- Email: [email protected]
Characterizing the Alzheimer's disease prefrontal cortex transcriptome by multiple RNA-sequencing
P574
Poster Presentations: P3
reprocessing. Transport mechanisms ferry cargoes over long distances along fragile highways, a process that appears inefficient and easily disrupted. In Alzheimer’s disease tau phosphorylation damages microtubules and axonal transport with disease propagation from entorhinal to other cortical regions and likely contributes to increased synaptic concentrations of aggregating amyloid beta (Braak H, et al. Curr Opin Neurol 2012;25:708). In the face of this vulnerable system, has evolutionary pressure generated better supply and waste management mechanisms? Results: Hypothesis Elaboration: Astrocytes far outnumber neurons and are principal candidates for this function. Astrocytic processes envelop neurons, make synaptic connections and act as intermediaries between blood and brain. Astrocytes may support neuronal networks through previously unappreciatedmechanisms, particularly local intercellular bidirectional exchange of organelles, RNA and other products essential for pre-synaptic integrity. This possibility is supported by t he recent discovery of organelle transfer between different cell types through tunneling nanotubes and in-vitro demonstration of nanotube connections between immature neurons and astrocytes (Abonunit S. J Cell Sci 2012;125:1089. Wang X et al. PLOS ONE 2012;7:e47429). Conclusions: Tunneling nanotubes and other intercellular transport mechanisms remain virtually unexplored in brain as potential local suppliers of essential products to protected neurons. Determination of the presence and nature of these mechanisms will be of great importance in understanding both normal brain function as well as contributions of the brain’s support system to the pathogenesis of Alzheimer’s disease. P3-064
CHARACTERIZING THE ALZHEIMER’S DISEASE PREFRONTAL CORTEX TRANSCRIPTOME BY MULTIPLE RNA-SEQUENCING
Paul Ryvkin1, Yuk Yee Leung2, Isabelle Dragomir1, Theresa Schuck1, Gerard Schellenberg1, Steven Arnold3, Alice Chen-Plotkin1, Vivianna Van Deerlin1, Virginia Lee3, John Trojanowski3, Brian Gregory1, Li-San Wang1, 1 University of Pennsylvania, Philadelphia, Pennsylvania, United States; 2 University of Pennsylvania, Philadelphia, Pennsylvania, United States; 3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States. Contact e-mail: [email protected] Background: The goal of this project is to elucidate what, if any, transcriptomic differences are associated with the Alzheimer’s disease state in the prefrontal cortex. Specifically, not only would we would like to determine which mRNAs are differentially expressed, but we would also like to examine differences in all non-coding RNAs (short and long), including those that are not currently annotated. Methods: Total RNA was extracted from human prefrontal cortex samples. Using a modified version of the Illumina strand-specific small RNA sequencing protocol, two types of cDNA libraries were produced. The first set of libraries are simply small RNAs as indicated by Illumina’s protocol. The second set comprise a whole transcriptome library, where total RNA was subjected to a Ribominus treatment in order to deplete ribosomal RNA, and then fragmented before being used in the strand-specific (RNA ligation) protocol. Results: We obtained a total of 31,000,000 unique reads covering the small RNA transcriptomes of 7 distinct samples. Among the small RNAs we were able to quantify are microRNAs, tRNA-fragments, and small RNAs derived from a variety of non-coding RNAs in the cell. Sixteen of the microRNAs are differentially expressed in the AD prefrontal cortex. We also found that many tRNA-derived small RNAs are differentially expressed in AD. Intriguingly, we found 42 completely unannotated small RNA loci that are differentially expressed in AD. In addition, we obtained70,000,000 unique reads covering the non-ribosomal RNA transcriptome across the same samples. Using this data we inferred that 121 proteincoding mRNAs and 66 non-coding RNAs are differentially expressed in AD. Overrepresented among the ncRNAs are molecules such as snoRNAs. Conclusions: The differential expression of tRNA-derived RNAs merits further investigation as these show the greatest degree of difference. The mRNAs found to be different in the whole transcriptome are highly consistent with what is known about the AD state, but there are several interesting new candidates that have not been reported, including several noncoding RNAs. P3-065
