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Neuropathological substrate of mild cognitive impairment
Oral
S198
pGiJ
NEUROPATHOLOGlCAL IMPAIRMENT
SUBSTRATE
OF MILD COGNITIVE
Ronald C Petersen. Dennis W Dickson, Joseph E Purisi, Kris A Johnson, Robert .I Ivnik, Glenn E Smith, Eric G Tang&s, Emre Kokmen, Mayo Clin, Rochester, MN Mild cognitive impawment (MCI) refers to the transitional state between normal aging and very mild Alzheimer’s disease (AD). Mild cognitive impairment subjects typically have a memory impairment beyond what would be expected for normal aging, yet they are not demented. The neuropathological substrate of MCI ic not known. As part of the Mayo Alzheimer’s Disease Research Center and the Alrheimer’s Disease Patient Registry over 14 years, we have evaluated 11 subjects who have died and come to autopsy when their clinical diagnosis was MCI. These wbjects (4 men, 7 women) ranged in age from 81 to 94 years (mean 90 years) with a Mini-Mental State Exam of 26 and a mean Dementia Rating Scale of 124 at the time of thetr last clinical evaluation which was within I2 months of death. The neuropathological results revealed AD-like pathology in 5 of I I subjects. Typically these subjects had frequent neurofibrillary tangles in the medial temporal lobe and moderate diffuse plaques with sparse neuritic plaques in the neocortex. typically in the temporal lobe. Three case> had argyrophdic grain disease, and two had neurofibrillary tangle-only involvement of the medial temporal lobe. These results indicate that all subjects who have the amnestic form of MCI have extensive neuropathological involvement of medial temporal lobe structures, and some subjects may also meet criteria for very early AD. Taken together, these results indicate that MCI is likely a transitional state between the changes of normal aging and AD for most subjects, but there may also be a subset of subjects who have non-AD pathology of the medial temporal lobe.
Antao Du, Dept of Veteran Affairs and Univ qf CA, San Francisco, CA; Norbert Schujj; Dime Amend, Vamc, San Francisco, CA; Yuan-Yi Hsu, Chang Gung Memorial Hmp, Tuivvun Taiwan Rot; Mikko P Laukso, Univ of Kuopio. Kuopio Finland; William Jugust, UCDavis, Davis, CA; Helena C Chui, Univ $Southrm CA, Los An&as, CA; Michael W Weiner, Vumc, San Francisco, CA Objective: To determine ERC and HP volume losses in AlLheimer’s disease (AD), subcortical ischemic vascular dementia (SIVD), and mixed dementia (MD). Method: Volumetric MRI measurements were performed on 33 AD, 14 SIVD, 17 MD, and 40 cognitively normal controls (CN). Results: Compared with CN, AD had both smaller ERC and HP volumes (piO.01). Compared with SIVD. AD had also smaller ERC and HP (p
HP (mm’)
ERC
(mn?)
CN
2720
2 606
6314
2 797
AD
165 I 2 480 (39%)*
4649
t
SIVD MD
2388 + 641 (12%) 1796 + 548 (34%)*
5621 -t 901 (II%)** 4602 i 704 (27%)*
988 (26%)*
Mean ? SD; percent change compared with CN in brackets * p < 0.01; ** p < 0.05 compared wiht CN.
Oral Presentation:
Beta Amyloid
EFFECT OF FAMILIAL ALZHEIMER’S DISEASE MUTATIONS ON THE FORMATION OF PRESENILIN AGGREGATES IN PROTEASOME INHIBITOR TREATED CELLS Dora M Kovacs. lmrr Kovacs. Kristen M Lentini, l*rura A MacKenzie Inguno, Luigi Puglielli, Rudolph E Tanzi. MA Gen Hasp und Harvard Med Sch, Churlestown, MA We and other\ have recently shown that PSI, like CFTR and PrP. can exit the ER by retrograde transport and form aggregates in the cytoplasm. PSI enters this pathway
Beta Amvloid
upon proteasomal inhibition or very htgh expression levels of the transgene. Cytoplasmic PSI accumulates in structures termed aggresomes. Aggresomes contain ubiquitinated aggregates of insoluble proteins and proteasomes, and colocalize with collapsed vimentin. We employed fluorescent confocal microscopy to study aggrerotne formation in stably transfected wild-type and FAD mutant PSI cell lines treated with the proteasome inhibitors MG132 and ALLN. We found that aggresomes stained positive for APP, but not for the Golgi marker GMl30. Moreover, aggresome formation correlated negatively with P-amyloid production and with induction of apoptosis in FAD mutant PSl CHO cells. The number of aggresome-containing cells was not affected by PSI mutations. Subcellular fractionation of both wild-type and FAD mutant PSI CHO cells on a 8.28% Opti-Prep gradient revealed high molecular weight PSI aggregates in the ER. Next we examined aggresome formation in primary rat hippocampal neurons using antibodles against PSI, vimentin and NF68. Although we did not see “classic” aggresome formation in primary neurons, we detected strong PS 1 staining in the nuclear membrane of neurons following treatment with MGI 32. Interestingly, none of other untransfected cell types (fibroblasts. lymphoblasts, CHO. H4 neuroglioma, SYSY neuroblastoma) treated with proteasome inhibitors showed increased PSI levels in the nuclear membrane/ER. These studies indicate that neucons may be more sensitive to formation of PSl aggregates m the nuclear membrane/ER as compared to other cell types. Collectively, these findings reveal an inverse correlation between aggresome formation and the effects of mutant preaenilins on ABand apoptosis. Furthermore, neurons appear to be more prone to forming nuclear/ER membrane aggregates than other cell types in response to proteasome inhibitors We are currently in the process of analyzing the effect of PSI FAD mutations on aggresome formation and on nuclear/ER membrane aggregate formation.
