Early β-amyloid deposition in hippocampal neurons in β-amyloid precursor protein 695 (APP695)...
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Poster Presentution: Animul Models IV s225 observed in the brams of non-transgenic litter-mates. This is the first demonstration of an association of JNK and SAPK expresalon with phosphorylated tat&eta-amyloid deposits in a tranrgenic mouse. EFFECTS OF ESTROGEN ON COGNITIVE FUNCTIONS AND AMYLOID METABOLISM IN AMYLOID PRECURSOR PROTEIN AND PRESENILIN-1 DOUBLE TRANSGENIC MICE Recent epidemiological studies suggest that estrogen replacement therapy (ERT) may slow down the memory decline in postmenopausal females and decrease the risk to develop Alzhenner’~ disease (AD). In this study we investigate the effects of ovariectomy (OVX) and chronic ERT on cognitive filnctions and amyloid metabohsm in amyloid precursor protein (APP) and presenilin-I (PSI) double transgenic mice (APP+PS-I). The APP+PS-I mu develop amyloid plaques at 9-12 months. Mice were ovariectomized at the age 4 months and were/will be treated with estrogen pellet5 (placebo. 0.18 or 0.72 mg/pellet) for 3 or 6 montha. The mice were/will be tested in water ma~e (WM) (visible platform: non-spatial navigation; hidden platform: spatial nawgation), radial arm maze (RAM) (allocentric spatial memory) and T-maze (egocentric spatial memory). The preliminary data suggest that APP+PS-I mice are cognitively impaired in WM and that estrogen treatment improves learning of APPfPSl mice m WM. RAM and T-mare but has no effect on hippocampal beta amyloid levels. The restdtb from this study will give us new information about the effects of estrogen on u$nitive functions and its interaction wth amyloid metabo- lism. CHARACTERIZATION OF AMYLOID DEPOSITS IN THE CERE- BRAL CORTEX OF TRANSGENIC MOUSE EXPRESSING HU- MAN APP695SWE: IMPLICATIONS FOR THE PATHOGENESIS OF ALZHEIMER’S DISEASE Kuzuhitx Twni, Akihiko lwai. Shigrki Kawahata. Yorhikazu Tnsnki, Tohru Wutancrbr. Keiji Miwto, Tokio Yarnuguchi. Yumanouchi Phurm Co Ltd, Tsukuhu Japun Recently, a tranagenic (tg) mouse expressing the human amyloid precursor protein with the Swedish mutation (APPswe), Tg2576, has been rstabhshed as an animal model of Alzheimer’s disease. In these mice, P-amyloid (AP) deposits in the brain are detected in addition to memory deficits by the age of 9-10 months. Although the amyloid pathology in these rmce is somewhat sunilar to that in Alzheimer’a disease, there is no detectable neuronal loss in the mice. To clarify the mechanism of amyloid deposltion, we characterized the% amyloid deposits biochemically and pathologi- cally A wrface-enhanced later drsorption/ionization affinity mass spectl-ometric study using the 6ElO monoclonal antibody demonstrated that the mayor specxs of AP in a formic acid extract of the tg mouse cerebral cortex were ADI -3X. API-40 and API-42. Immunohi\tochemistry using antibodies for the carboxy-terminal epitopes of API-40 and Apl-42 (kindly provided by Dr. P.D. Mehta, IBR, NY) as well as antibody 6EIO showed that plaques containing API-42 were more widely distributed than those containing ApI- throughout the cerebral cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that API-40 was preferentially located in the central part of the API-42 positive plaques. ELISA measurements of API-40 and Apl-42 showed that there was several times more API-40 than API-42. These data suggest that API-42 deposition may precede Afil-40 deposition, while Apl-40 begins to deposit in the central part of the plaques and accumulate\. Congophilic structures corresponded almwt exactly with API -40 immunostaining in \erial brain sections of the tg mouse. Aberrant swollen synapses related to the congophilic structures were detected by synaptophyain and wsynuclein immunostain- ing. Furthermore. congophilic plaques were surrounded by reactive glial cell;. These data Indicate that congophilic amyloid deposition, which conzaponds to accumulated AD l-40 in the central part of the plnquea, result\ m dystrophic synapses dwxtly or via gliosis. Those aberrant synapses may result in the memory de&t in this mouse. (10241 ALTERED IP3-DEPENDENT INTRACELLULAR CA’+POOLS OF HIPPOCAMPAL NEURONS IN PRESENILIN-1 MUTANT TRANS- GENlC MICE Altered neuronal Ca* ’ homeostaai\ ha& been implicated m the pathogenesic and neuronal cell-death in Alzheimer‘s disease (AD). Here. we have analyzed transgenic mice that habor a mutant human presendin-I tranagene, i.e. PS-l[A246EI linked to early onset AD, and studied neuronal Ca ” homeostasis m detail. Microfluorimetric