WITHDRAWN
P3-066
THE INCREASE OF BRI2 IN EARLY STAGES OF ALZHEIMER’S DISEASE CORRELATES WITH REDUCED LEVELS OF ADAM10 AND MAY PREVENT ITS BINDING TO APP
Marta Del Campo Milan1, Jeroen Hoozemans2, Carsten Korth3, Andreas M€uller-Schiffmann3, Philip Scheltens4, Marinus A. Blankenstein4, Connie Jimenez4, Rob Veerhuis4, Charlotte Teunissen5, 1VU Medical Center, Amsterdam, Netherlands; 2Vrije University Medisch Centrum, Amsterdam, Netherlands; 3Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany; 4VU University Medical Center, Amsterdam, Netherlands; 5Alzheimer Center, VU Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: BRI2 mutations are responsible of familial British and Danish dementias (FBD and FDD) causing amyloid deposits and neurofibrillary tangles similar to those observed in Alzheimer’s disease (AD). BRI2 binds APP downregulating the production of amyloid- b. We previously observed increased levels of BRI2 and deposition of a 50kDa wild-type BRI2 form in sporadic AD human hippocampus compared to controls suggesting involvement of BRI2 in AD pathology. Here, we further analyzed in which stage of the disease BRI2 is increased as well as the possible causes of BRI2 accumulation by analyzing the levels of furin, ADAM10 and SPPL2b, the enzymes involved in BRI2 processing. Additionally, we investigated whether BRI2-APP binding is preserved in human AD hippocampus. Methods: Post-mortem human hippocampus homogenates from AD patients (n ¼ 14) and controls (n¼14) were analyzed by western blot. Polyclonal and monoclonal antibodies against a BRI2 140-153 were produced by Biogenes (Germany) and their specificity has been fully characterized. Anti-FurinB6 (Santa Cruz), anti-ADAM10 ab1997 (Abcam), anti-SPPL2b (Aviva System Biology), anti-APP-22C11 (Millipore) were used. Immunopurification was performed using anti- BRI2 140-153 and anti-BRI2 113-231 (Dr. J.Presto). Results: BRI2 was increased in early pathological stages (Braak-III; Thal-2/3)of AD. While the levels of furin and ADAM10 were decreased in AD human hippocampus, the levels of SPPL2b were increased up to 10-times in AD patients compare to controls. The data revealed a correlation between the levels of ADAM10 with the levels both furin (r¼0.67,p<0.0001) and BRI2 (r¼0.42,p ¼0.02). BRI2-APP complexes were observed in the hippocampus homogenates from control cases (n¼4) but not in AD patients (n¼4). Conclusions: Our data show that BRI2 is increased in early stages of AD, supporting the potential involvement of BRI2 in AD pathology development. The observed inverse correlation between ADAM10 and BRI2 suggest that reduced ADAM10 levels and activity determines BRI2 increased. Processing by ADAM10 leads to the release of the BRI2 ectodomain and may thus promote the observed BRI2 deposition in plaques. Deposition of BRI2 may be the cause of the lack of BRI2-APP complexes in AD. These results strongly suggest that human BRI2 is a relevant player in the early steps of the amyloid cascade and thus, on AD etiology. P3-067
NEURONAL LEPTIN RESISTANCE IN ALZHEIMER’S DISEASE
David Bonda1, Jeremy Stone1, Sandy Richardson1, Sandra Siedlak1, George Perry2, Gemma Casadesus1, Mark Smith3, Xiongwei Zhu3, Hyoung-gon Lee1, 1Case Western Reserve University, Cleveland, Ohio, United States; 2University of Texas at San Antonio, San Antonio, Texas, United States; 3Case Western Reserve University, Cleveland, Ohio, United States. Contact e-mail: [email protected] Background: Recently, leptin signaling has received considerable attention in the Alzheimer disease (AD) field with its pathophysiological implication and therapeutic potential. Previously, leptin has been demonstrated to attenuate tau hyperphosphorylation in neuronal cells and has been shown to be modulated by amyloid-b Moreover, its role in neuroprotection and neurogenesis within the hippocampus has been shown in animal models. Therefore, the signaling pathway induced by leptin may play an important role in the pathogenesis of AD while its mechanism is unclear. To understand the mechanism, in this study, we investigated the status of leptin signaling pathway in AD brains. Methods: To further characterize the association between leptin signaling and vulnerable neurons in AD, we assessed the profile
