mice. Our data suggest that activation of p75NTR is one of the mechanisms crucial for the neurotoxic effect of gp120. These data indicate that p75NTR antagonists could provide an adjunct therapy against synaptic simplification caused by HIV.

Age-Related Decrease in Tyrosine Hydroxylase Immunoreactivity in the Substantia Nigra and Region-Specific Changes in Microglia Morphology in HIV-1 Tg Rats.

Animal models have been used to study cellular processes related to human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). The HIV-1 transgenic (Tg) rat expresses HIV viral genes except the gag-pol replication genes and exhibits neuropathological features similar to HIV patients receiving combined antiretroviral therapy (cART). Using this rat, alterations in dopaminergic function have been demonstrated; however, the data for neuroinflammation and glial reactivity is conflicting. Differences in behavior, tyrosine hydroxylase (TH) immunoreactivity, neuroinflammation, and glia reactivity were assessed in HIV-1 Tg male rats. At 6 and 12 weeks of age, rotarod performance was diminished, motor activity was not altered, and active avoidance latency performance and memory were diminished in HIV-1 Tg rats. TH+ immunoreactivity in the substantia nigra (SN) was decreased at 8 months but not at 2-5 months. At 5 months, astrocyte and microglia morphology was not altered in the cortex, hippocampus, or SN. In the striatum, astrocytes were unaltered, microglia displayed slightly thickened proximal processes, mRNA levels for Iba1 and Cd11b were elevated, and interleukin (Il)1α,Cxcr3, and cell adhesion molecule, Icam, decreased. In the hippocampus, mRNA levels for Tnfa and Cd11b were slightly elevated. No changes were observed in the cortex or SN. The data support an age-related effect of HIV proteins upon the nigrostriatal dopaminergic system and suggest an early response of microglia in the terminal synaptic region with little evidence of an associated neuroinflammatory response across brain regions.

Helix-A peptide prevents gp120-mediated neuronal loss.

AIM: The human-immunodeficiency virus (HIV) envelope protein gp120 promotes synaptic damage similar to that observed in people living with HIV who have neurocognitive disorders. The neurotoxic effect of gp120 appears to occur through the α-helix motif that binds to neuronal microtubules (MTs). In this study, we examined the ability of short peptide derivatives from Helix-A, a peptide synthesized based on α-helix structure of gp120, to displace gp120 from binding to MTs and prevent its neurotoxic effects. METHODS: Surface plasmon resonance was used to determine the binding of Helix-A and its modifications to MTs. Helix-A peptide and derivatives were delivered inside rat primary cortical neurons by mesoporous silica nanoparticles (MSN). Neuronal processes and survival were evaluated by microtubule associated protein 2-immunostaining and Hoechst/Propidium iodide, respectively. RESULTS: Surface plasmon resonance analysis revealed that Helix-A but not its modifications binds to MTs. Also, only Helix-A MSN but not other peptides prevented the ability of gp120 to reduce neuronal processes as well as neuronal survival. Thus, the amino acid structure of Helix-A is key for its neuroprotective activity.

HIV-1 Tat promotes astrocytic release of CCL2 through MMP/PAR-1 signaling.

The HIV-1 protein Tat is continually released by HIV-infected cells despite effective combination antiretroviral therapies (cART). Tat promotes neurotoxicity through enhanced expression of proinflammatory molecules from resident and infiltrating immune cells. These molecules include matrix metalloproteinases (MMPs), which are pathologically elevated in HIV, and are known to drive central nervous system (CNS) injury in varied disease settings. A subset of MMPs can activate G-protein coupled protease-activated receptor 1 (PAR-1), a receptor that is highly expressed on astrocytes. Although PAR-1 expression is increased in HIV-associated neurocognitive disorder (HAND), its role in HAND pathogenesis remains understudied. Herein, we explored Tat's ability to induce expression of the PAR-1 agonists MMP-3 and MMP-13. We also investigated MMP/PAR-1-mediated release of CCL2, a chemokine that drives CNS entry of HIV infected monocytes and remains a significant correlate of cognitive dysfunction in the era of cART. Tat exposure significantly increased the expression of MMP-3 and MMP-13. These PAR-1 agonists both stimulated the release of astrocytic CCL2, and both genetic knock-out and pharmacological inhibition of PAR-1 reduced CCL2 release. Moreover, in HIV-infected post-mortem brain tissue, within-sample analyses revealed a correlation between levels of PAR-1-activating MMPs, PAR-1, and CCL2. Collectively, these findings identify MMP/PAR-1 signaling to be involved in the release of CCL2, which may underlie Tat-induced neuroinflammation.

HIV-associated neurodegeneration: exploitation of the neuronal cytoskeleton.

