- Email: [email protected]
O4-02-04
Oral O4-02: Disease Mechanisms (Others) WEDNESDAY, JULY 19, 2006 ORAL O4-02 DISEASE MECHANISMS (OTHERS) O4-02-01
ALTERED EXPRESSION OF APP GENE FAMILY MEMBERS IN DROSOPHILA CAUSE DEFECTS IN AXONAL TRANSPORT AND AFFECT SYNAPTIC PLASTICITY
Stefan Kins1, Patricia Rusu1, Anna Jansen2, Peter Soba1, Gunther Merdes1, Yung-Hui Kuan1, Anita Jung2, Konrad Beyreuther1, Ole Kjaerulff2, 1Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), Heidelberg, Germany; 2Division of Neurophysiology, Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark. Contact e-mail: [email protected] Alzheimer’s disease (AD) is characterised by neurofibrillary tangles and extracellular plaques, which consist mainly of -amyloid derived from the -amyloid precursor protein (APP). Additional features of AD are axonal transport defects, which might contribute to impairment of cognitive functions. Axonal transport defects have also been reported in AD animal models, including mice and flies that overexpress APP and Tau. Here we demonstrate that APP-induced traffic jam of vesicles in peripheral nerves of Drosophila larvae depends on the NPTY motif in the APP intracellular domain. Furthermore, heterologous expression of JIP1b, an adaptor protein, which is able to bind to the NPTY-motif, also perturbs axonal transport indicating that JIP1b may be involved in the APP-induced axonal transport defect. Based on the assumption that APP-mediated transport defects might affect synaptic function, we have characterised neurotransmission at the neuromuscular junction in larvae that overexpress human APP, and in larvae missing APPL (the Drosophila homolog of APP), which display very similar axonal transport defects. We found that evoked responses were reduced and paired-pulse facilitation enhanced in APPL-deficient mutants but not in APP overexpressing flies. Moreover, post-tetanic potentiation was prolonged both by loss of APPL and overexpression of APP, possibly reflecting a defect in the homeostasis of intracellular Ca2⫹. Our results support the hypothesis that the APP gene family has essential functions in neurotransmission and that normal synaptic plasticity is affected by the APP/APPL-perturbed axonal transport. Thus, functional synaptic changes resulting from APP-related transport defects might affect cognitive function early in the course of AD. O4-02-02
PRESENILIN’S (PS) ROLE IN KINESIN AND AMYLOID PRECURSOR PROTEIN (APP) MEDIATED TRANSPORT PATHWAYS
Shermali Gunawardena, Larry Goldstein, University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Our previous work suggests that both APP and Drosophila APPL function as kinesin receptors in axonal transport, and that excess APP causes neuronal cell death. Recently, secretases (BACE and PS) responsible for the generation of pathogenic A were suggested to be present within APP vesicles transported by kinesin. Perhaps neuronal death results from aberrant cleavage of APP within blockages resulting in A toxicity. Biochemical analysis indicates that APP can undergo cleavage in Drosophila. Strikingly, PS appears to influence both the kinesin and APP-mediated transport pathways. Reducing the dose of PS with excess APP leads to the suppression of APP-mediated axonal blockages and neuronal death. Surprisingly, reducing the dose of nicastrin or APH-1 with APP did not suppress the APP-mediated transport phenotype, suggesting that the PS dependent suppression phenotype may not solely be due to PS’s gamma secretase activity. In vivo analysis of APP transport in PS reduced axons show increased anterograde transport of APP vesicles, and the velocities of
S77
APP vesicles traveling anterogradely and retrogradely increase compared to the transport of synaptotagmin vesicles, suggesting that reduction of PS directly affects APP transport. One possibility is that PS mediates the transport of APP by aberrant cleavage of APP. Alternatively, PS may independently influence the axonal transport of APP by directly affecting the transport machinery. To test this we increased the dose of PS with human APP. Surprisingly, increasing PS also led to the suppression of APP-mediated axonal blockages and neuronal death. Although excess PS alone had no effect, reducing the dose of kinesin (not dynein) with excess PS caused axonal blockages, but not neuronal death, suggesting that PS also affects the kinesin-mediated transport pathway. In vivo analysis of APP transport in PS increased axons also shows increased anterograde transport of APP vesicles, but a broad distribution of velocities for APP vesicles traveling anterograde or retrograde is observed. Perhaps excess PS may cleave APP to generate C-terminal fragments, which may be moving at different speeds. Thus we propose that while PS may also function in kinesin-mediated transport, APP cleavage may play a vital role in regulating APP vesicle movement within axons. O4-02-03
