Fig 2: Synaptosomal expression of PLD1 (A, C), not PLD2 (B, D), is increased in the hippocampal and temporal cortex in AD patients (black bars) compared to age-matched controls (hollow bars). After the synaptosomes were prepared from the appropriate brain regions of the hippocampus (A, B), temporal cortex (C, D), and frontal cortex (E, F), the samples were coded and provided to the experimenter to perform the Western blots, resulting in the patterns of loading seen in each panel. (A) Hippocampal PLD1 expression of the AD group (2.791 ± 0.093, n = 4) was increased ~50% (*P < .05) compared to controls (1.387 ± 0.115, n = 4). Likewise, (C) AD temporal cortex PLD1 levels (1.597 ± 0.358, n = 3) were significantly increased (*P < .05) compared to controls (0.571 ± 0.038, n = 3). Interestingly, (E) AD frontal cortex PLD1 levels (0.022 ± 0.003, n = 4) were significantly lowered ~50% (*P < .05) compared to controls (0.046 ± 0.005, n = 4). In contrast, PLD2 levels in the (B) hippocampus (AD: 0.079 ± 0.005, n = 4, vs. control: 0.136 ± 0.120, n = 4; P = .34) and (D) temporal cortex (AD: 0.672 ± 0.219, n = 3, vs. control: 1.081 ± 0.241, n = 3; P = .21) were not significantly different, whereas the (F) frontal cortex (AD: 0.004 ± 0.001, n = 4, vs. control: 0.014 ± 0.002, n = 4; *P < .05) expressed low levels similar to frontal cortex PLD1 expression. Each lane in the blots above the graph represents the protein sample from one human subject brain area. A total of 11 AD and 8 control brain regions were used (details provided in Table 1). * indicates P < .05 compared to controls. Statistical significance was assessed using nonparametric analysis to account for the sample size and distribution using Mann-Whitney U post hoc analysis. Abbreviations: AD, Alzheimer's disease; PLD1, phospholipase D isoform 1; PLD2, phospholipase D isoform 2.

Fig 3: The PLD1 inhibitor injected ip following oAß (A, B) or otau (C, D) icv prevents the respective oligomer-induced memory deficits. Male and female C57Bl/6 mice were habituated, trained, and tested as shown in the gray panel. NOR memory for 2 hours (A, C) and 24 hours (B, D) was tested using (A and B) either vehicle (0.9% saline, 1 mg/mL ip) or PLD1 inhibitor (VU0155069, 10 mg/kg ip) injected 2 hours after oAß (3 µL of a 95-µM stock icv) or vehicle (ACSF, 3 µL icv) and (C and D) either vehicle (0.9% saline, 1 mg/mL ip) or PLD1 inhibitor (VU0155069, 10 mg/kg ip) injected 24 hours after otau (3 µL of a 1-µM stock icv) or vehicle (ACSF, 3 µL icv). (A) After 2 hours of training, animals injected with saline and given ACSF icv (white bar, 0.811 ± 0.087, n = 6) spent more time with the novel object, compared to the older object, whereas the animals treated with oAß (cross-hatched bar: 0.497 ± 0.0551, n = 6) showed no discrimination between the two objects. While the PLD1 inhibitor–treated animals (horizontal bars, 0.820 ± 0.086, n = 6) showed no deficits, the PLD1 inhibitor treatment before oAß icv (vertical bars, 0.821 ± 0.051, n = 6) was effective in preventing the memory deficit mediated by oAß. (B) The same group of animals were tested 24 hours after their training session, using a new (round) novel object—the control group of animals still showed increased time in the novel object zone (vehicle + saline: 0.852 ± 0.055, n = 6), while the oAß group showed no discrimination between the new and the old object (oAß + saline: 0.446 ± 0.093, n = 6). There was no difference in the NOR memory at 24 hours following PLD1 inhibitor treatment with (oAß + PLD1 inhibitor: 0.864 ± 0.076, n = 6) or without oAß (vehicle + PLD1 inhibitor: 0.870 ± 0.041, n = 6). (C) After 2 hours of training, animals injected with saline and given ACSF icv (gray bar, 0.621 ± 0.0432, n = 6) spent more time with the novel object, compared to the older object, whereas the animals treated with otau (cross-hatched bar: 0.494 ± 0.016, n = 6) showed no discrimination between the two objects. Again, PLD1 inhibitor treatment, by itself, did not affect NOR (horizontal bars: 0.605 ± 0.004, n = 6) but was effective in preventing otau-mediated NOR deficits (vertical bars: 0.600 ± 0.040, n = 6). (D) After 24 hours to their training session, the control group of animals still spent more time in the novel object zone (vehicle + saline: 0.607 ± 0.052, n = 6), whereas the otau group showed no discrimination between the new and the old object (otau + saline: 0.489 ± 0.048, n = 6). Once again, PLD1 inhibitor treatment was effective after 24 hours in preventing the otau-driven memory deficits (otau + PLD1 inhibitor: 0.598 ± 0.036, n = 6) while having no detrimental effect when applied without any otau (vehicle + PLD1 inhibitor: 0.602 ± 0.006, n = 6). For each data point, n = 6 animals (3 males and 3 females), and there were no sex differences observed. Statistical significance was assessed using nonparametric one-way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test. * indicates P < .05 compared to control/PLD1 Inhibitor treated/oligomer + PLD1 Inhibitor treated. Abbreviations: Aß, amyloid ß; ANOVA, analysis of variance; ACSF, artificial cerebrospinal fluid; icv, intracerebroventricularly; ip, intraperitoneal; NOR, novel object recognition; oAß, oligomeric Aß; otau, oligomeric tau species; PLD1, phospholipase D isoform 1.