Poster Presentations: P3
reprocessing. Transport mechanisms ferry cargoes over long distances along fragile highways, a process that appears inefficient and easily disrupted. In Alzheimer’s disease tau phosphorylation damages microtubules and axonal transport with disease propagation from entorhinal to other cortical regions and likely contributes to increased synaptic concentrations of aggregating amyloid beta (Braak H, et al. Curr Opin Neurol 2012;25:708). In the face of this vulnerable system, has evolutionary pressure generated better supply and waste management mechanisms? Results: Hypothesis Elaboration: Astrocytes far outnumber neurons and are principal candidates for this function. Astrocytic processes envelop neurons, make synaptic connections and act as intermediaries between blood and brain. Astrocytes may support neuronal networks through previously unappreciatedmechanisms, particularly local intercellular bidirectional exchange of organelles, RNA and other products essential for pre-synaptic integrity. This possibility is supported by t he recent discovery of organelle transfer between different cell types through tunneling nanotubes and in-vitro demonstration of nanotube connections between immature neurons and astrocytes (Abonunit S. J Cell Sci 2012;125:1089. Wang X et al. PLOS ONE 2012;7:e47429). Conclusions: Tunneling nanotubes and other intercellular transport mechanisms remain virtually unexplored in brain as potential local suppliers of essential products to protected neurons. Determination of the presence and nature of these mechanisms will be of great importance in understanding both normal brain function as well as contributions of the brain’s support system to the pathogenesis of Alzheimer’s disease. P3-064
CHARACTERIZING THE ALZHEIMER’S DISEASE PREFRONTAL CORTEX TRANSCRIPTOME BY MULTIPLE RNA-SEQUENCING
Paul Ryvkin1, Yuk Yee Leung2, Isabelle Dragomir1, Theresa Schuck1, Gerard Schellenberg1, Steven Arnold3, Alice Chen-Plotkin1, Vivianna Van Deerlin1, Virginia Lee3, John Trojanowski3, Brian Gregory1, Li-San Wang1, 1 University of Pennsylvania, Philadelphia, Pennsylvania, United States; 2 University of Pennsylvania, Philadelphia, Pennsylvania, United States; 3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States. Contact e-mail: [email protected] Background: The goal of this project is to elucidate what, if any, transcriptomic differences are associated with the Alzheimer’s disease state in the prefrontal cortex. Specifically, not only would we would like to determine which mRNAs are differentially expressed, but we would also like to examine differences in all non-coding RNAs (short and long), including those that are not currently annotated. Methods: Total RNA was extracted from human prefrontal cortex samples. Using a modified version of the Illumina strand-specific small RNA sequencing protocol, two types of cDNA libraries were produced. The first set of libraries are simply small RNAs as indicated by Illumina’s protocol. The second set comprise a whole transcriptome library, where total RNA was subjected to a Ribominus treatment in order to deplete ribosomal RNA, and then fragmented before being used in the strand-specific (RNA ligation) protocol. Results: We obtained a total of 31,000,000 unique reads covering the small RNA transcriptomes of 7 distinct samples. Among the small RNAs we were able to quantify are microRNAs, tRNA-fragments, and small RNAs derived from a variety of non-coding RNAs in the cell. Sixteen of the microRNAs are differentially expressed in the AD prefrontal cortex. We also found that many tRNA-derived small RNAs are differentially expressed in AD. Intriguingly, we found 42 completely unannotated small RNA loci that are differentially expressed in AD. In addition, we obtained70,000,000 unique reads covering the non-ribosomal RNA transcriptome across the same samples. Using this data we inferred that 121 proteincoding mRNAs and 66 non-coding RNAs are differentially expressed in AD. Overrepresented among the ncRNAs are molecules such as snoRNAs. Conclusions: The differential expression of tRNA-derived RNAs merits further investigation as these show the greatest degree of difference. The mRNAs found to be different in the whole transcriptome are highly consistent with what is known about the AD state, but there are several interesting new candidates that have not been reported, including several noncoding RNAs. P3-065
