Opioid and chemokine regulation of cortical synaptodendritic damage in HIV-associated neurocognitive disorders

Abstract

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) persist despite effective antiretroviral therapies (ART). Evidence suggests that modern HAND is driven by subtle synaptodendritic damage in select brain regions, as ART-treated patients do not display overt neuronal death in postmortem brain studies. HAND symptoms are also aggravated by drug abuse, particularly with injection opioids. Opioid use produces region-specific synaptodendritic damage in similar brain regions, suggesting a convergent mechanism that may enhance HAND progression in opioid-using patients. Importantly, studies indicate that synaptodendritic damage and cognitive impairment in HAND may be reversible. Activation of the homeostatic chemokine receptor CXCR4 by its natural ligand CXCL12 positively regulates neuronal survival and dendritic spine density in cortical neurons, reducing functional deficits. However, the molecular mechanisms that underlie CXCR4, as well as opioid-mediated regulation of dendritic spines are not completely defined. Here, we will consolidate studies that describe the region-specific synaptodendritic damage in the cerebral cortex of patients and animal models of HAND, describe the pathways by which opioids may contribute to cortical synaptodendritic damage, and discuss the prospects of using the CXCR4 signaling pathway to identify new approaches to reverse dendritic spine deficits. Additionally, we will discuss novel research questions that have emerged from recent studies of CXCR4 and µ-opioid actions in the cortex. Understanding the pathways that underlie synaptodendritic damage and rescue are necessary for developing novel, effective therapeutics for this growing patient population.

Keywords: CXCL12; CXCR4; Dendritic spines; HAND; Opioids; neuroHIV.

Figure 1.

Dendritic spine morphologies A. Grayscale micrograph of a dendrite from the rat layer 2/3 medial prefrontal cortex that was stained with DiI, a lipophilic fluorescent dye that labels dendritic spines (Seabold et al., 2010). B. The spines on this dendrite can be characterized into different morphological classes using Neurolucida 360 software (Rodriguez et al., 2008): Dendrite (red), filopodia (white), thin spines (cyan), mushroom spines (magenta), and stubby spines (yellow).

Figure 2.. Synaptodendritic damage in motor and somatosensory cortices in animal models of HAND

Studies in A and B were completed using adult (4–5 months-old) male F344 HIV-1 transgenic rats and wild-type F344 controls (n=4 rats per group). The preparation of brain tissue, dendritic spine staining (Seabold et al., 2010), and the analysis of layer 2/3 cortical neurons (Festa et al., 2015) were performed as previously described. A. HIV-Tg rat motor cortex. The overall dendritic spine density in motor cortex layer 2/3 neurons of HIV-Tg rats was significantly reduced compared to wild-type controls (t[14] = 4.183, p=0.0009). Dendritic spine morphology analysis showed that the percentage of immature filopodia are significantly increased in these same neurons (filopodia t[434] = 6.094, p<0.0001). Further, Sholl analysis showed the HIV-Tg rats have significantly fewer intersections at 100 μM (p=0.0172) from the soma in this region (distance F[9,100]= 41.98, p<0.0001; group F[1,100]= 7.590, p=0.007). B. HIV-Tg rat somatosensory cortex. There were no significant changes in dendritic spine density (t[14] = 1.044, p=0.3142), morphology, or Sholl analysis (distance F[9,100] = 66.68, p<0.0001; group F[1, 100] = 2.93, p=0.0901) in HIV-Tg rats compared to wild-type. Studies in C and D were completed using adult (4–5 months-old) male Sprague Dawley rats. Each rat was stereotaxically implanted with a cannula targeting the lateral ventricle, infused with either gp120_IIIB (50 ng/μL in 0.1% BSA, n=5) or vehicle (n=6) once daily for 7 days, and sacrificed 28 days after the final infusion, as previously reported (Festa et al., 2015). The preparation of brain tissue, dendritic spine staining, and analysis of layer 2/3 cortical neurons were also performed as previously reported (Festa et al., 2015). C. gp120-infused rat motor cortex. The overall dendritic spine density in motor cortex layer 2/3 neurons of gp-120 infused rats was significantly decreased (t[20] = 4.896, p<0.0001). Dendritic spine morphology analysis showed that the percentage of thin spines was significantly decreased (thin t[84] = 4.027, p=0.0009), and the percentage of filopodia was significantly increased in these same neurons (filopodia t[84] = 3.856, p=0.0013). Further, Sholl analysis showed that gp-120 infused rats had significantly fewer dendritic intersections at 80 μm (p=0.0006), 100 μm (p=0.0029), and 120 μm (p=0.0197) from the soma in this region (distance F[9, 200] = 23.98, p<0.0001; group F[1, 200] = 50.64, p<0.0001). D. gp120 infused rat somatosensory cortex. There were no significant differences in overall dendritic spine density in layer 2/3 somatosensory cortex neurons of either group (t[20] = 1.046, p=0.3082), but spine morphology analysis showed that the percentage of mushroom spines were significantly decreased in gp-120 infused rats (mushroom t[126] = 4.059, p=0.0006). Further, Sholl analysis did not show any changes of dendritic intersections in this region (distance F[9, 200] = 66.38, p<0.0001; group F[1, 200] = 3.978, p= 0.0475). Data in all figures represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Spine density data was analyzed with two-tailed Students t-test; each point in HIV-Tg studies is one dendrite, and each point in gp120 studies is the average spine density of one neuron (two points per animal). Spine morphology data was analyzed with multiple t-tests and Holm Sidak correction. Sholl analysis was analyzed with two-way ANOVA and Sidak’s multiple comparisons test. All animals in these studies were housed in Association for Assessment and Accreditation of Laboratory Animal Care-accredited facilities in accordance with the National Institutes of Health guidelines and institutional approval by the Drexel University Institutional Animal Care and Use Committee.

Figure 3.. Potential network-level interactions of μOR and CXCR4 leading to dendritic spine deficits

Although μOR activation could lead to CXCR4 inhibition and dendritic spine deficits all in the same excitatory pyramidal neuron (PN), μOR regulation of CXCR4 in GABAergic interneurons may also regulate dendritic spine density in PNs by altering the excitatory/inhibitory balance of the local circuit through distinct mechanisms. Some possibilities include A. Reduced GABAergic innervation of PNs; B. an overall decrease in neuronal activity of GABAergic interneurons and PNs; or C. Reduced activity of GABAergic disinhibition circuits.

Figure 4.

μOR regulation of CXCR4 working model Morphine or other μ-opioids binding to μORs on cortical neurons activates a Gαi-protein pathway that leads to de-acidification of endolysosomes, and efflux of endolysosomal iron to the cytoplasm. This increases labile iron levels in the neuron, resulting in increased production of the iron storage proteins ferritin heavy chain (FHC) and ferritin light chain (FLC) through a post-transcriptional mechanism. In addition to its iron storage functions, FHC also associates with the homeostatic chemokine receptor CXCR4 and blocks its downstream signaling. Normally, CXCR4 activation by its natural ligand CXCL12 increases dendritic spine density in cortical neurons, which may occur through activation of several pathways. First, CXCR4 activation leads to HDAC4 translocation to the nucleus, which regulates several genes involved in synaptic activity. Specific knockdown of HDAC4 prevents CXCL12 from increasing dendritic spine density, suggesting that CXCL12/CXCR4 signaling regulates synaptic protein expression. Second, CXCL12 may activate the Rac1/PAK pathway in cortical neurons, resulting in phosphorylation of the actin severing protein cofilin. Phosphorylated cofilin is unable to break down the actin cytoskeleton, which may result in stabilization of actin-rich dendritic spines.