pig ENTORHINAL CORTEX AND HIPPOCAMPUS IN ALZHEIMER’S DISEASE, SUBCORTICAL ISCHEMIC VASCULAR DEMENTIA AND MIXED DEMENTIA
Presentation:
EARLY EVENTS IN THE FOLDING OF APP.
Dmitr? Goldgubrr, E/mu M Znitsevn, Christi A Weston. Jouv M Privrs. Stufe Unit, of New York. Stony Rmok. NY Functions of proteins critically depend on their proper folding. Newly formed nabcent glycoproteins are co-translationally tramlocated through the endoplasmic reticulum (ER) membrane and interact with molecular chaperones. This interaction is easentlal for the proper folding and subsequent modifications of glycoproteins. ER molecular chaperones also play a key role in quality control, i.e. nascent glycoproteins that are misfolded or mutated are recognized, retro-translocated back from the ER into the cytosol, and degraded by the ubiquitin-proteasome degradation pathway. We investigated the mteraction of endogenous and overexpressed APP with the key ER molecular chaperones, BiP, calnexin, and calreticulin, in HEK293 cells. Western blotting of cell extracts reveals endogenous APP as a doublet contaming immature (lower band) and mature (a tight upper band) forma. lmmunoprecipitation experiments demonstrated that BiP binds only to the immature form of APP. The use of BiP mutant with impaired ability to release bound protein produced essentially the same results. Unexpectedly, similar immunoprecipitafion experiments determined that clan&n and calreticulin bind to both the immature and mature forms of APP. Overexpression of APP by transfection with APP cDNA impairs the maturation process and results in the appearance of a diffuse upper band corresponding to APP that probably did not complete the maturation process. Co-transfection of BiP with APP did not improve the maturation of overexpressed APP. However, co-transfection of calnexin with APP resulted m the appearance of a tight mature band of APP, ruggesting that calnexin restored the maturation process of overexpressed APP. Additional experiments were conducted with monoclonal antibody P2-I that recognizs an aminoterminal epitope of APP with fully formed disulfide bonds. Immature form of APP in complexes with ER molecular chaperones was recognized with mAb P2-I. This augge\ts that disulfide bond formatlo” is a very early event in the folding of APP.