SYNAPSE FORMATION AND FUNCTION IS MODULATED BY THE AMYLOID PRECURSOR PROTEIN
Jochen W. Herms, Christina Priller, Thomas Bauer, Michael Mansour, Hans Kretzschmar, University Munich, 81377 Munich, Germany. Contact e-mail: [email protected] Background: APP processing has been shown to have a physiological role in synaptic transmission. A has been found to suppress excitatory synaptic transmission and it has been suggested that A generation may be part of a negative feedback mechanism that controls neuronal excitability (Kamenetz et al. Neuron 37:925-37; 2003). Objective: Here we aim to get to know, if lack of APP and consequently a total lack in the production of A peptides affect synaptic transmission as well. Methods: Patch-clamp analysis of excitatory synaptic transmission in autaptic hippocampal cultures from APP deficient mice. Results: Hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor mediated excitatory postsynaptic currents. Moreover, the size of the readily releasable synaptic vesicle pool was increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP while the amplitude of miniature synaptic currents was not found to be altered. Taken together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunhohistochemical analysis revealing an increase of synaptophysin positive puncta per cultured APP deficient neuron. Conclusion: Lack of APP affects synapse formation and transmission in cultured hippocampal neurons. O4-02-04
OPPOSING ROLES OF APP AND ABETA ON SYNAPTIC PLASTICITY
Matthew Townsend1, Dennis J. Selkoe2, 1Brigham & Women’s Hospital, Boston, MA, USA; 2Brigham & Women’s Hospital, Boston, USA. Contact e-mail: [email protected] Background: The normal function of APP is incompletely understood. Previous studies have shown that APP may have neurotrophic effects and injection of APPs can enhance memory function in mice. Moreover, the mechanisms by which A inhibits certain forms of synaptic plasticity remain unknown. Objectives: To study the acute effects of soluble APP and A on synaptic function and signal transduction cascades in wild-type mouse hippocampal neurons. Methods: We have used biochemical and electrophsyiological techniques on acute hippocampal slices or cultured primary neurons. Results: We now report that APP processing plays an essential role in the normal synaptic function of hippocampal neurons. Application of exogenous APPs to mouse hippocampal slices caused a
S78
Oral O4-02: Disease Mechanisms (Others)
rapid potentiation of glutamatergic synapses. APPs increased the amplitudes of evoked AMPA receptor currents and miniature EPSPs. This effect was blocked 1) by preclearing the perfusate with anti-APPs antibody; 2) by the G-protein antagonist, NF023; and 3) by secreted A. In view of these data, we tested multiple ␣, , and ␥-secretase inhibitors for effects on LTP, and found that acute application of several BACE antagonists inhibited LTP. This was rescued with exogenous APPs. Since oligomers of A inhibit the potentiation of synapses by APPs or high-frequency stimulation, we examined how A affects the signal transduction cascades that mediate LTP. While some kinases such as PKA and PKC were activated normally by an LTP-inducing stimulus, A prevented the activation of the Erk/ MAPK, CaMKII, and PI3K pathways. Using a panel of antagonists, we examined whether inhibition of any single cell-surface receptor could replicate the signal-transduction pattern seen with A. To date, antagonists to the mGluR and insulin receptor appear to replicate the pattern Amediated signal transduction inhibition. Conclusions: These data show that proteolytic processing of APP by BACE, is necessary for the normal expression of LTP. Furthermore, our results suggest that small, soluble A oligomers interfere selectively with certain signal transduction pathways necessary for LTP, offering a mechanism by which A could lead to subtle amnestic deficits early in AD. O4-02-05
TOTAL EXCHANGEABLE CU2ⴙ PROTEIN BINDING SITES ARE ELEVATED IN ALZHEIMER’S DISEASE
