Fig 2: Autophagy associated gene expression from microarrays in three brain regions of HIV-infected subjects. Heat maps are shown for the indicated genes from (A) frontal lobe white matter, (B) frontal cortex, and (C) basal ganglia of subjects that were HIV-negative (A1–A6), HIV-positive (B1–B6), HIV-positive with neurocognitive impairment (NCI; C1–C7), and HIV-positive with combined NCI and HIV encephalitis (HIVE; D1–D5). IDs of individual probesets included in the array for each gene are given in parenthesis. (D) A significant difference of RMA probeset intensity values from the microarray data analyses was found in the basal ganglia for LAMP1 (201553_s_at) between HIV-negative (Group A) and NCI-HIVE (Group D) subjects; *p < 0.05. Error bars show the SEM.

Fig 3: Autophagy associated protein immunoreactivity in HIV-infected brain tissue. (A) Representative images from five randomly selected fields of cells each examined in duplicate frontal lobe white matter sections for the indicated subject groups. The indicated proteins were labeled red and microglia with the cell-type-specific marker Iba1 (green). Blue staining indicates cell nuclei. Arrow heads indicate examples of higher Iba1 immunoreactivity whereas arrows indicate more focal (punctal) vs. diffuse (filamentous) patterns of autophagy associated protein expression. Scale bar = 10 µm. (B) Quantification of relative Iba1 immunoreactivity from (A). F(3,20) = 6.450, p = 0.0031; *p < 0.05 when compared to all other subject groups. Error bars show the SEM for the average values of 2–6 regions from each subject group across the six autophagy associated proteins examined. (C) Quantification of the indicated autophagy associated protein relative immunoreactivity from (A). Beclin 1: F(3,12) = 11.29, p = 0.0008; LC3B: F(3,12) = 1.994, p = 0.1687; APG7/ATG7: F(3,12) = 84.20, p = < 0.0001; ATG5: F(3,12) = 6.218, p = 0.0086; p62/SQSTM1: F(3,12) = 87.04, p = < 0.0001; LAMP1: F(3,12) = 8.317, p = 0.0029. *p < 0.05 when compared to HIV-negative; #p < 0.05 when compared to HIV-positive; and Op < 0.05 when compared to HIV-positive/NCI subjects. Error bars show the SEM for four regions from each subject group.

Fig 4: Macrophage nanoparticle uptake and subcellular distribution. Uptake and subcellular distribution of nanoparticles was determined in human MDM (monocyte-derived macrophage). EuCF-DTG and FA-EuCF-DTG nanoparticles were detected in cells at 2 h. EuCF-DTG and FA-EuCF-DTG nanoparticles were added to MDM culture at a concentration of 5 µg/mL iron (cytotoxicity tests available in Figure S6). (A) Iron concentrations in MDM following nanoparticle uptake over 12 h and (B) corresponding DTG levels; data represent mean ± SEM (n = 3). Statistical differences were determined using one-way ANOVA among groups followed by Student's t-test for differences between groups at each time-point, ****p < 0.0001. (C) Intracellular nanoparticles were detected by confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510/520 nm (see Figure S7 for time-dependent uptake of nanoparticles). (D) For subcellular distribution analysis, MDM were treated with EuCF-DTG nanoparticles (5 µg/mL based on iron; green) for 8 h and then immunostained with Rab7, Rab11, Rab14 and LAMP-1 antibodies and Alexa Fluor 594-labeled secondary antibody (red) to visualize nanoparticle and organelle co-registration. The yellow (merged) shows overlap of nanoparticles and Rab compartments. DAPI (blue) stain indicates cell nuclei. Images were captured with 63X objective on a Zeiss LSM 710 confocal microscope. Scale bars = 20 µm (low-power images are available in Figure S8). (E-H) TEM ultrastructural evaluation of macrophage nanoparticle uptake and subcellular distribution. Nanoparticles were added to MDM cultures for 8 h. Cells were fixed and processed for TEM. (E) Typical internal morphology of control macrophages is shown. Detailed evaluation of membrane-bound intracellular structures at areas of interest is presented in magnified panel ii and iii. (F-H) Intracellular uptake of (F) PCL-DTG (without EuCF), (G) EuCF-DTG and (H) FA-EuCF-DTG nanoparticles. Areas of interest bordered with dotted red lines are presented in corresponding high-resolution images (ii-iii) and illustrate nanoparticles within membrane-bound intracellular structures. All nanoparticle types were internalized and entrapped in endosomal vesicles in the macrophages. Images of macrophages treated with FA-functionalized nanoparticles reveal a higher number of nanoparticles internalized in vesicles compared to non-decorated particles. EuCF-DTG and FA-EuCF-DTG nanoparticles are seen as black punctate structures encapsulated in white polymeric nanoparticles within membrane-bound endosomes (Figure 2G-H and Figure S4 show surface morphology by SEM of cells after nanoparticle treatment).

Fig 5: Locked N-terminal domain NPC1 rescues cholesterol export from lysosomes.(A) Domain structure of NPC1 protein. The N-terminal domain (residues 23–259 including the polyproline linker), middle lumenal domain (MLD, 372–620), and C-terminal domain (CTD, 854–1098) are colored red, blue, and yellow, respectively. (B) NPC1 residues mutated to Cys for disulfide bond formation between the polyproline linker and CTD (see inset). The location of the sterol-sensing domain is shown in orange; P691 faces the back. (C) Confocal immunofluorescence microscopy of mouse NPC1 P251C/L929C and LAMP1 proteins expressed in HeLa cells (bar, 20 µm). (D) Confocal immunofluorescence microscopy of cholesterol accumulation rescue. NPC1-/- HeLa cells were transfected with GFP-mouse NPC1-wild type or P251C/L929C plasmids for 48 h. Thirty-two hours post transfection, cells were incubated with 1 µM U18666a for 16 h; cells were briefly incubated with 10 mM methylamine hydrochloride and chased for cholesterol export for 1 h in 5% LPDS medium, followed by immediate fixation. Intrinsic GFP fluorescence and AF647-PFO* labeling are shown (bar, 20 µm). Images represent maximum intensity projections. (E) Flow cytometry of the experiment shown in (D). GFP-positive cells of similar intensity were analyzed: NPC1-/-, 497 cells; NPC1 wild type, 478 cells; P251/L929C, 1486 cells. Cell numbers were normalized for comparison. (F) Flow cytometry of a rescue experiment using the indicated constructs, carried out as in (E). GFP-positive cells were analyzed: NPC1-/-, 2968 cells; NPC1 wild type, 2358 cells; P251/L929C/P249K/P259K, 3906 cells. Flow cytometry analyzed only GFP-positive cells of similar intensity.