The cell-cell adhesion mechanisms to form plaques.

<p>(A) Scheme of PrP-MuLV-Gal interaction in cell membrane. (B) Based on our data between the cells PrP-Gal3 has binding activity and Gal3-CAgag-Gal6 shows binding activity. To produce the plaques, Gal3 is suggested to combine between PrP and MuLV. Gal6 is suggested to combine two different viruses to form plaques.</p

Different susceptibility to MuLV infection of MoPrP^<i>KO</i>, MoPrP^<i>wild</i> and MoPrP^<i>mut</i> neuronal cells.

<p>Neuronal cells expressing PrP^C, regardless of their type, were shown to have higher susceptibility to MuLV infection in illumination microscopy. Neuronal cells expressing MoPrP^<i>wild</i>, ZW, or PrP^C with the 3F4 epitope, or MoPrP^<i>mut</i> with the octarepeat deletion, PrPΔ, showed intense staining of both PrP and CAgag at a similar level. The location of PrP in these cells was primarily in cytosol and membrane before MuLV infection. After MuLV infection, PrP staining was observed in the nucleus, cytosol, and also membranes. MuLV infection was observed mainly in cytosol by detection of CAgag. Neuronal cells expressing P101L mutant type of PrP^C, MoPrP^<i>mut</i>, were also susceptible to MuLV infection. The PrP^C of P101L was mainly located in the nuclear portion of the cells, thus the overlapping between PrP^C and CAgag was not observed clearly through illumination microscopy. MuLV infections in astroglial cells were not affected by PrP^C. Different from neuronal cells, astroglial cells were largely resistant to infection by MuLV. Green, PrP; Red, CAgag; Blue, DAPI; Yellow, Merge. Scale bar = 20 μm.</p

Expression and binding activity of PrP^C with galectin-1,-3, and -6 mRNAs and proteins.

<p>(A) Expression of mRNA levels of galectin-1, -3, and -6 was observed by quantitative RT-PCR method. Binding activity of PrP^C with galectin-1, -3, and -6 mRNAs was investigated by immunoprecipitation of mRNA-protein complex method using anti-PrP antibody (anti-3F10). Galectin-1 mRNA is constitutively expressed regardless of no infection or MuLV infection. Galectin-3 mRNA expression is closely related with PrP^C expression, thus it is detected in PrP^C-expressing neuronal cells but not in PrP^-/- neuronal cells. Galectin-6 mRNA expression is related to MuLV infection in PrP^C-expressing cells but not in PrP^-/- cells, P101L cells and PrP^+/+ astroglial cells. Binding activity of PrP^C to galectin mRNAs was closely related to MuLV infection in PrP^+/+ cells. For P101L cells, the binding activity to galectin-1 and -6 was unusual in that binding occurred in non-infected cells. This may have contributed to the large plaque size seen in P101L cells. (B) Anti-PrP (3F10) antibody was used to proceed immunoprecipitation then anti-PrP (3F10) (27–33 kDa), -galectin-1 (Gal1)(14 kDa), -galectin-3 (Gal3)(31 kDa), -galectin-6 (Gal6)(32 kDa), and –CAgag (30 kDa) antiboties were used to detect expression level of each protein. PrP was detected in all PrP expressing cell lines: ZW, 3F4, PrPΔ, P101L, and ICR cell lines regardless of infected or non-infected. CAgag and Gal3 was detected in MuLV-infected neuronal cell lines: ZW, 3F4, PrPΔ, and P101L cell lines. Gal1 and Gal6 was not detected in any of cell lines regardless of infected or non-infected. (C) Anti-CAgag antibody was used to proceed immunoprecipitation then anti-PrP (3F10) (27–33 kDa), -galectin-1 (Gal1)(14 kDa), -galectin-3 (Gal3)(31 kDa), -galectin-6 (Gal6)(32 kDa), and –CAgag (30 kDa) antiboties were used to detect expression level of each protein. CAgag was detected in all MuLV infected cells and Gal1 was not detected. PrP, Gal3, and Gal6 was detected at MuLV-infected PrP^+/+ cells.</p

Correlation of PrP^C and MuLV infection in neuronal and astroglial cells.