ACCUMULATION GENT-INSOLUBLE
OF AMYLOID B PROTEIN IN THE DETERMEMBRANE COMPARTMENT
Takeshr Kawarabayashi, Suzanne Wahrle, Linda H Yomkin, Wen-Lang Lin, Dmnis W Dickson, Steven G Yuunkin, Mayo Clin, Jacksonville, FL We have examined the amyloid p protein (Ap) in detergent-insoluble glycolipidenriched membrane domains (DIGS) from human and Tg2576 mouse brains. Brains were homogenized with MES-buffered saline containing I% Triton X-100 and protease inhibitors. The homogenate was fractionated by discontinuous sucrose gradient centrifugation. DIGS floated, as expected, to the interface between the 38% and 5% sucrose layers based on the immunoreactivity of tlotillin, a marker for brain DIGS. In mouse and human brains, amyloid p protein precursor (PAPP), C-temunal fragments of BAPP (CTF), apolipoprotein E, presenilin I, and AP were all found to be concentrated in DlGs. Analysis by ELISA showed that -25% of the total Ab in control brain is in DIGS. Immunoprecipitation followed by immunoblotting confirmed
S198
pGiJ
NEUROPATHOLOGlCAL IMPAIRMENT
SUBSTRATE
OF MILD COGNITIVE
Ronald C Petersen. Dennis W Dickson, Joseph E Purisi, Kris A Johnson, Robert .I Ivnik, Glenn E Smith, Eric G Tang&s, Emre Kokmen, Mayo Clin, Rochester, MN Mild cognitive impawment (MCI) refers to the transitional state between normal aging and very mild Alzheimer’s disease (AD). Mild cognitive impairment subjects typically have a memory impairment beyond what would be expected for normal aging, yet they are not demented. The neuropathological substrate of MCI ic not known. As part of the Mayo Alzheimer’s Disease Research Center and the Alrheimer’s Disease Patient Registry over 14 years, we have evaluated 11 subjects who have died and come to autopsy when their clinical diagnosis was MCI. These wbjects (4 men, 7 women) ranged in age from 81 to 94 years (mean 90 years) with a Mini-Mental State Exam of 26 and a mean Dementia Rating Scale of 124 at the time of thetr last clinical evaluation which was within I2 months of death. The neuropathological results revealed AD-like pathology in 5 of I I subjects. Typically these subjects had frequent neurofibrillary tangles in the medial temporal lobe and moderate diffuse plaques with sparse neuritic plaques in the neocortex. typically in the temporal lobe. Three case> had argyrophdic grain disease, and two had neurofibrillary tangle-only involvement of the medial temporal lobe. These results indicate that all subjects who have the amnestic form of MCI have extensive neuropathological involvement of medial temporal lobe structures, and some subjects may also meet criteria for very early AD. Taken together, these results indicate that MCI is likely a transitional state between the changes of normal aging and AD for most subjects, but there may also be a subset of subjects who have non-AD pathology of the medial temporal lobe.
Antao Du, Dept of Veteran Affairs and Univ qf CA, San Francisco, CA; Norbert Schujj; Dime Amend, Vamc, San Francisco, CA; Yuan-Yi Hsu, Chang Gung Memorial Hmp, Tuivvun Taiwan Rot; Mikko P Laukso, Univ of Kuopio. Kuopio Finland; William Jugust, UCDavis, Davis, CA; Helena C Chui, Univ $Southrm CA, Los An&as, CA; Michael W Weiner, Vumc, San Francisco, CA Objective: To determine ERC and HP volume losses in AlLheimer’s disease (AD), subcortical ischemic vascular dementia (SIVD), and mixed dementia (MD). Method: Volumetric MRI measurements were performed on 33 AD, 14 SIVD, 17 MD, and 40 cognitively normal controls (CN). Results: Compared with CN, AD had both smaller ERC and HP volumes (piO.01). Compared with SIVD. AD had also smaller ERC and HP (p
HP (mm’)
ERC
(mn?)
CN
2720
2 606
6314
2 797
AD
165 I 2 480 (39%)*
4649
t
SIVD MD
2388 + 641 (12%) 1796 + 548 (34%)*
5621 -t 901 (II%)** 4602 i 704 (27%)*
988 (26%)*
Mean ? SD; percent change compared with CN in brackets * p < 0.01; ** p < 0.05 compared wiht CN.
Oral Presentation:
Beta Amyloid
EFFECT OF FAMILIAL ALZHEIMER’S DISEASE MUTATIONS ON THE FORMATION OF PRESENILIN AGGREGATES IN PROTEASOME INHIBITOR TREATED CELLS Dora M Kovacs. lmrr Kovacs. Kristen M Lentini, l*rura A MacKenzie Inguno, Luigi Puglielli, Rudolph E Tanzi. MA Gen Hasp und Harvard Med Sch, Churlestown, MA We and other\ have recently shown that PSI, like CFTR and PrP. can exit the ER by retrograde transport and form aggregates in the cytoplasm. PSI enters this pathway
Beta Amvloid
upon proteasomal inhibition or very htgh expression levels of the transgene. Cytoplasmic PSI accumulates in structures termed aggresomes. Aggresomes contain ubiquitinated aggregates of insoluble proteins and proteasomes, and colocalize with collapsed vimentin. We employed fluorescent confocal microscopy to study aggrerotne formation in stably transfected wild-type and FAD mutant PSI cell lines treated with the proteasome inhibitors MG132 and ALLN. We found that aggresomes stained positive for APP, but not for the Golgi marker GMl30. Moreover, aggresome formation correlated negatively with P-amyloid production and with induction of apoptosis in FAD mutant PSl CHO cells. The number of aggresome-containing cells was not affected by PSI mutations. Subcellular fractionation of both wild-type and FAD mutant PSI CHO cells on a 8.28% Opti-Prep gradient revealed high molecular weight PSI aggregates in the ER. Next we examined aggresome formation in primary rat hippocampal neurons using antibodles against PSI, vimentin and NF68. Although we did not see “classic” aggresome formation in primary neurons, we detected strong PS 1 staining in the nuclear membrane of neurons following treatment with MGI 32. Interestingly, none of other untransfected cell types (fibroblasts. lymphoblasts, CHO. H4 neuroglioma, SYSY neuroblastoma) treated with proteasome inhibitors showed increased PSI levels in the nuclear membrane/ER. These studies indicate that neucons may be more sensitive to formation of PSl aggregates m the nuclear membrane/ER as compared to other cell types. Collectively, these findings reveal an inverse correlation between aggresome formation and the effects of mutant preaenilins on ABand apoptosis. Furthermore, neurons appear to be more prone to forming nuclear/ER membrane aggregates than other cell types in response to proteasome inhibitors We are currently in the process of analyzing the effect of PSI FAD mutations on aggresome formation and on nuclear/ER membrane aggregate formation.