Caz+measurements were performed on cultured hippocampal neutrons of PSI mutant animals and non-transgeic mice. In PS-1 mutant neurons glutamate induced a [Ca*+li increase that was significantly enhanced compared to wild-type neurons. Application of NMDA did not reveal differences between transgenic PSI mutant neurons and wild-type cells while the rise in[&+]i induced by depolarization with SO mM K+ was also not different in PS-I mutant cells. This indicated that neither Ca’+ influx through voltage-gated Ca” channels nor @+-induced Ca” release from intracel- lular storea was affected by the PSI mutant. On the other hand, however, appbcation of bradykinin (I 0 FM), known to activate the IP3 cascade, provoked a significantly enhanced effect, similar to that of glutamate, on [Ca* ‘Ii in PS-I mutant hippocampal neurons. These rerults clearly indicate that endoplasmatic IP3 dependent Ca*+ poolc are severely affected by the expression of the mutant PSI transgene. piF%j MEASUREMENT OF CHOLINERGIC RECEPTORS IN APP- OVEREXPRESSING TRANSGENIC MICE The Tg(HuApp695,KM670/671)2576 transgenic mouse model of Alzheimer’s dw ease (AD) has been shown to develop AD like pathology between 6-9 months of age and to display performance impairment in number of memory and learning tasks. However, as yet no changes in “euroreceptor levels have been reported. In this study we examined the density of 1 ‘2sll abungarotoxin (aBTX) binding site> in the brains of these mice between 4-18 months of age (n=5-7) compared to age matched control animals using in vitro autoradlography. Age related decreases in l’*‘I] aBTX binding were observed in a number of cortical regiona such as frontal, par&l and piriform cortica of Tg2576 mice but not in control mice. Additionally, I’ rsI] aBTX binding in these brain regions in control animals was sigmficantly greater than in Tg2576 animals at 9 and II months. Interestingly, [“‘I] uBTX binding in a number of auhcortical regions of Tg2576 mice, such as the hippocampus CAI-3 are significantly greater than in age matched controls and show no age related decline. No age related reductions in bmding were observed in CAI-3 of control animal\. The reductionc in cortical [‘2511 aBTX binding observed in Tg2576 mice in this study we consistent with the temporal and regional appearance of AD pathology as previously observed m thiq mouse strain and suggest a loss of rr7 nicotinic receptor% The elevation 01 [“‘ I/ aBTX bindlng Gtes in subcortical regmn\ may result from the interaction of high levels of Pamyloid with a7 mcotinic receptors reaultmg in receptor upregulation or prevention of the characteristic postnatal decline in receptor number. Currently autoradiographic studies with [‘HI cytisine, I’Hl AFDX 384 and r7Hl pirenzepmr Imeawring a4p2 mcotinic receptors and Ml and M2 mubcarimc receptors we 011 going and will provide more information on the cholinrrgic receptors in this mouw model The loss of cortical nicotimc receptor\ compared to age matchrd controla i\ consistent with the nicotinic receptor IOI~ observed in AD and provides further validation of this mouw stram as an animal model for AD. (10261 EARLY B-AMYLOID DEPOSITION IN HIPPOCAMPAL NEU- RONS IN B-AMYLOID PRECURSOR PROTEIN 695 (APP695) TRANSGENIC MICE IncreaGng c\idence jugpats that the modulation of b-amylold (Afi) production and depositIon may be an early event Ieadmg to Alrhcimer’\ diww (AD). Recently. tran\genic mouse models have shown AD-like plaque formation. However. behavior deficits in these mice are typically observed much earlier than Apdeposition can br detected. Apstaioing in APP695 transgrnuz mice i\ normally undetectable until I I months of age. Using our Improved immunohistochemlcal procedure, we report here that punctate APstaining in the hippocampw was observed as early aa 4 months of age in these mice. The small hippocampal Apdepositions dramatically increaxd with age. The depositions were distributed mostly in the CAI field and essentially absent in the dentate gyrw. No positive staining wa, observed in wild-type control\. Thioflavin-S aining suggested that the small deposltions were P-pleated \heet structures. Confocal mxroscopy demonstrated that these Apdepositiona could be mtracellular or membrane associated. Our results are conrirtent with findings in human brain, where early APinmmuno-reactivity i\ observed within neurons most vulnerable to the earl&t AD pathology. These findings suggest that early Apdepositlon in the hippocampus may contrihutc to neuronal dysfunction and may play an important role in later neuritic plaque formation.
