Introduction:The levels and distribution of amyloid deposits in the brain does not correlate well with Alzheimer's disease (AD) progression. Therefore, it is likely that amyloid precursor protein and its proteolytic fragments other than amyloid b (Ab) contribute to the onset of AD. Methods:We developed a sensitive assay adapted to the detection of C99, the direct precursor of b-amyloid. Three postmortem groups were studied: control with normal and stable cognition; patients with moderate AD, and individuals with severe AD. The amount of C99 and A was quantified and correlated with the severity of AD.Results: C99 accumulates in vulnerable neurons, and its levels correlate with the degree of cognitive impairment in patients suffering from AD. In contrast, A levels are increased in both vulnerable and resistant brain areas. Discussion:These results raise the possibility that C99, rather than A plaques, is responsible for the death of nerve cells in AD.A deposits in AD brains has a poor correlation with dementia severity, loss of neural function, and cognitive impairment. 4-9 Therefore, it is possible that APP metabolites other than A make a significant contribution to AD pathophysiology. In addressing this issue, a challenge has been the inability to determine the distribution and levels of specific APP metabolites in intact cells and tissues. We have approached this problem by adapting the proximity ligation assay (PLA) for the detection of C99. In this assay, two primary antibodies recognize the target epitopes on the protein of interest. If the epitopes are in close proximity, secondary antibodies covalently bound to complementary DNA strands participate in rolling circle DNA synthesis. The DNA synthesis reaction results in a one thousand-fold amplification of the DNA circle. Fluorescently labeled complementary oligonucleotide Alzheimer's Dement. 2020;16:273-282.wileyonlinelibrary.com/journal/alz 273

Introduction The levels and distribution of amyloid deposits in the brain does not correlate well with Alzheimer's disease (AD) progression. Therefore, it is likely that amyloid precursor protein and its proteolytic fragments other than amyloid b (Ab) contribute to the onset of AD. Methods We developed a sensitive assay adapted to the detection of C99, the direct precursor of b‐amyloid. Three postmortem groups were studied: control with normal and stable cognition; patients with moderate AD, and individuals with severe AD. The amount of C99 and Aβ was quantified and correlated with the severity of AD. Results C99 accumulates in vulnerable neurons, and its levels correlate with the degree of cognitive impairment in patients suffering from AD. In contrast, Aβ levels are increased in both vulnerable and resistant brain areas. Discussion These results raise the possibility that C99, rather than Aβ plaques, is responsible for the death of nerve cells in AD.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by the accumulation of beta-amyloid (Aβ), C99, and Tau in vulnerable areas of the brain. Despite extensive research, current strategies to lower Aβ levels have shown limited efficacy in slowing the cognitive decline associated with AD. Recent findings suggest that C99 may also play a crucial role in the pathogenesis of AD. Our laboratory has discovered that CK1γ2 phosphorylates Presenilin 1 at the γ-secretase complex, leading to decreased C99 and Aβ levels. Thus, CK1γ2 activation appears as a promising therapeutic target to lower both C99 and Aβ levels. In this study, we demonstrate that CK1γ2 is inhibited by intramolecular autophosphorylation and describe a high-throughput screen designed to identify inhibitors of CK1γ2 autophosphorylation. We hypothesize that these inhibitors could lead to CK1γ2 activation and increased PS1-Ser367 phosphorylation, ultimately reducing C99 and Aβ levels. Using cultured cells, we investigated the impact of these compounds on C99 and Aβ concentrations and confirmed that CK1γ2 activation effectively reduces their levels. Our results provide proof of concept that CK1γ2 is an attractive therapeutic target for AD. Future studies should focus on the identification of specific compounds that can inhibit CK1γ2 autophosphorylation and evaluate their efficacy in preclinical models of AD. These studies will pave the way for the development of novel therapeutics for the treatment of AD.