pig ENTORHINAL CORTEX AND HIPPOCAMPUS IN ALZHEIMER’S DISEASE, SUBCORTICAL ISCHEMIC VASCULAR DEMENTIA AND MIXED DEMENTIA
Presentation:
EARLY EVENTS IN THE FOLDING OF APP.
Dmitr? Goldgubrr, E/mu M Znitsevn, Christi A Weston. Jouv M Privrs. Stufe Unit, of New York. Stony Rmok. NY Functions of proteins critically depend on their proper folding. Newly formed nabcent glycoproteins are co-translationally tramlocated through the endoplasmic reticulum (ER) membrane and interact with molecular chaperones. This interaction is easentlal for the proper folding and subsequent modifications of glycoproteins. ER molecular chaperones also play a key role in quality control, i.e. nascent glycoproteins that are misfolded or mutated are recognized, retro-translocated back from the ER into the cytosol, and degraded by the ubiquitin-proteasome degradation pathway. We investigated the mteraction of endogenous and overexpressed APP with the key ER molecular chaperones, BiP, calnexin, and calreticulin, in HEK293 cells. Western blotting of cell extracts reveals endogenous APP as a doublet contaming immature (lower band) and mature (a tight upper band) forma. lmmunoprecipitation experiments demonstrated that BiP binds only to the immature form of APP. The use of BiP mutant with impaired ability to release bound protein produced essentially the same results. Unexpectedly, similar immunoprecipitafion experiments determined that clan&n and calreticulin bind to both the immature and mature forms of APP. Overexpression of APP by transfection with APP cDNA impairs the maturation process and results in the appearance of a diffuse upper band corresponding to APP that probably did not complete the maturation process. Co-transfection of BiP with APP did not improve the maturation of overexpressed APP. However, co-transfection of calnexin with APP resulted m the appearance of a tight mature band of APP, ruggesting that calnexin restored the maturation process of overexpressed APP. Additional experiments were conducted with monoclonal antibody P2-I that recognizs an aminoterminal epitope of APP with fully formed disulfide bonds. Immature form of APP in complexes with ER molecular chaperones was recognized with mAb P2-I. This augge\ts that disulfide bond formatlo” is a very early event in the folding of APP.
ACCUMULATION GENT-INSOLUBLE
OF AMYLOID B PROTEIN IN THE DETERMEMBRANE COMPARTMENT
Takeshr Kawarabayashi, Suzanne Wahrle, Linda H Yomkin, Wen-Lang Lin, Dmnis W Dickson, Steven G Yuunkin, Mayo Clin, Jacksonville, FL We have examined the amyloid p protein (Ap) in detergent-insoluble glycolipidenriched membrane domains (DIGS) from human and Tg2576 mouse brains. Brains were homogenized with MES-buffered saline containing I% Triton X-100 and protease inhibitors. The homogenate was fractionated by discontinuous sucrose gradient centrifugation. DIGS floated, as expected, to the interface between the 38% and 5% sucrose layers based on the immunoreactivity of tlotillin, a marker for brain DIGS. In mouse and human brains, amyloid p protein precursor (PAPP), C-temunal fragments of BAPP (CTF), apolipoprotein E, presenilin I, and AP were all found to be concentrated in DlGs. Analysis by ELISA showed that -25% of the total Ab in control brain is in DIGS. Immunoprecipitation followed by immunoblotting confirmed