Transcript of Early β-amyloid deposition in hippocampal neurons in β-amyloid precursor protein 695 (APP695)...
Poster Presentution: Animul Models IV s225
observed in the brams of non-transgenic litter-mates. This is the first demonstration of an association of JNK and SAPK expresalon with phosphorylated tat&eta-amyloid deposits in a tranrgenic mouse.
EFFECTS OF ESTROGEN ON COGNITIVE FUNCTIONS AND AMYLOID METABOLISM IN AMYLOID PRECURSOR PROTEIN AND PRESENILIN-1 DOUBLE TRANSGENIC MICE
Recent epidemiological studies suggest that estrogen replacement therapy (ERT) may slow down the memory decline in postmenopausal females and decrease the risk to develop Alzhenner’~ disease (AD). In this study we investigate the effects of ovariectomy (OVX) and chronic ERT on cognitive filnctions and amyloid metabohsm in amyloid precursor protein (APP) and presenilin-I (PSI) double transgenic mice (APP+PS-I). The APP+PS-I mu develop amyloid plaques at 9-12 months. Mice were ovariectomized at the age 4 months and were/will be treated with estrogen pellet5 (placebo. 0.18 or 0.72 mg/pellet) for 3 or 6 montha. The mice were/will be tested in water ma~e (WM) (visible platform: non-spatial navigation; hidden platform: spatial nawgation), radial arm maze (RAM) (allocentric spatial memory) and T-maze (egocentric spatial memory). The preliminary data suggest that APP+PS-I mice are cognitively impaired in WM and that estrogen treatment improves learning of APPfPSl mice m WM. RAM and T-mare but has no effect on hippocampal beta amyloid levels. The restdtb from this study will give us new information about the effects of estrogen on u$nitive functions and its interaction wth amyloid metabo- lism.
CHARACTERIZATION OF AMYLOID DEPOSITS IN THE CERE- BRAL CORTEX OF TRANSGENIC MOUSE EXPRESSING HU- MAN APP695SWE: IMPLICATIONS FOR THE PATHOGENESIS OF ALZHEIMER’S DISEASE
Kuzuhitx Twni, Akihiko lwai. Shigrki Kawahata. Yorhikazu Tnsnki, Tohru Wutancrbr.
Keiji Miwto, Tokio Yarnuguchi. Yumanouchi Phurm Co Ltd, Tsukuhu Japun
Recently, a tranagenic (tg) mouse expressing the human amyloid precursor protein with the Swedish mutation (APPswe), Tg2576, has been rstabhshed as an animal model of Alzheimer’s disease. In these mice, P-amyloid (AP) deposits in the brain are detected in addition to memory deficits by the age of 9-10 months. Although the amyloid pathology in these rmce is somewhat sunilar to that in Alzheimer’a disease, there is no detectable neuronal loss in the mice. To clarify the mechanism of amyloid deposltion, we characterized the% amyloid deposits biochemically and pathologi- cally A wrface-enhanced later drsorption/ionization affinity mass spectl-ometric study using the 6ElO monoclonal antibody demonstrated that the mayor specxs of AP in a formic acid extract of the tg mouse cerebral cortex were ADI -3X. API-40 and API-42. Immunohi\tochemistry using antibodies for the carboxy-terminal epitopes of API-40 and Apl-42 (kindly provided by Dr. P.D. Mehta, IBR, NY) as well as antibody 6EIO showed that plaques containing API-42 were more widely distributed than those containing ApI- throughout the cerebral cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that API-40 was preferentially located in the central part of the API-42 positive plaques. ELISA measurements of API-40 and Apl-42 showed that there was several times more API-40 than API-42. These data suggest that API-42 deposition may precede Afil-40 deposition, while Apl-40 begins to deposit in the central part of the plaques and accumulate\. Congophilic structures corresponded almwt exactly with API -40 immunostaining in \erial brain sections of the tg mouse. Aberrant swollen synapses related to the congophilic structures were detected by synaptophyain and wsynuclein immunostain- ing. Furthermore. congophilic plaques were surrounded by reactive glial cell;. These data Indicate that congophilic amyloid deposition, which conzaponds to accumulated AD l-40 in the central part of the plnquea, result\ m dystrophic synapses dwxtly or via gliosis. Those aberrant synapses may result in the memory de&t in this mouse.
