Estrogen enhances phagocytosis of amyloid β protein by microglia derived from human cortex

S182

Poster

@ziJ Jill

M

ROLE OF TESTICULAR RECEPTOR OF ALZHEIMER’S DISEASE

Weimer,

Rochester

Med

Loretta Ctr.

Collins,

Rochester,

Nienwen

NY;

Paul

Chow,

2 IN THE PATHOGENESIS

Chawnshang

D Coleman,

Univ

Chang,

r~f Rochester.

Univ

of

Rochester.

NY

Though extensive amounts of research have focused on the role of steroid hormone,, most notably estrogen and glucocorticoids, in Alrheimer’s disease (AD), little research has been done on the role of the receptors which these hormone activate. Research has shown that increased activation of the glucocorticoid receptor enhanced cells susceptibihty to amylotd beta-protein and oxidative stress and that, ifcholinergic innervation is eliminated by electrolytic lesion, there is a marked increase in the expression of message for mineralocorticoid and glucocorticoid receptors in the CAI pyramidal cells of the htppocampus, the area most severally effected by AD. These rewlts suggest that increased expression of these receptors message and mcreased levels of hormone lead to higher rates of neuronal loss. Little research has been done to examine other steroid receptors, specifically androgen, prostoglandin or orphan receptors, in AD. There has been a sigmficant amount of work on the effects of altered levels of estrogen in AD, but httle investigation of the receptor. Rewarch on the effects of the hormone suggest that by increasing estrogen levels in patients, one might protect neurons from oxidative stress induced cell death. These findings suggest that an alteration in steroid receptor expression might lead to an increase in the vulnerability of neurons in the brain to cellular stress as well as an increase in neuron death. We describe the protein of a novel orphan receptor, testicular receptor 2 (TR2), in post-mortem AD tissue. Data will be presented showing double immunohistochemistry for localization of TR2 protein with early markers for neurofibtillary changes in areas of human brain most severely effected by AD. Western blot analyses explore quantttative differences in the TR2 protein expression between AD and age matched control samples. Detailing changes in the expression of TR2 will lend insight into further understanding the significance of alterations in receptor expression in the pathogenesis of thi? disease.

EXPRESSION OF THE MITOTIC CDCUCYCLIN B 18251ECTOPIC KINASE IN POSTMITOTIC NEURONS TRIGGERS DEGENERATION Ine? Vincent, Li. Univ

Yuluon Mu, Janice

of Washington,

Seattle,

Hollows,

Jucob

Hu.wernun,

Andrei

Tomushevski,

Jin

WA

Neurodegeneration is a relentless outcome of age-associated dementing illnesses. irrespective of genetic or \poradtc etiology. We and others have shown that the cell cycle cdc2/cyclin B kinase and certain downstream indices of mitosis, none of which are detectable in normal postmitotic neurons, mark early stages of the neurodegenrrntive process in Alrheimer’s direaae. and a subset of disorders including Down syndrome, Progressive Supranuclear Palsy, Fronto-temporal dementia, and Pick’s disease. We have hypothesized that induction of cdc2/cyclin B is a convergent point in the neurodegenerative mechanism of these diseases. To determine whether mitotic kinase causes degeneration of mnture neurons, we have generated neuronal cell lines with conditional expression of cdc2/cyclin B. The genes were inserted into a bidrectional vector allowing simultaneous stochiometric expression driven by a single tetracycline-inducible promoter. Neuronal cells were first differentiated, and then treated with the tetracycline analog, doxycycline, to induce cdc2/cyclin B expression. Within four days post-treatment, the cells underwent degeneration, which was preceded by the appearance of mitotic indices, including production of the TG-3 and MPM-2 phosphoepitopes and the phosphorylation of RNA polymerase 11, a regulator of cellular transcription. These alterations lead to a repression of messenger RNA rynthesis and to cytoskeletal rearrangement, followed by degeneration of neuronal processes, and eventual death. In contrast to these data, expression of the cd&related cdk4 kinase in similar neurons did not result in such changes. Encouraged by this lethal effect of cdc2/cyclin B in vitro, we have developed transgenic mice with temporally-regulated, targeted expression of cdc21cyclm B in hippocampal and forebrain neurons. These mice are presently being characterized, and the results wdl be discussed at the presentation of this work. Given the dramatic effects of cdc2/cyclin B expression in differentiated neuronal cultures, new intervention strategies targeting this early degenerative stage may be sought through the application of a swath of candidate mitotic inhibitors from cancer research.

