Advanced Assay Monitoring APP-Carboxyl-Terminal Fragments as Markers of APP Processing in Alzheimer Disease Mouse Models

Abstract

The 99-amino-acid-long APP-carboxy-terminal fragment, named C99, is a membrane-bound peptide generated from the amyloid precursor protein (APP) by β-secretase cleavage and is the direct precursor of amyloid beta (Aβ). Here we describe a method for the quantification of C99. The amount of C99 is an indicative value of the amyloid pathology in an Alzheimer's disease (AD) model, and could be used as a marker to study AD progression in comprehensive experiments, including screening for new compounds and repurposing of drugs to treat AD.

Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats

“…We next examined the formation of amyloid plaques, another marker of neurodegeneration, by Congo red staining or immunoblot analysis using the anti-CT19 antibody recognizing the amyloid precursor protein (APP) carboxy-terminal C99 and C83 fragments [25] in the brains of HIV-1 Tg rats or WT. The number and size of amyloid plaques, stained with Congo red, were significantly elevated in the cerebral cortex of HIV-1 Tg rats compared to those of WT (Fig 4A and Figures E-G in S1 File), despite a relatively small number of amyloid plaques, compared to the amyloid plaques frequently observed in autopsied brain specimens from Alzheimer disease patients.…”

“…The number and size of amyloid plaques, stained with Congo red, were significantly elevated in the cerebral cortex of HIV-1 Tg rats compared to those of WT (Fig 4A and Figures E-G in S1 File), despite a relatively small number of amyloid plaques, compared to the amyloid plaques frequently observed in autopsied brain specimens from Alzheimer disease patients. In addition, the immuno-reactive levels of amyloid C-terminal fragment C99, which is critical for producing toxic amyloid-β peptides (e.g., Aβ1–42) [25, 26], were significantly elevated (i.e., by more than 5-fold) in the brains of HIV-1 Tg rats compared to WT (Fig 4B). However, under our conditions, the relatively less-toxic C83 amyloid fragment [26] was not clearly observed in the HIV-1 Tg and WT rats.…”

“…Scale bar, 200 μm. (B) Representative immunoblots for detecting APP and toxic amyloid C-terminal C99 fragment [25, 26] with the anti-CT19 antibody are shown. The densitometric results for the immunoblots (n = 4/group) are presented below.…”

“…Increased oxidative stress and inflammatory cytokines such as such as TNF-α are also known to activate (phosphorylate) MAPKs such as cell-death associated JNK and p38K [23, 40] possibly through suppression of their respective phosphatases [78]. JNKs and p38K are the major brain MAPKs and critically important in neuronal cell death and neurodegeneration through phosphorylation of tau [35, 79, 80] and amyloid precursor protein followed by the production of a membrane-associated amyloid C99 fragment [25]. In theory, activated p-JNK or p-P38K can phosphorylate tau at different amino acids such as Thr181, Ser199, Ser202, Thr205, Thr231, Ser396, Ser404, etc [35].…”

A First-in-Class Small-Molecule that Acts as a Dual Inhibitor of HDAC and PDE5 and that Rescues Hippocampal Synaptic Impairment in Alzheimer’s Disease Mice

“…For hAPP processing and tau phosphorylation (pTau), neurons were treated for 2 days at 100 nM and collected at day 3, 24 h after the last treatment. The effect of CM-414 on hAPP processing by the amyloidogenic pathway was analyzed by western blot analysis measuring the 99-amino-acid-long APP-carboxyterminal fragments (APP-CTFs) designated C99, the precursor of Aβ 42 (Garcia-Osta and Cuadrado-Tejedor, 2016). We next analyzed the effect of CM-414 on levels of phosphorylated tau in the same samples using a phosphospecific antibody (AT8) that recognizes hyperphosphorylated epitopes on Ser202/Thr205.…”