(10241 ALTERED IP3-DEPENDENT INTRACELLULAR CA’+POOLS OF HIPPOCAMPAL NEURONS IN PRESENILIN-1 MUTANT TRANS- GENlC MICE
Altered neuronal Ca* ’ homeostaai\ ha& been implicated m the pathogenesic and neuronal cell-death in Alzheimer‘s disease (AD). Here. we have analyzed transgenic mice that habor a mutant human presendin-I tranagene, i.e. PS-l[A246EI linked to
early onset AD, and studied neuronal Ca ” homeostasis m detail. Microfluorimetric Caz+measurements were performed on cultured hippocampal neutrons of PSI mutant animals and non-transgeic mice. In PS-1 mutant neurons glutamate induced a [Ca*+li increase that was significantly enhanced compared to wild-type neurons. Application of NMDA did not reveal differences between transgenic PSI mutant neurons and wild-type cells while the rise in[&+]i induced by depolarization with SO mM K+ was also not different in PS-I mutant cells. This indicated that neither Ca’+ influx through voltage-gated Ca” channels nor @+-induced Ca” release from intracel- lular storea was affected by the PSI mutant. On the other hand, however, appbcation of bradykinin (I 0 FM), known to activate the IP3 cascade, provoked a significantly enhanced effect, similar to that of glutamate, on [Ca* ‘Ii in PS-I mutant hippocampal neurons. These rerults clearly indicate that endoplasmatic IP3 dependent Ca*+ poolc are severely affected by the expression of the mutant PSI transgene.
piF%j MEASUREMENT OF CHOLINERGIC RECEPTORS IN APP- OVEREXPRESSING TRANSGENIC MICE
The Tg(HuApp695,KM670/671)2576 transgenic mouse model of Alzheimer’s dw ease (AD) has been shown to develop AD like pathology between 6-9 months of age and to display performance impairment in number of memory and learning tasks. However, as yet no changes in “euroreceptor levels have been reported. In this study we examined the density of 1 ‘2sll abungarotoxin (aBTX) binding site> in the brains of these mice between 4-18 months of age (n=5-7) compared to age matched control animals using in vitro autoradlography. Age related decreases in l’*‘I] aBTX binding were observed in a number of cortical regiona such as frontal, par&l and piriform cortica of Tg2576 mice but not in control mice. Additionally, I’rsI] aBTX binding in these brain regions in control animals was sigmficantly greater than in Tg2576 animals at 9 and II months. Interestingly, [“‘I] uBTX binding in a number of auhcortical regions of Tg2576 mice, such as the hippocampus CAI-3 are significantly greater than in age matched controls and show no age related decline. No age related reductions in bmding were observed in CAI-3 of control animal\. The reductionc in cortical [‘2511 aBTX binding observed in Tg2576 mice in this study we consistent with the temporal and regional appearance of AD pathology as previously observed m thiq mouse strain and suggest a loss of rr7 nicotinic receptor% The elevation 01 [“‘I/ aBTX bindlng Gtes in subcortical regmn\ may result from the interaction of high levels of Pamyloid with a7 mcotinic receptors reaultmg in receptor upregulation or prevention of the characteristic postnatal decline in receptor number. Currently autoradiographic studies with [‘HI cytisine, I’Hl AFDX 384 and r7Hl pirenzepmr Imeawring a4p2 mcotinic receptors and Ml and M2 mubcarimc receptors we 011 going and will provide more information on the cholinrrgic receptors in this mouw model The loss of cortical nicotimc receptor\ compared to age matchrd controla i\ consistent with the nicotinic receptor IOI~ observed in AD and provides further validation of this mouw stram as an animal model for AD.
(10261 EARLY B-AMYLOID DEPOSITION IN HIPPOCAMPAL NEU- RONS IN B-AMYLOID PRECURSOR PROTEIN 695 (APP695) TRANSGENIC MICE
IncreaGng c\idence jugpats that the modulation of b-amylold (Afi) production and depositIon may be an early event Ieadmg to Alrhcimer’\ diww (AD). Recently. tran\genic mouse models have shown AD-like plaque formation. However. behavior deficits in these mice are typically observed much earlier than Apdeposition can br detected. Apstaioing in APP695 transgrnuz mice i\ normally undetectable until I I months of age. Using our Improved immunohistochemlcal procedure, we report here that punctate APstaining in the hippocampw was observed as early aa 4 months of age in these mice. The small hippocampal Apdepositions dramatically increaxd with age. The depositions were distributed mostly in the CAI field and essentially absent in the dentate gyrw. No positive staining wa, observed in wild-type control\. Thioflavin-S aining suggested that the small deposltions were P-pleated \heet structures. Confocal mxroscopy demonstrated that these Apdepositiona could be mtracellular or membrane associated. Our results are conrirtent with findings in human brain, where early APinmmuno-reactivity i\ observed within neurons most vulnerable to the earl&t AD pathology. These findings suggest that early Apdepositlon in the hippocampus may contrihutc to neuronal dysfunction and may play an important role in later neuritic plaque formation.