Simon A. James1,2, Jean-Francois Monty1, Paul A. Adlard1, Victor Villemagne1, Anthony G. Wedd2,3, Ashley I. Bush1,4, 1The Mental Health Research Institute of Victoria and the Department of Pathology, The University of Melbourne, Parkville, Australia; 2The School of Chemistry, The University of Melbourne, Parkville, Australia; 3 Bio21 Insitiute, Parkville, Australia; 4Genetics and Aging Unit, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA. Contact e-mail: [email protected] Background: Oxidative stress is central to many neurodegenerative conditions including Alzheimer’s Disease (AD). Emerging evidence indicates that inappropriate redox reactivity between copper/iron and molecular oxygen is responsible for the pathological production of ROS in AD. A is one established protein factor that fosters such pro-oxidant activity. Neurofibrillary tangles (NFT), composed of hyperphosphorylated tau, also coordinate Cu and Fe in a pro-oxidant manner. Essential copper-dependent enzyme activities (eg SOD1, cytochrome c oxidase, PAM) are decreased in AD. Because of the accumulation of copper binding proteins in AD, copper homeostasis may be significantly dysregulated. Objective: To interrogate interactions of total brain proteins with Cu(II) and Zn(II) in post-mortem brain samples from AD and age-matched controls. To explore any modification in the metal binding properties of tau between the AD and control samples. Methods: Brain samples from well-characterized donors were homogenized, and separated into soluble and pellet fractions. The soluble fraction was applied to Immobilised Metal Affinity Chromatography (IMAC) in order to separate proteins on the basis of affinity. The proteins were eluted using an imidazole gradient. The protein concentration of each fraction was measured. Western blots of each fraction were performed to probe for both total tau and phosphorylated tau before and after the removal of endogenous metals. Results and Conclusions: The total protein elution profile differs between AD and control brain samples (soluble fraction), indicating the greater abundance of higher affinity copper binding proteins in AD. Incubation of the homogenates with metal chelators prior to the application of the samples to the column produced a significant change in the elution profile for all samples, with further differences between the AD and control samples. Western blots documented a change in the elution profile of phosphorylated tau between the AD and controls.
O4-02-06
UPREGULATION OF RAB6A IN ALZHEIMER’S DISEASE IS CONNECTED TO ENDOPLASMIC RETICULUM STRESS
Wiep Scheper1, Jeroen J.M. Hoozemans1, Rob Zwart1, Casper C. Hoogenraad2, Annemieke Rozemuller1, Piet Eikelenboom1, Frank Baas1, 1Academic Medical Center, Amsterdam, The Netherlands; 2 Erasmus Medical Center, Rotterdam, The Netherlands. Contact e-mail: [email protected] Background: A major hallmark of Alzheimer’s disease (AD) is the accumulation of misfolded proteins. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in induction of the unfolded protein response, an initially protective response aimed to restore the homeostasis in the ER. However, prolonged stress leads to cell death and may thus be an important factor in neuronal loss during AD progression. We previously observed activation of the unfolded protein response in neurons in the temporal cortex and hippocampus of AD patients. The small GTPase Rab6a is involved in transport route from the Golgi back to the ER. We hypothesise that this trafficking route involves a post-ER quality control mechanism, by which aberrant proteins that have escaped ER quality control are transported back to the ER. Objective(s): In this study we investigated the role of Rab6A trafficking in connection to ER stress in AD pathogenesis. Methods: We used immuno-histochemistry and semi-quantitative Western blotting to determine Rab6A and ER stress levels in post-mortem brain samples of AD patients and non-demented controls. Mechanistic aspects of the relation between Rab6A trafficking and ER stress were studied in human neuroblastoma cells and an ER stress reporter cell line. Results: Rab6A is predominantly expressed in the pyramidal neurons in the temporal cortex and hippocampus. Western blot analysis of a panel of AD and control patients shows that Rab6A is strongly upregulated in AD brain from Braak stage III. Our data indicate that Rab6A upregulation precedes tangle formation. We show that the levels of the ER chaperone BiP/GRP78 correlate closely with those of Rab6A in human temporal cortex, suggesting a functional connection between Rab6A and ER stress. This is supported by evidence provided by experiments in cell culture, where increased Rab6A levels suffice to induce ER stress. Conclusions: Our data support a role of Rab6A and ER stress relatively early in AD pathogenesis. Targeting protein quality control therefore creates an interesting therapeutic window of opportunity. O4-02-07
