UB Breakthroughs Summer 2016

The UB Breakthroughs newsletter for summer of 2016. This issue contains articles discussing Dr. Faezipour's research into a smartphone app for skin cancer detection, Dr. Katsifis' research into the mutagenic and carcinogenic effects of heavy metals, Dr. Oberleitner’s research into the link between social isolation and exclusion and physical and emotional pain, Dr. Lee’s classes and camps teaching college and high school students big data analytics, professor Good’s study into teaching chiropractic warm-up with resistance bands, professor Brett’s research into the safety and efficacy of electro-acupuncture, Dr. Picardi’s research into employee and employer perceptions and how to create better matches in employment, Dr. Richmond’s new book examining African-American student activism in the northeast from the 1960s through 2015, Dr. Xiong’s new MEMS-based sensor for detecting miniscule air pollutants, UB’s 3-D Printing and Advanced Manufacturing Center, Dr. Wei’s study of China and international relations regarding the South China Sea, and Dr. Pallis’ support of the UB CanSat Competition team

The Concerted Regulation of Intracellular Signaling by Amyloid Precursor Protein and Aβ Peptide

It is widely accepted that A-beta (Aβ) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in the Alzheimer’s disease (AD) brain, yet little is known about the contribution of APP preceding AD pathogenesis. Our data presented here suggest that APP has a functional role in cell cycle regulation and proliferation. First, we demonstrat that APP is pathologically phosphorylated at Thr668 and that P-APP localizes to the centrosomes. Furthermore, P-APP is proteolytically processed in a cell cycle -dependent manner to generate its pathogenic metabolites. Using Stable Isotope Labeling by Amino Acids in Culture (SILAC) and mass spectrometry analyses, we also show that expression of APP results in the expression of proliferation-associated proteins and the phosphorylation of proteins associated with cell cycle regulation and transcription. Here, we demonstrate that APP expression and oligomeric Aβ42 elicit Ras/ERK signaling cascade and glycogen synthase kinase3 (GSK3) activation. Both ERK and GSK3 are known to induce hyperphosphorylation of tau and of APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human brain samples show increased expression of Ras, activation of GSK3 and phosphorylation of APP and tau, which correlate with Aβ levels in the AD brains. Furthermore, treatment of primary rat neurons with Aβ recapitulate these events and show enhanced Ras-ERK signaling, GSK3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Aβ induces Thr668 phosphorylation on APP, which we show enhances APP proteolysis and Aβ generation, denotes a vicious feed-forward mechanism by which APP and Aβ promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and an inhibition of this would impede the mitotic catastrophe and neurodegeneration observed in AD

The Concerted Regulation of Intracellular Signaling by Amyloid Precursor Protein and Aβ Peptide

It is widely accepted that A-beta (Aβ) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in the Alzheimer’s disease (AD) brain, yet little is known about the contribution of APP preceding AD pathogenesis. Our data presented here suggest that APP has a functional role in cell cycle regulation and proliferation. First, we demonstrat that APP is pathologically phosphorylated at Thr668 and that P-APP localizes to the centrosomes. Furthermore, P-APP is proteolytically processed in a cell cycle -dependent manner to generate its pathogenic metabolites. Using Stable Isotope Labeling by Amino Acids in Culture (SILAC) and mass spectrometry analyses, we also show that expression of APP results in the expression of proliferation-associated proteins and the phosphorylation of proteins associated with cell cycle regulation and transcription. Here, we demonstrate that APP expression and oligomeric Aβ42 elicit Ras/ERK signaling cascade and glycogen synthase kinase3 (GSK3) activation. Both ERK and GSK3 are known to induce hyperphosphorylation of tau and of APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human brain samples show increased expression of Ras, activation of GSK3 and phosphorylation of APP and tau, which correlate with Aβ levels in the AD brains. Furthermore, treatment of primary rat neurons with Aβ recapitulate these events and show enhanced Ras-ERK signaling, GSK3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Aβ induces Thr668 phosphorylation on APP, which we show enhances APP proteolysis and Aβ generation, denotes a vicious feed-forward mechanism by which APP and Aβ promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and an inhibition of this would impede the mitotic catastrophe and neurodegeneration observed in AD

Altered Processing of Amyloid Precursor Protein in Cells Undergoing Apoptosis

<div><p>Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25–35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease.</p> </div

Potential Role of PCTAIRE-2, PCTAIRE-3 and P-Histone H4 in Amyloid Precursor Protein-dependent Alzheimer Pathology

Amyloid Precursor Protein (APP) is regulated in a mitosis-specific manner and plays a role in proliferative signaling in cells. Though APP-derived Aβ generation has a well-established role in neurodegeneration, the mechanistic role of APP in this process is not fully understood. Here, we performed an unbiased, comprehensive analysis of the phosphoproteome signature in APP-null neuroblastoma cells (B103) compared to those expressing APP-695 isoform (B103-695) to determine if APP expression affects protein phosphorylation. Stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry-based phosphoproteomic analysis with PolyMAC identified a total of 2,478 phosphopeptides in the B103 and B103-695 cell culture model system. We observed that phosphorylation of PCTAIRE-2 (CDK17), PCTAIRE-3 (CDK18), and Histone H4 are significantly elevated in B103-695 cells; western blot analysis confirmed overexpression of PCTAIREs and increased phosphorylation of Histone H4. More importantly, analysis of primary neurons treated with Aβ, as well as brain samples from MCI (mild cognitive impaired) and AD patients recapitulated these results, showing increased levels of PCTAIREs and P-Histone H4. These novel findings identify a hitherto uncharacterized mechanism by which APP and/or Aβ may promote AD neurodegeneration, and raises the possibility that their inhibition may protect against pathology development in AD

CPT induced apoptosis of H4-APP cells is associated with the formation of novel APP fragments.

