Preventing Pathology in a Transgenic Mouse Model of Alzheimer's Disease Following Over-Expression of BCL-2

Publication Date

12-2007

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Biology

Department

Biology

Supervisory Committee Chair

Troy Rohn

Supervisory Committee Member

Julia Oxford

Supervisory Committee Member

Juliette Tinker

Abstract

Alzheimer's disease (AD) is characterized by the accumulation of senile plaques containing beta-amyloid (Aβ) and neurofibrillary tangles (NFTs), manifesting in symptoms involving memory loss as well as difficulties with language and other cognitive impairments. Although Aβ deposition is thought to precede the formation of NFTs in AD and represent one of the earliest known steps in the disease process, the molecular steps connecting these two pathologies is not known. Recent studies have suggested caspase activation and the cleavage of tau as an interconnecting step linking senile plaques with neurofibrillary tangles in Alzheimer's disease.

To test the hypothesis that caspase activation plays a central role in the pathology associated with AD, a triple transgenic Alzheimer's mouse model (3xTg-AD) overexpressing the anti-apoptotic protein, Bcl-2, was generated. To determine whether caspases cleave tau in vivo, fixed tissue sections from 3xTg-AD mice and 3xTg-AD mice overexpressing Bcl-2 (3xTg-AD/Bcl-2 OE) were examined by immunohistochemistry employing an antibody that specifically detects caspase-cleaved tau (Tau-CCP). In 3xTg- AD mice overexpressing Bcl-2, reduced labeling with TauCCP was observed which was limited primarily to the apical dendrites, whereas neuronal CA1 labeling was evident within the cytoplasm and apical dendrites of 3xTg-AD mice. Western blot analysis confirmed the immunohistochemical findings. Tissue sections were also examined for the accumulation of full length human tau usmg HT7 antibody. 3xTg-AD mice overexpressmg Bcl-2 showed an enhanced intraneuronal labeling of human tau as compared to 3xTg-AD mice alone. Western blot analysis of total mouse brain lysates from 12 month-old animals showed increased levels of human tau in 3xTg-AD mice overexpressing Bcl-2 following application ofHT7.

Studies were extended to 18 month-old animals to determine if the formation of NFT's is prevented in 3xTg-AD mice overexpressing Bcl-2. Tissue sections from 18 month-old cohorts were processed for immunohistochemistry using a panel of tangle markers. Immuno-positive neurons were identified in the CA1 region of the hippocampus of 3xTg-AD mice, whereas no neuronal staining was observed in 3xTg-AD mice overexpressing Bcl-2.

There is also evidence that the amyloid precursor protein (APP), the precursor molecule for Aβ, is also a target for caspase-mediated cleavage. To test this hypothesis, immunohistochemical analysis was performed in 3xTg-AD mice alone and 3xTg-AD mice overexpressing Bcl-2 using monoclonal antibody 1560, which detects both full-length APP as well as the Aβ fragment. The 3xTg-AD mice showed immunolabeling for APP limited to the cytoplasm of cortical neurons. In contrast, intense staining was observed in the neocortex in animals overexpressing Bcl-2, with an apparent accumulation of human APP within the cytoplasm and apical dendrites. To determine if intracellular labeling represented primarily APP or Aβ, an anti-APP specific antibody, 22C11 was employed. In 3xTg-AD mice overexpressing Bcl-2, there was strong immunoreactivity to 22C11 in the neocortex compared to 3xTg-AD mice alone. Finally, to confirm these findings, an antibody specific for Aβ (1-42) detected intracellular Aβ immunoreactivity in neurons within the neocortex of 3xTg-AD mouse whereas lower intracellular levels of Aβ were present in 3xTg-AD mice overexpressing Bcl-2.

In summary, my results indicate that 3xTg-AD mice overexpressing Bcl-2 are not only capable of preventing the caspase cleavage of tau, but also block the formation of NFT's and the Aβ plaques. Age based study of 3xTg-AD and 3xTg-AD/Bcl-2 OE mice indicate that caspase cleavage of tau is an early event in the pathology of AD. Overall, the results indicate an important role of caspases in the pathology of AD.

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