MITOCHONDRIAL DYSFUNCTION AND ALZHEIMER’S DISEASE IN DOWN’S SYNDROME
Jorge Busciglio, Pablo Helguera, Jackeline Seiglie, UCI, Irvine, CA, USA. Contact e-mail: [email protected] Down’s Syndrome (DS) is the most frequent genetic cause of mental retardation. Most DS patients develop Alzheimer’s disease (AD) by middle age. The evidence available indicates that mitochondrial dysfunction is a critical element of DS: 1) mitochondrial dysfunction exists in DS neurons and astrocytes, which leads to aberrant amyloid precursor protein (APP) metabolism and intracellular amyloid beta accumulation; 2) there is increased oxidative stress in DS cells which promotes neuronal death by overexpression of the transcription factor Ets-2 through a p53-dependent, mitochondrial apoptotic pathway; and 3) there are structural and functional alterations in mitochondria of DS cells that may directly affect cell function and survival. We have initiated studies to determine the molecular elements that are involved and to delineate the specific nature of the mitochondrial disorder present in DS. These studies are focused on the role of intracellular amyloid beta in mitochondrial function, alterations in the activity of uncoupling proteins, and the role of Down Syndrome Critical Region 1 (DSCR1), which has been recently implicated as a key regulator of mitochondrial function. The results support the emerging view that different pathological mechanisms may converge into a chronic state of mitochondrial dysfunction in DS, leading to a persistent systemic deficit on cell capabilities and viability. Supported by NIH, The Alzheimer’s Association and UCI.
ALTERED EXPRESSION OF APP GENE FAMILY MEMBERS IN DROSOPHILA CAUSE DEFECTS IN AXONAL TRANSPORT AND AFFECT SYNAPTIC PLASTICITY
Stefan Kins1, Patricia Rusu1, Anna Jansen2, Peter Soba1, Gunther Merdes1, Yung-Hui Kuan1, Anita Jung2, Konrad Beyreuther1, Ole Kjaerulff2, 1Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), Heidelberg, Germany; 2Division of Neurophysiology, Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark. Contact e-mail: [email protected] Alzheimer’s disease (AD) is characterised by neurofibrillary tangles and extracellular plaques, which consist mainly of -amyloid derived from the -amyloid precursor protein (APP). Additional features of AD are axonal transport defects, which might contribute to impairment of cognitive functions. Axonal transport defects have also been reported in AD animal models, including mice and flies that overexpress APP and Tau. Here we demonstrate that APP-induced traffic jam of vesicles in peripheral nerves of Drosophila larvae depends on the NPTY motif in the APP intracellular domain. Furthermore, heterologous expression of JIP1b, an adaptor protein, which is able to bind to the NPTY-motif, also perturbs axonal transport indicating that JIP1b may be involved in the APP-induced axonal transport defect. Based on the assumption that APP-mediated transport defects might affect synaptic function, we have characterised neurotransmission at the neuromuscular junction in larvae that overexpress human APP, and in larvae missing APPL (the Drosophila homolog of APP), which display very similar axonal transport defects. We found that evoked responses were reduced and paired-pulse facilitation enhanced in APPL-deficient mutants but not in APP overexpressing flies. Moreover, post-tetanic potentiation was prolonged both by loss of APPL and overexpression of APP, possibly reflecting a defect in the homeostasis of intracellular Ca2⫹. Our results support the hypothesis that the APP gene family has essential functions in neurotransmission and that normal synaptic plasticity is affected by the APP/APPL-perturbed axonal transport. Thus, functional synaptic changes resulting from APP-related transport defects might affect cognitive function early in the course of AD. O4-02-02
PRESENILIN’S (PS) ROLE IN KINESIN AND AMYLOID PRECURSOR PROTEIN (APP) MEDIATED TRANSPORT PATHWAYS