<p>(A) <i>Prnp</i> expression levels in <i>Prnp</i>^+/+ neuronal and glial cells. <i>Prnp</i>^+/+ neuronal cells showed up-regulation after MuLV infection. <i>Prnp</i>^+/+ astroglial cells showed no statistically significant differences from uninfected cells. (B) Densitometry analysis of <i>Prnp</i> expression showed significant differences between infected and control neuronal cells (*<i>p</i> < 0.05). There was no difference in expression of <i>Prnp</i>^+/+ between control and infected ICR-A3. (C, D) In neuronal cells, significant correlation was observed between PrP^C expression and MuLV antigen. PrP^C protein levels were up-regulated in MuLV-infected neuronal cells, but expression was down-regulated in astroglial cells. PrP^+/+ cells showed significantly higher levels of CAgag than PrP^-/- cells (*<i>p</i> < 0.01). Cells expressing P101L mutant form showed significantly higher levels of CAgag than wild-type and octarepeat deletion forms of PrP^C (*<i>p</i> < 0.05). Astroglial cells showed significantly lower levels of MuLV infection than either PrP^-/- or PrP^+/+ neuronal cells (†<i>p</i> < 0.01). (E) The biochemical character of PrP^C in non-infected and MuLV-infected cells was investigated using PK treatment (20 μg/mL). Expressed PrP showed no PK-resistant form in either non-infected or MuLV-infected neuronal and astroglial cells.</p

Expression levels of the PrP^C genes and proteins in neuronal and astroglial cell lines.

<p>(A) Analysis of expression levels of <i>Prnp</i> in <i>Prnp</i>^-/-, Zpl, Vec, and Za, and <i>Prnp</i>^+/+, ZW, 3F4, PrPΔ, P101L, and ICR cell lines. ZW, 3F4, P101L, and ICR-A cell lines contained full-length of <i>Prnp</i> (789 bp). Cell lines expressing wild-type PrP^C are called the ZW cell line (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.t001" target="_blank">Table 1</a>). PrPΔ cell line contained shorter length <i>Prnp</i> (663 bp). Zpl, Vec, and Za cell lines were negative for <i>Prnp</i> detection. (B) Protein levels of PrP in cell lines were consistent with the results of RT-PCR analysis. PrPΔ cell line showed shorter length PrP. (C) Densitometry analysis of PrP protein expression showed no significant difference between wild-type cells and PrP-transfected cells. Relative values are represented as the mean±SEM. Cell lines were assessed by three separate experiments. ZW 13–2, 100±7.82; 3F4-A3, 87.8±9.53; PrPΔP1-3, 93.8±9.11; P101L-C4, 83.94±9.41; ICR-A3, 88.75±10.23.</p

Primer sequences and conditions for RT-PCR.

<p>Primer sequences and conditions for RT-PCR.</p

Association of PrP^C and morphological appearance of plaques in PrP^-/- and PrP^+/+ neuronal and astroglial cells.

<p>The XC/UV plaque assay was applied to each of the cell lines listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.s005" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.s006" target="_blank">S2</a> Tables. (A) The average plaques numbers of each cell lines were 35.6±5.68 from Zpl, 144.3±28.43 from ZW, 24.6±1.52 from Vec, 112±6.08 from 3F4, 107±3.21 from PrPΔ, 363±15.17 from P101L, and 1.5±1 from each of Za and ICR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.s005" target="_blank">S1 Table</a>). The shape and size of the plaques were similar in the 2 <i>Prnp</i>^-/- lines (MoPrP^<i>KO</i>) and in ZW, 3F4 (MoPrP^<i>wild</i>) and PrPΔ cell lines (MoPrP^<i>mut</i>). In P101L cells (MoPrP^<i>mut</i>), the plaques were approximately 4 times larger (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.s006" target="_blank">S2 Table</a>), and the shape was more round and floral than seen in the other lines. (B) Astroglial cells did not form plaques like neuronal cells. The number of plaques was very low, and the average size was 25-fold smaller than those seen in neuronal cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167293#pone.0167293.s005" target="_blank">S1 Table</a>). MoPrP^<i>KO</i> or MoPrP^<i>wild</i> did not affect plaque formation in astroglial cells. Scale bar = 0.1 mm. (C-D) Quantity of plaque numbers and size were measured. There is a significant difference in plaque numbers and size of P101L (MoPrP^<i>mut</i>) (**<i>p</i> < 0.001) and <i>Prnp</i>^-/- lines (MoPrP^<i>KO</i>) and in ZW, 3F4 (MoPrP^<i>wild</i>) and PrPΔ cell lines (MoPrP^<i>mut</i>)(*<i>p</i> < 0.01).</p

Colocalization of PrP^C and CAgag proteins in MuLV-infected cells.