Human immunodeficiency virus-1 (HIV) infection of the central nervous system damages synapses and promotes axonal injury, ultimately resulting in HIV-associated neurocognitive disorders (HAND). The mechanisms through which HIV causes damage to neurons are still under investigation. The cytoskeleton and associated proteins are fundamental for axonal and dendritic integrity. In this article, we review evidence that HIV proteins, such as the envelope protein gp120 and transactivator of transcription (Tat), impair the structure and function of the neuronal cytoskeleton. Investigation into the effects of viral proteins on the neuronal cytoskeleton may provide a better understanding of HIV neurotoxicity and suggest new avenues for additional therapies.

The orphan G-protein-coupled receptor 75 signaling is activated by the chemokine CCL5.

The chemokine CCL5 prevents neuronal cell death mediated both by amyloid ß, as well as the human immunodeficiency virus viral proteins gp120 and Tat. Because CCL5 binds to CCR5, CCR3 and/or CCR1 receptors, it remains unclear which of these receptors plays a role in neuroprotection. Indeed, CCL5 also has neuroprotective activity in cells lacking these receptors. CCL5 may bind to a G-protein-coupled receptor 75 (GPR75), which encodes for a 540 amino-acid orphan receptor of the Gqα family. In this study, we have used SH-SY5Y human neuroblastoma cells to characterize whether CCL5 could activate a Gq signaling through GPR75. Both qPCR and flow cytometry show that these cells express GPR75 but do not express CCR5, CCR3 or CCR1 receptors. SY-SY5Y cells were then used to examine CCL5-mediated signaling. We report that CCL5 promotes a time- and concentration-dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3ß, and extracellular signal-regulated kinase (ERK) 1/2. Specific antagonists of CCR5, CCR3, and CCR1 did not prevent CCL5 from increasing phosphorylated AKT or ERK. Moreover, CCL5 promotes a time-dependent internalization of GPR75. Lastly, knocking down GPR75 expression by a CRISPR-Cas9 approach inhibited the ability of CCL5 to activate pERK in SH-SY5Y cells. Therefore, we propose that GPR75 is a novel receptor for CCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small molecule GPR75 agonists that mimic CCL5. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Human immunodeficiency virus Tat impairs mitochondrial fission in neurons.

Human immunodeficiency virus-1 (HIV) infection of the central nervous system promotes neuronal injury that culminates in HIV-associated neurocognitive disorders. Viral proteins, including transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanisms remain unclear. Tat transgenic mice or neurons exposed to Tat, which show neuronal loss, exhibit smaller mitochondria as compared to controls. To provide an experimental clue as to which mechanisms are used by Tat to promote changes in mitochondrial morphology, rat cortical neurons were exposed to Tat (100 nM) for various time points. Within 30 min, Tat caused a significant reduction in mitochondrial membrane potential, a process that is regulated by fusion and fission. To further assess whether Tat changes these processes, fission and fusion proteins dynamin-related protein 1 (Drp1) and mitofusin-2 (Mfn2), respectively, were measured. We found that Drp1 levels increased beginning at 2 h after Tat exposure while Mfn2 remained unchanged. Moreover, increased levels of an active form of Drp1 were found to be present following Tat exposure. Furthermore, Drp1 and calcineurin inhibitors prevented Tat-mediated effects on mitochondria size. These findings indicate that mitochondrial fission is likely the leading factor in Tat-mediated alterations to mitochondrial morphology. This disruption in mitochondria homeostasis may contribute to the instability of the organelle and ultimately neuronal cell death following Tat exposure.

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells.

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1ß, TNFα, TGFß, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels.

Morphine Withdrawal Increases Brain-Derived Neurotrophic Factor Precursor.

Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of morphine withdrawal on BDNF and its precursor protein, or proBDNF, which induces neuronal apoptosis. In this work, we examined whether BDNF and proBDNF levels change in rats chronically injected with escalating doses of morphine and those who undergo spontaneous withdrawal for 60 h. We observed, in the frontal cortex and striatum, that the ratio of BDNF to proBDNF changed depending upon the experimental paradigm. Morphine treatment and morphine withdrawal increased both BDNF and proBDNF levels. However, the increase in proBDNF immunoreactivity in withdrawal rats was more robust than that observed in morphine-treated rats. proBDNF is processed either intracellularly by furin or extracellularly by the tissue plasminogen activator (tPA)/plasminogen system or matrix metalloproteases (MMPs). To examine the mechanisms whereby chronic morphine treatment and morphine withdrawal differentially affects BDNF/proBDNF, the levels MMP-3 and MMP-7, furin, and tPA were analyzed. We found that morphine increases tPA levels, whereas withdrawal causes a decrease. To confirm the involvement of tPA in the morphine-mediated effect on BDNF/proBDNF, we exposed cortical neurons to morphine in the presence of the tPA inhibitor plasminogen activator inhibitor-1 (PAI-1). This inhibitor reversed the morphine-mediated decrease in proBDNF, supporting the hypothesis that morphine increases the availability of BDNF by promoting the extracellular processing of proBDNF by tPA. Because proBDNF could negatively influence synaptic repair, preventing withdrawal is crucial for reducing neurotoxic mechanisms associated with opioid abuse.