Shermali Gunawardena, Larry Goldstein, University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Our previous work suggests that both APP and Drosophila APPL function as kinesin receptors in axonal transport, and that excess APP causes neuronal cell death. Recently, secretases (BACE and PS) responsible for the generation of pathogenic A were suggested to be present within APP vesicles transported by kinesin. Perhaps neuronal death results from aberrant cleavage of APP within blockages resulting in A toxicity. Biochemical analysis indicates that APP can undergo cleavage in Drosophila. Strikingly, PS appears to influence both the kinesin and APP-mediated transport pathways. Reducing the dose of PS with excess APP leads to the suppression of APP-mediated axonal blockages and neuronal death. Surprisingly, reducing the dose of nicastrin or APH-1 with APP did not suppress the APP-mediated transport phenotype, suggesting that the PS dependent suppression phenotype may not solely be due to PS’s gamma secretase activity. In vivo analysis of APP transport in PS reduced axons show increased anterograde transport of APP vesicles, and the velocities of
S77
APP vesicles traveling anterogradely and retrogradely increase compared to the transport of synaptotagmin vesicles, suggesting that reduction of PS directly affects APP transport. One possibility is that PS mediates the transport of APP by aberrant cleavage of APP. Alternatively, PS may independently influence the axonal transport of APP by directly affecting the transport machinery. To test this we increased the dose of PS with human APP. Surprisingly, increasing PS also led to the suppression of APP-mediated axonal blockages and neuronal death. Although excess PS alone had no effect, reducing the dose of kinesin (not dynein) with excess PS caused axonal blockages, but not neuronal death, suggesting that PS also affects the kinesin-mediated transport pathway. In vivo analysis of APP transport in PS increased axons also shows increased anterograde transport of APP vesicles, but a broad distribution of velocities for APP vesicles traveling anterograde or retrograde is observed. Perhaps excess PS may cleave APP to generate C-terminal fragments, which may be moving at different speeds. Thus we propose that while PS may also function in kinesin-mediated transport, APP cleavage may play a vital role in regulating APP vesicle movement within axons. O4-02-03
SYNAPSE FORMATION AND FUNCTION IS MODULATED BY THE AMYLOID PRECURSOR PROTEIN
Jochen W. Herms, Christina Priller, Thomas Bauer, Michael Mansour, Hans Kretzschmar, University Munich, 81377 Munich, Germany. Contact e-mail: [email protected] Background: APP processing has been shown to have a physiological role in synaptic transmission. A has been found to suppress excitatory synaptic transmission and it has been suggested that A generation may be part of a negative feedback mechanism that controls neuronal excitability (Kamenetz et al. Neuron 37:925-37; 2003). Objective: Here we aim to get to know, if lack of APP and consequently a total lack in the production of A peptides affect synaptic transmission as well. Methods: Patch-clamp analysis of excitatory synaptic transmission in autaptic hippocampal cultures from APP deficient mice. Results: Hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor mediated excitatory postsynaptic currents. Moreover, the size of the readily releasable synaptic vesicle pool was increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP while the amplitude of miniature synaptic currents was not found to be altered. Taken together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunhohistochemical analysis revealing an increase of synaptophysin positive puncta per cultured APP deficient neuron. Conclusion: Lack of APP affects synapse formation and transmission in cultured hippocampal neurons. O4-02-04
OPPOSING ROLES OF APP AND ABETA ON SYNAPTIC PLASTICITY
Matthew Townsend1, Dennis J. Selkoe2, 1Brigham & Women’s Hospital, Boston, MA, USA; 2Brigham & Women’s Hospital, Boston, USA. Contact e-mail: [email protected] Background: The normal function of APP is incompletely understood. Previous studies have shown that APP may have neurotrophic effects and injection of APPs can enhance memory function in mice. Moreover, the mechanisms by which A inhibits certain forms of synaptic plasticity remain unknown. Objectives: To study the acute effects of soluble APP and A on synaptic function and signal transduction cascades in wild-type mouse hippocampal neurons. Methods: We have used biochemical and electrophsyiological techniques on acute hippocampal slices or cultured primary neurons. Results: We now report that APP processing plays an essential role in the normal synaptic function of hippocampal neurons. Application of exogenous APPs to mouse hippocampal slices caused a