EXPRESSION DISEASE.

OF ADAPTOR

PROTEIN

AP-2 IN ALZHEIMER’S

Adaptor protein AP-2 is a specific component that determines whether clathrm coated vewle\ wdl form endocytic transport vesicles at the plasma membrane. Previously,

Presentation:

Molecular

and

Cellular

Biology

III

using immunoblot analysis we observed a moderate reduction of AP-2 protein in Alzheimer’s disease (AD) neocortex. In this study, we examined immunohistochemitally the expression of AP-2 protein in three regions of pot-r murfem brains: hippocampus, cerebellum and superior frontal gyrus. Alrheimer’samples were compared with neurological normal controls. AP-2 expression appeared as imunoreactive punctate granules that were ubiquitously expressed in neuronal cell bodies, their processes and in the neuropil. In the hippocampus and cerebellum, neither the expression pattern nor intenstty of AP-2 immunoreactivity m AD is readily distinguishable from the control. In superior frontal gyrus, expression of AP-2 in most layers of AD cortex appears to be unchanged, but, in layer II, immunreactivity for this protein was decreased significantly. These rewlts provide anatomical and spatial information on AP-2 expression in AD brains. The results suggest that the loss of AP-2 in AD may occur only in certain types of nerve termmals.

ENHANCES PHAGOCYTOSIS 1827] ESTROGEN TEIN BY MICROGLIA DERIVED FROM Rena Li. Sun Health

Research

Institute,

OF AMYLOID B PROHUMAN CORTEX

Sun Ciry. AZ

In the recent years inflammatory mechanisms have been became increasingly appreciated as important steps in the pathology of Alzheimer‘s disease (AD). Microglia are a key component of the immune system in the brain associated with AD plaques containing amyloid-b-peptide (Ap). Although there is accumulating evidence that microglia activation is important for clearing AP deposits in the AD brain, the phagocytic mechanism of microglia remains obscure. Recent findings indicate that estrogen plays an important role in delaying the development of AD and even protecting against AD. An understanding of cellular and molecular basis of estrogen’s actions is still limited. The present study was undertaken to investigate the relationship between estrogen treatment and internalization of Al3 by microglia. Human cortical microglia cultures were incubated with Apl-42 and AP uptake was measured Microghal uptake of A@ was found to be dose- and time-dependent. At high doses, AP uptake increased at 1.5 hours and continued to increase for 24 hours thereafter. Pretreatment with estrogen enhanced this effect. Estrogen receptor (ER-beta) expression also increased after estrogen treatment. Cells co-treated with ICI 182,780, an estrogen receptor antagonist, showed significantly reduction of internalization of AP by microglia cultures. These results show that microglia express estrogen receptora that may play a significant role in the clearance of AD plaques. Stimulation of the estrogen receptor might contribute to the therapeutic action of estrogen in the treatment of AD.

pig

LOCALIZATION

OF PRESENILINl

AT SYNAPTIC

SITES.

We have shown in epithelial cells that PSI IS localized at cell-cell adhesion sites where it forms complexes with components of the cadherin/catenin adheston system (Georgakopoulos et al. Mol.Cell 1999). Since PSI 1s localized in neuronal procesw (Elder et al. J.Neurosci.Res. 1996) and the components of the cadherin/catenin adhesion system are present at the neuronal synapse we tested weather PSI is also present at the synapse. Double labeling of primary neuronal cultures from rat hippocampus with antibodies against PSI/NTF and the synaptic marker synaptophysin showed that PSI localizes at the synapse. Further analysis of the colocalization of PSl with other specific pre- and post-synaptic markers is currently being carried om. Electron microscopy of mouse cerebral cortex using either immunogold or immunoperoxidase showed strong staining for both PSllNTF and PSI/CTF at synaptic areas suggesting that PSI may be a part of the cadherin/catenin synaptic complex. Indeed immunoprecipitates from mouse brain homogenates with anti-PSI antibody contained E- and N-cadhetins as well ar p-catenin but no Transferrin receptor. Consistently PSUNTF, PSIICTF, E- and N-cadherins and p-catenin comigrated in glycerol gradient centrifugation experiments indicating that they form complexes of Mr-400 KDa. The biochemical characterization of PSI fragments in mouse brain synaptosomal preparations will be further studied. Our data show that brain PSl is concentrated at synaptic adhesion sites, forms complexes with componenta of the synaptic cadherin/catenin system and suggest that this system may be a potential target for PSI FAD mutations.