REVERSAL OF LYSOSOMAL DYSFUNCTION RESCUES BETA-AMYLOID PATHOGENESIS IN ALZHEIMER’S DISEASE

Abstract

Amyloid beta (Aβ) deposit is a major neuropathological hallmark of Alzheimer's disease (AD). It has been identified that intracellular Aβ (iAβ) accumulation is one of the crucial events in AD pathogenesis. Amount of evidence suggest that insufficient cellular clearance of iAβ results from lysosomal dysfunctions and enhancement of lysosomal function restores AD pathology. Therefore, identifying genes that promote the clearance of iAβ by reinforcing lysosomal activity and discovering the underlying mechanism are vital for AD intervention. For cell-based functional screening, cells were transfected with cDNA encoding lysosome-associated proteins and then further incubated with synthetic oligomeric Aβ1–42. Cytotoxicity was measured by staining with EthD. Hippocampal homogenates from AD patients with Braak stage V or VI and non-AD patients were provided by the Harvard Brain Tissue Resource Center (McLean Hospital, Belmont, Massachusetts, USA). For detection of intracellular Aβ1–42, membrane-associated Aβ1–42were washed with trypsin-EDTA and harvested cells were subjected to immunoblotting using 4G8 and 6E10 antibodies. Activity-defective mutant HYAL D129A/E131A was generated by mutagenesis. For lysosomal activity assay using fusion protein degraded by lysosome, GFP-CINCCKVL was amplified from pEGFP-C1 vector using synthesized primers (EGFP-5′-HindIII, EGFP-CINCCKVL-3′-EcoRI) and subcloned into pcDNA3.0. HYAL was isolated as the most effective gene that suppressed Aβ-induced cell death. Compared to normal controls, HYAL expression was significantly decreased in AD brains. Downregulation of HYAL aggravated Aβ-induced cell death and increased iAβ level. In overexpression assay, HYAL dramatically decreased Aβ-neurotoxicity and reduced iAβ level. An activity-defective mutant altered neither Aβ-induced toxicity nor the iAβ level, indicating that catalytic activity is critical for its protective role. Degradation of iAβ was halted by lysosomal inhibitors but not by proteasomal inhibitor, indicating that iAβ is degraded mostly by lysosomal pathway. In lysosomal activity assay, ectopically expressed HYAL hindered abnormal accumulation of lysosomal substrates due to Aβ1–42 exposure, suggesting that HYAL serve as positive lysosomal regulators in Aβ pathogenesis. These results suggest that HYAL exerts a protective role by facilitating Aβ degradation, possibly through enhancing lysosomal function. Reference: (1) Frank M. et al. Nat. Rev. Neurosci. 2007; 430(7000):499–509. (2) Nixon, R.A., and Yang, D.-S. Neurobiol. Dis. 2011; 43, 38–45.