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Adeno-associated virus (AAV) gene therapy for neurological disease has gained traction due to stunning advances in capsid evolution for CNS targeting. With AAV brain delivery now in focus, conventional improvements in viral expression vectors offer a complementary route for optimizing gene delivery. We previously introduced a novel AAV gene therapy to slow amyloid aggregation in the brain based on neuronal release of an Aβ sequence variant that inhibited fibrilization of wild-type Aβ. Here we explore three coding elements of the virally delivered DNA plasmid in an effort to maximize the production of therapeutic peptide in the brain. We demonstrate that simply replacing the Gaussia luciferase signal peptide with the mouse immunoglobulin heavy chain signal peptide increased release of variant Aβ by ∼5-fold. Sequence modifications within the expressed minigene further increased peptide release by promoting γ-secretase cleavage. Addition of a cytosolic fusion tag compatible with γ-secretase interaction allowed viral transduction to be tracked by immunostaining, independent from the variant Aβ peptide. Collectively these construct modifications increased neuronal production of therapeutic peptide by 10-fold upon intracranial AAV injection of neonatal mice. These findings demonstrate that modest changes in expression vector design can yield substantial gains in AAV efficiency for therapeutic applications.

Epidemiological studies have linked type 2 diabetes mellitus (T2DM) with an increased risk of developing Alzheimer's disease (AD). In T2DM, the elevated blood glucose level promotes formation of advanced glycation end products (AGEs). The receptor for AGEs (RAGE) is a type I membrane-protein and is also able to import amyloid-β (Aβ) from the blood across the blood-brain-barrier into the brain. Oligomeric Aβ peptides disturb synaptic function in the brain and are believed to contribute to the development of AD. Aβ peptides are released from the amyloid-β protein precursor (AβPP) after sequential proteolysis by β- and γ-secretases but α-secretase-mediated cleavage of AβPP prevents Aβ generation. Insulin influences Aβ production by modulating α-secretase activity and Aβ degradation. Recent publications demonstrate that RAGE is subjected to protein ectodomain shedding. Proteolysis of RAGE occurs constitutively and is inducible by activation of protein kinase C. Alpha-secretase-like enzymes release the ligand binding domain of RAGE from the cell surface and after that γ-secretase processes the membrane-remaining part of RAGE. Proteolysis of RAGE may represent a regulatory mechanism in RAGE signal transduction and in addition may prevent Aβ peptide transport across the blood-brain-barrier. Current data suggest that the sequential proteolysis of RAGE is homologous to AβPP processing. [ABSTRACT FROM AUTHOR]

Abstract: Here we show that prolyl isomerase Pin1 is involved in the Aβ production central to the pathogenesis of Alzheimer’s disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Aβ40 and Aβ42 was lower than that of the wild-type mice, indicating that Pin1 promotes Aβ production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1 −/− MEF transfected with C99, Pin1 co-transfection enhanced the levels of Aβ40 and Aβ42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Aβ40 and Aβ42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its γ-cleavage, generating Aβ40 and Aβ42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Aβ generation, explaining the inhibitory effect of lithium on Aβ generation. [Copyright &y& Elsevier]

Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aβ metabolism through multiple mechanisms.