RAC1 Inhibition Targets Amyloid Precursor Protein Processing by γ-Secretase and Decreases Aβ Production in Vitro and in Vivo

Abstract

␤-Amyloid peptides (A␤) that form the senile plaques of Alzheimer disease consist mainly of 40-and 42-amino acid (A␤ 40 and A␤ 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of A␤ involves ␤-secretase and ␥-secretase activities and is regulated by membrane trafficking of the proteins involved in A␤ production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks A␤ 40 and A␤ 42 production but does not impact sAPP␣ levels and does not inhibit ␤-secretase. Rather, EHT 1864 modulates APP processing at the level of ␥-secretase to prevent A␤ 40 and A␤ 42 generation. This effect does not result from a direct inhibition of the ␥-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces A␤ 40 and A␤ 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting A␤ formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease. Alzheimer disease (AD)2 is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterized by the presence of amyloid plaques (1), intracellular neurofibrillary tangles, and pronounced cell death. The ␤-amyloid peptide (A␤) (2) is the main constituent of senile plaques found in AD brains. Furthermore, extracellular A␤ 42 appears toxic to neurons in vitro and in vivo (reviewed in Ref.3). A␤ is generated by proteolysis of an integral membrane protein, the amyloid precursor protein (APP), via at least two post-translational pathways. The amyloidogenic cleavage of APP is a sequential processing of APP initiated by ␤-secretase (BACE), which cleaves APP within the luminal domain or at the cell surface, generating the N terminus of A␤ (4). This cleavage generates several membrane-bound proteolytic C-terminal fragments (CTFs), such as the 99-residue ␤-CTF (also called C99), as well as the secreted APP ectodomain sAPP␤. The C terminus of A␤ is subsequently generated by intramembranous cleavage of CTFs by ␥-secretase, producing either A␤ 40 or A␤ 42. The cleavages at residues 40 -42 are referred to as ␥-cleavage, and the cleavages at residues 49 -52 are referred to as ⑀-cleavage (5). The nonamyloidogenic cleavage of APP, which precludes A␤ generation, is mediated by ␣-secretase, a disintegrin and metalloproteinase 10, and a disintegrin and metalloproteinase 17, in a reaction believed to occur primarily on the plasma membrane. This proteolytic cleavage by ␣-secretase occurs within the A␤ region and produces soluble APP (sAPP␣), the dominant processing product, and the residual membrane-bound 10-kDa CTF (CTF␣, also called C83). Like C99, C83 is a subs...

Transcription Factors NRF2 and NF-κB Are Coordinated Effectors of the Rho Family, GTP-binding Protein RAC1 during Inflammation

“…In Fragile X syndrome, the most common hereditary form of mental retardation, lack of Fragile X mental retardation protein (FMRP) induced an overactivation of RAC1 in the mouse brain, indicating that regulation of RAC1 may provide a functional link among deficient neuronal morphology, aberrant synaptic plasticity, and cognition impairment in this disease (31). Interestingly, in Alzheimer disease it has been found that RAC1 inhibition targets amyloid precursor protein processing by ␥-secretase and decreases A␤ production in vitro and in vivo (32). But RAC1 is not only involved in neurodegenerative disorders.…”

“…-To investigate the implication of the canonical pathway of NF-B in the effects mediated by RAC1 Q61L , we used a dominant-negative mutant of IB␣ (IB␣ S32A/S36A ) in which Ser 32 and Ser 36 residues had been changed to Ala to block their phosphorylation and, consequently, their degradation. IB␣ S32A/S36A produced a dose-dependent inhibition of NF-B by RAC1 Q61L (Fig.…”

Direct Modulation of Small GTPase Activity and Function

“…[53] Another study identified EHT1864 (3)asaRac1 inhibitor,which effectively reduces Ab peptide levels in models of Alzheimers diesease in vitro (Figure 3a). [54] Further investigation of the mode of action led to the postulate that the compound tightly binds Rac1 with low nanomolar affinity (dissociation constant K d = 40 nm). Compound 3 acts through an oncompetitive mechanism, as observed for compound 2,which induces nucleotide release upon binding to Rac1.…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…67 Therefore, we hypothesized that electrotransfection could lead to actin remodeling and enhance macropinocytosis through activation of Cdc42 and Rac1. To test this hypothesis, we performed experiments with two pharmacological inhibitors, ML141 68 and EHT1864, 69 that targeted Cdc42 and Rac1, respectively. We chose these inhibitors because they are potent, selective, and most importantly reversible, which allowed us to observe the inhibitory effects without permanently interfering with cell signaling.…”