Fig 4: GRA7-I and -III showed anti-mycobacterial activity against MTB challenge in vivo.Schematic of the TB model treated with rGRA7 or vehicle (A, upper). (A) Bacterial loads in lung, liver, and spleen in PKCa+/+ and PKCa-/- mice. n = 10. (B) Histopathology scores were obtained from H&E stained lung sections (left), as described in Methods. Number of granulomas observed in 10 different lung sections per mouse (right). Scale bar, 500 µm. (C) Bacterial loads in lung, liver, and spleen in PLD1+/+ and PLD1-/- mice. n = 10. (D) Number of granulomas observed in 10 different lung sections per mouse. Black bars represent the median values. Student’s t-test and Grubbs’ outlier test were used for statistical analysis. The data are representative of two independent experiments with similar results. Significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with PKCa+/+ and PLD1+/+.

Fig 5: Amygdala crude synaptosomal membrane phospholipase D (PLD)1/PLD2 isoforms show elevated phosphorylation states in the Day-1, while both phosphorylation and expression levels are increased in the Day-14 drug-free states in the paired group following conditioned hyperactivity (CH). 5-HT2CR expression remains unchanged in Day-1 and increases in the Day-14 drug-free state in the paired group following CH. Results represent the percent change in expression of the unpaired and paired compared to control group (±SEM). For both Day-1 (clear background) and Day-14 (dark background) drug-free states, increased levels of phosphorylation were observed in the paired group of animals (cross-hatch bars) compared to unpaired (slanted bars) at the [B] pPLD1T147 (middle panel), pPLD1S561 [last panel] in PLD1 isoform and [C] pPLD2Y169 (last panel) in the PLD2 isoform. Interestingly, crude synaptosomal levels for both PLD1 (B, first panel) and PLD2 (C, first panel) were increased by 50% in Day-14 (dark background), but not Day-1 (clear background) drug-free states in the paired (hatched bars) compared to the unpaired (slanted bars) group. 5-Hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expression in the paired group (A, hatched bars) is elevated only in the Day-14 (dark background), not in the Day-1 (clear background), drug-free state compared with the unpaired group (slanted bars). # P<.05 vs paired; *P<.05 vs unpaired; n=8 animals/group.