S78
Oral O4-02: Disease Mechanisms (Others)
rapid potentiation of glutamatergic synapses. APPs increased the amplitudes of evoked AMPA receptor currents and miniature EPSPs. This effect was blocked 1) by preclearing the perfusate with anti-APPs antibody; 2) by the G-protein antagonist, NF023; and 3) by secreted A. In view of these data, we tested multiple ␣, , and ␥-secretase inhibitors for effects on LTP, and found that acute application of several BACE antagonists inhibited LTP. This was rescued with exogenous APPs. Since oligomers of A inhibit the potentiation of synapses by APPs or high-frequency stimulation, we examined how A affects the signal transduction cascades that mediate LTP. While some kinases such as PKA and PKC were activated normally by an LTP-inducing stimulus, A prevented the activation of the Erk/ MAPK, CaMKII, and PI3K pathways. Using a panel of antagonists, we examined whether inhibition of any single cell-surface receptor could replicate the signal-transduction pattern seen with A. To date, antagonists to the mGluR and insulin receptor appear to replicate the pattern Amediated signal transduction inhibition. Conclusions: These data show that proteolytic processing of APP by BACE, is necessary for the normal expression of LTP. Furthermore, our results suggest that small, soluble A oligomers interfere selectively with certain signal transduction pathways necessary for LTP, offering a mechanism by which A could lead to subtle amnestic deficits early in AD. O4-02-05
TOTAL EXCHANGEABLE CU2ⴙ PROTEIN BINDING SITES ARE ELEVATED IN ALZHEIMER’S DISEASE
Simon A. James1,2, Jean-Francois Monty1, Paul A. Adlard1, Victor Villemagne1, Anthony G. Wedd2,3, Ashley I. Bush1,4, 1The Mental Health Research Institute of Victoria and the Department of Pathology, The University of Melbourne, Parkville, Australia; 2The School of Chemistry, The University of Melbourne, Parkville, Australia; 3 Bio21 Insitiute, Parkville, Australia; 4Genetics and Aging Unit, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA. Contact e-mail: [email protected] Background: Oxidative stress is central to many neurodegenerative conditions including Alzheimer’s Disease (AD). Emerging evidence indicates that inappropriate redox reactivity between copper/iron and molecular oxygen is responsible for the pathological production of ROS in AD. A is one established protein factor that fosters such pro-oxidant activity. Neurofibrillary tangles (NFT), composed of hyperphosphorylated tau, also coordinate Cu and Fe in a pro-oxidant manner. Essential copper-dependent enzyme activities (eg SOD1, cytochrome c oxidase, PAM) are decreased in AD. Because of the accumulation of copper binding proteins in AD, copper homeostasis may be significantly dysregulated. Objective: To interrogate interactions of total brain proteins with Cu(II) and Zn(II) in post-mortem brain samples from AD and age-matched controls. To explore any modification in the metal binding properties of tau between the AD and control samples. Methods: Brain samples from well-characterized donors were homogenized, and separated into soluble and pellet fractions. The soluble fraction was applied to Immobilised Metal Affinity Chromatography (IMAC) in order to separate proteins on the basis of affinity. The proteins were eluted using an imidazole gradient. The protein concentration of each fraction was measured. Western blots of each fraction were performed to probe for both total tau and phosphorylated tau before and after the removal of endogenous metals. Results and Conclusions: The total protein elution profile differs between AD and control brain samples (soluble fraction), indicating the greater abundance of higher affinity copper binding proteins in AD. Incubation of the homogenates with metal chelators prior to the application of the samples to the column produced a significant change in the elution profile for all samples, with further differences between the AD and control samples. Western blots documented a change in the elution profile of phosphorylated tau between the AD and controls.