<p>(A) Lysates from H4-APP cells were separated on 10–20% tricine gel and western blot analysis was performed using 6E10 antibodies. The blot shows a time-dependent increase in the level of APP fragments in apoptotic cells (top panel labeled APP fragments). A short exposure of the blot shows that levels of full length APP are decreased in a time-dependent manner in CPT treated cells (second panel). The blot was re-probed with caspase-cleaved APP antibody, which shows a time-dependent increase in generation of the cleaved fragments in apoptotic cells. The lower panel shows the reprobe of the blot with an antibody against β-actin performed to show protein loading. (B) Lysates from H4-APP cells treated with CPT for one, three, and six hours were separated on a 15% tris-glycine gel and analyzed by western blot using 6E10 (top and second panel) as well as caspase-cleaved APP antibodies (third panel). 6E10 antibody shows the appearance of the cleaved fragment of APP (second panel) with a concomitant decrease in the levels of full length APP (top panel) after CPT treatment. Caspase-cleaved APP antibody shows a much stronger immunoreactivity to the proteolytic fragments (third panel). Probing for β-actin (lower panel) showed equal amount of protein loading on the gels. (C) Quantification of the bottom band detected by caspase-cleaved APP antibody, with normalization to actin, revealed a significant induction in formation of this band after six hours of CPT treatment, p = 0.018. (D) Quantification of the number of cells undergoing apoptosis in the presence and absence of caspase inhibitors and CPT, as analyzed by flow cytometry with propidium iodide staining. Asterisks indicate a significant difference between groups, p<0.05. (E) Western blot analysis of the H4-APP cells showed a strong reduction in the formation of the new fragments in the presence of Z-DEVD-FMK. In the presence of Z-VAD-FMK, the formation of these bands was completely abolished. Reprobe of these blots with an antibody against β-actin shows protein loading on gels. (F) Quantification of the bottom band detected by the caspase-cleaved APP antibody, normalized to actin, from three independent experiments, reveals significant attenuation in the formation of the fragments by caspase inhibitors, with the six hour CPT treated sample showing a significant difference from all other groups, p<0.05. Note that a logarithmic scale is used in this panel.</p

Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.

<p>(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057979#pone-0057979-g002" target="_blank">Figure 2A and 2B</a>.</p

Potential Role of PCTAIRE-2, PCTAIRE-3 and P-Histone H4 in Amyloid Precursor Protein-dependent Alzheimer Pathology

Amyloid Precursor Protein (APP) is regulated in a mitosis-specific manner and plays a role in proliferative signaling in cells. Though APP-derived Aβ generation has a well-established role in neurodegeneration, the mechanistic role of APP in this process is not fully understood. Here, we performed an unbiased, comprehensive analysis of the phosphoproteome signature in APP-null neuroblastoma cells (B103) compared to those expressing APP-695 isoform (B103-695) to determine if APP expression affects protein phosphorylation. Stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry-based phosphoproteomic analysis with PolyMAC identified a total of 2,478 phosphopeptides in the B103 and B103-695 cell culture model system. We observed that phosphorylation of PCTAIRE-2 (CDK17), PCTAIRE-3 (CDK18), and Histone H4 are significantly elevated in B103-695 cells; western blot analysis confirmed overexpression of PCTAIREs and increased phosphorylation of Histone H4. More importantly, analysis of primary neurons treated with Aβ, as well as brain samples from MCI (mild cognitive impaired) and AD patients recapitulated these results, showing increased levels of PCTAIREs and P-Histone H4. These novel findings identify a hitherto uncharacterized mechanism by which APP and/or Aβ may promote AD neurodegeneration, and raises the possibility that their inhibition may protect against pathology development in AD

UB Breakthroughs Summer 2016

The UB Breakthroughs newsletter for summer of 2016. This issue contains articles discussing Dr. Faezipour's research into a smartphone app for skin cancer detection, Dr. Katsifis' research into the mutagenic and carcinogenic effects of heavy metals, Dr. Oberleitner’s research into the link between social isolation and exclusion and physical and emotional pain, Dr. Lee’s classes and camps teaching college and high school students big data analytics, professor Good’s study into teaching chiropractic warm-up with resistance bands, professor Brett’s research into the safety and efficacy of electro-acupuncture, Dr. Picardi’s research into employee and employer perceptions and how to create better matches in employment, Dr. Richmond’s new book examining African-American student activism in the northeast from the 1960s through 2015, Dr. Xiong’s new MEMS-based sensor for detecting miniscule air pollutants, UB’s 3-D Printing and Advanced Manufacturing Center, Dr. Wei’s study of China and international relations regarding the South China Sea, and Dr. Pallis’ support of the UB CanSat Competition team

Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.

<p>(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.</p