<p>(A) Cells infected with MuLV were labeled with immunogold particles to observe colocalization of PrP^C and MuLV using electron microscopy. PrP^C and MuLV were conjugated with 10 nm-gold particle and 15 nm-gold particle, respectively. Particles due to colocalization were not observed in PrP^-/- cells, regardless of the cell type. In PrP^+/+ neuronal cells, ZW, 3F4, PrPΔ, and P101L, PrP^C-CAgag colocalization was easily observed, whereas colocalization was very rare in PrP^+/+ astroglial cells, ICR-A. Black arrows indicate the colocaloization of PrP^C and CAgag. Scale bar = 500 nm. (B, C) Quantitation of the experiment shown in (A). Colocalization was assessed in whole sections (B) and in nuclear and cytosol fractions separately (C) by counting the paired immunogold particles. PrP^+/+ neuronal cells showed markedly higher numbers of pairings of PrP^C-CAgag gold particles compared to PrP^-/- cells and non-infected cells (*<i>p</i> < 0.001). Particles in the latter 2 groups were non-specific deposits. P101L PrP^+/+ cells had significantly more PrP^C-CAgag gold particles pairings than other PrP^+/+ neuronal cells, ZW, 3F4, and PrPΔ (*<i>p</i> < 0.001). The small number of particles seen in PrP^-/- cells are background immunogold deposits.</p

Image_1_Buprenorphine reverses neurocognitive impairment in EcoHIV infected mice: A potential therapy for HIV-NCI.jpeg

Thirty-eight million people worldwide are living with HIV, PWH, a major public health problem. Antiretroviral therapy (ART) revolutionized HIV treatment and significantly increased the lifespan of PWH. However, approximately 15-50% of PWH develop HIV associated neurocognitive disorders (HIV-NCI), a spectrum of cognitive deficits, that negatively impact quality of life. Many PWH also have opioid use disorder (OUD), and studies in animal models of HIV infection as well as in PWH suggest that OUD can contribute to HIV-NCI. The synthetic opioid agonist, buprenorphine, treats OUD but its effects on HIV-NCI are unclear. We reported that human mature inflammatory monocytes express the opioid receptors MOR and KOR, and that buprenorphine reduces important steps in monocyte transmigration. Monocytes also serve as HIV reservoirs despite effective ART, enter the brain, and contribute to HIV brain disease. Using EcoHIV infected mice, an established model of HIV infection and HIV-NCI, we previously showed that pretreatment of mice prior to EcoHIV infection reduces mouse monocyte entry into the brain and prevents NCI. Here we show that buprenorphine treatment of EcoHIV infected mice with already established chronic NCI completely reverses the disease. Disease reversal was associated with a significant reduction in brain inflammatory monocytes and reversal of dendritic injury in the cortex and hippocampus. These results suggest that HIV-NCI persistence may require a continuing influx of inflammatory monocytes into the brain. Thus, we recommend buprenorphine as a potential therapy for mitigation of HIV brain disease in PWH with or without OUD.</p

Infected mice lacking T cells develop cognitive impairment and have elevated number of monocytes/macrophages in the brain.

<p><b>A.</b> Control and EcoHIV-infected nude mice with or without ART were tested 24 h after fear conditioning training for contextual fear response and 24 h later for cued fear response. *P<0.05, **P<0.01, EcoHIV vs. EcoHIV+ART; #P<0.05, EcoHIV vs. PBS. <b>B.-D.</b> Flow cytometry analysis of macrophage or monocytes in brains of nude mice with EcoHIV, EcoHIV with cART or PBS showing representative dot plots. Cell populations were gated based on isotype control antibodies. <b>B.</b> Staining for CD45 and CD11b and negative for Ly-6G/C. <b>C.</b> Staining for CD45, CD11b and Ly-6C and negative for Ly-6G. <b>D.</b> As determined by flow cytometry using 5 mice per group, the number of cells in each population per total mouse brain are shown. <b>E.</b> Total and integrated vDNA was measured by QPCR and nested QPCR, respectively, in infiltrating leukocytes isolated from the brain of EcoHIV-infected mice with and without cART. *P<0.05, **P<0.01, ***P<0.001.</p