O4-02-06
UPREGULATION OF RAB6A IN ALZHEIMER’S DISEASE IS CONNECTED TO ENDOPLASMIC RETICULUM STRESS
Wiep Scheper1, Jeroen J.M. Hoozemans1, Rob Zwart1, Casper C. Hoogenraad2, Annemieke Rozemuller1, Piet Eikelenboom1, Frank Baas1, 1Academic Medical Center, Amsterdam, The Netherlands; 2 Erasmus Medical Center, Rotterdam, The Netherlands. Contact e-mail: [email protected] Background: A major hallmark of Alzheimer’s disease (AD) is the accumulation of misfolded proteins. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in induction of the unfolded protein response, an initially protective response aimed to restore the homeostasis in the ER. However, prolonged stress leads to cell death and may thus be an important factor in neuronal loss during AD progression. We previously observed activation of the unfolded protein response in neurons in the temporal cortex and hippocampus of AD patients. The small GTPase Rab6a is involved in transport route from the Golgi back to the ER. We hypothesise that this trafficking route involves a post-ER quality control mechanism, by which aberrant proteins that have escaped ER quality control are transported back to the ER. Objective(s): In this study we investigated the role of Rab6A trafficking in connection to ER stress in AD pathogenesis. Methods: We used immuno-histochemistry and semi-quantitative Western blotting to determine Rab6A and ER stress levels in post-mortem brain samples of AD patients and non-demented controls. Mechanistic aspects of the relation between Rab6A trafficking and ER stress were studied in human neuroblastoma cells and an ER stress reporter cell line. Results: Rab6A is predominantly expressed in the pyramidal neurons in the temporal cortex and hippocampus. Western blot analysis of a panel of AD and control patients shows that Rab6A is strongly upregulated in AD brain from Braak stage III. Our data indicate that Rab6A upregulation precedes tangle formation. We show that the levels of the ER chaperone BiP/GRP78 correlate closely with those of Rab6A in human temporal cortex, suggesting a functional connection between Rab6A and ER stress. This is supported by evidence provided by experiments in cell culture, where increased Rab6A levels suffice to induce ER stress. Conclusions: Our data support a role of Rab6A and ER stress relatively early in AD pathogenesis. Targeting protein quality control therefore creates an interesting therapeutic window of opportunity. O4-02-07
MITOCHONDRIAL DYSFUNCTION AND ALZHEIMER’S DISEASE IN DOWN’S SYNDROME
Jorge Busciglio, Pablo Helguera, Jackeline Seiglie, UCI, Irvine, CA, USA. Contact e-mail: [email protected] Down’s Syndrome (DS) is the most frequent genetic cause of mental retardation. Most DS patients develop Alzheimer’s disease (AD) by middle age. The evidence available indicates that mitochondrial dysfunction is a critical element of DS: 1) mitochondrial dysfunction exists in DS neurons and astrocytes, which leads to aberrant amyloid precursor protein (APP) metabolism and intracellular amyloid beta accumulation; 2) there is increased oxidative stress in DS cells which promotes neuronal death by overexpression of the transcription factor Ets-2 through a p53-dependent, mitochondrial apoptotic pathway; and 3) there are structural and functional alterations in mitochondria of DS cells that may directly affect cell function and survival. We have initiated studies to determine the molecular elements that are involved and to delineate the specific nature of the mitochondrial disorder present in DS. These studies are focused on the role of intracellular amyloid beta in mitochondrial function, alterations in the activity of uncoupling proteins, and the role of Down Syndrome Critical Region 1 (DSCR1), which has been recently implicated as a key regulator of mitochondrial function. The results support the emerging view that different pathological mechanisms may converge into a chronic state of mitochondrial dysfunction in DS, leading to a persistent systemic deficit on cell capabilities and viability. Supported by NIH, The Alzheimer’s Association and UCI.