WITHDRAWN
P3-066
THE INCREASE OF BRI2 IN EARLY STAGES OF ALZHEIMER’S DISEASE CORRELATES WITH REDUCED LEVELS OF ADAM10 AND MAY PREVENT ITS BINDING TO APP
Marta Del Campo Milan1, Jeroen Hoozemans2, Carsten Korth3, Andreas M€uller-Schiffmann3, Philip Scheltens4, Marinus A. Blankenstein4, Connie Jimenez4, Rob Veerhuis4, Charlotte Teunissen5, 1VU Medical Center, Amsterdam, Netherlands; 2Vrije University Medisch Centrum, Amsterdam, Netherlands; 3Heinrich-Heine-Universit€at D€usseldorf, D€usseldorf, Germany; 4VU University Medical Center, Amsterdam, Netherlands; 5Alzheimer Center, VU Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: BRI2 mutations are responsible of familial British and Danish dementias (FBD and FDD) causing amyloid deposits and neurofibrillary tangles similar to those observed in Alzheimer’s disease (AD). BRI2 binds APP downregulating the production of amyloid- b. We previously observed increased levels of BRI2 and deposition of a 50kDa wild-type BRI2 form in sporadic AD human hippocampus compared to controls suggesting involvement of BRI2 in AD pathology. Here, we further analyzed in which stage of the disease BRI2 is increased as well as the possible causes of BRI2 accumulation by analyzing the levels of furin, ADAM10 and SPPL2b, the enzymes involved in BRI2 processing. Additionally, we investigated whether BRI2-APP binding is preserved in human AD hippocampus. Methods: Post-mortem human hippocampus homogenates from AD patients (n ¼ 14) and controls (n¼14) were analyzed by western blot. Polyclonal and monoclonal antibodies against a BRI2 140-153 were produced by Biogenes (Germany) and their specificity has been fully characterized. Anti-FurinB6 (Santa Cruz), anti-ADAM10 ab1997 (Abcam), anti-SPPL2b (Aviva System Biology), anti-APP-22C11 (Millipore) were used. Immunopurification was performed using anti- BRI2 140-153 and anti-BRI2 113-231 (Dr. J.Presto). Results: BRI2 was increased in early pathological stages (Braak-III; Thal-2/3)of AD. While the levels of furin and ADAM10 were decreased in AD human hippocampus, the levels of SPPL2b were increased up to 10-times in AD patients compare to controls. The data revealed a correlation between the levels of ADAM10 with the levels both furin (r¼0.67,p<0.0001) and BRI2 (r¼0.42,p ¼0.02). BRI2-APP complexes were observed in the hippocampus homogenates from control cases (n¼4) but not in AD patients (n¼4). Conclusions: Our data show that BRI2 is increased in early stages of AD, supporting the potential involvement of BRI2 in AD pathology development. The observed inverse correlation between ADAM10 and BRI2 suggest that reduced ADAM10 levels and activity determines BRI2 increased. Processing by ADAM10 leads to the release of the BRI2 ectodomain and may thus promote the observed BRI2 deposition in plaques. Deposition of BRI2 may be the cause of the lack of BRI2-APP complexes in AD. These results strongly suggest that human BRI2 is a relevant player in the early steps of the amyloid cascade and thus, on AD etiology. P3-067
NEURONAL LEPTIN RESISTANCE IN ALZHEIMER’S DISEASE
David Bonda1, Jeremy Stone1, Sandy Richardson1, Sandra Siedlak1, George Perry2, Gemma Casadesus1, Mark Smith3, Xiongwei Zhu3, Hyoung-gon Lee1, 1Case Western Reserve University, Cleveland, Ohio, United States; 2University of Texas at San Antonio, San Antonio, Texas, United States; 3Case Western Reserve University, Cleveland, Ohio, United States. Contact e-mail: [email protected] Background: Recently, leptin signaling has received considerable attention in the Alzheimer disease (AD) field with its pathophysiological implication and therapeutic potential. Previously, leptin has been demonstrated to attenuate tau hyperphosphorylation in neuronal cells and has been shown to be modulated by amyloid-b Moreover, its role in neuroprotection and neurogenesis within the hippocampus has been shown in animal models. Therefore, the signaling pathway induced by leptin may play an important role in the pathogenesis of AD while its mechanism is unclear. To understand the mechanism, in this study, we investigated the status of leptin signaling pathway in AD brains. Methods: To further characterize the association between leptin signaling and vulnerable neurons in AD, we assessed the profile