Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits

Abstract

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimer's disease.

Blocking IGF Signaling in Adult Neurons Alleviates Alzheimer's Disease Pathology through Amyloid-β Clearance

“…Meanwhile, inactivation of IGF-1R did not cause any noticeable motor or balance deficits in rotarod test (data not shown). As regards the observed sex differences in cognitive amelioration, our data are in line with studies revealing more advanced AD phenotype in females (Wang et al, 2003;Hirata-Fukae et al, 2008;Minami et al, 2010), and not indicative of sex-specific effects of IGF-1R inactivation on disease progression. Thus, our findings clearly indicated that cognitive and behavioral defects in female APP/PS1 mice were significantly alleviated by IGF-1R inactivation induced at 2 months of age.…”

“…Regardless of genotype, males displayed 28 -58% fewer plaques and half the levels of A␤ peptides found in females (Fig. 3 A, B), consistent with studies reporting higher amyloid burden and plaque prevalence in female APP/ PS1 mice (Wang et al, 2003;Hirata-Fukae et al, 2008;Minami et al, 2010). We next measured A␤ in cerebral cortex homogenates from 23-month-old mice.…”

“…In macroautophagy, damaged organelles and proteins are sequestered in autophagosomes that fuse with lysosomes for hydrolysis. Accumulation of autophagosomes has been described in AD brain as a result of disturbed autophagy-lysosomal degradation (Nixon, 2013). Interestingly, we found a 26% decrease in autophagosome marker protein LC3-II in cortical homogenates of ADINKO mice compared with controls ( Fig.…”

“…8A, top), indicating sustained downregulation of neuronal PI3K-Akt-mTOR signaling. As IGF-1R controls protein synthesis and autophagy through downstream PI3K-Akt-mTOR pathways, known to be defective in AD Nixon, 2013), we assessed selected autophagy markers. In macroautophagy, damaged organelles and proteins are sequestered in autophagosomes that fuse with lysosomes for hydrolysis.…”

Esterification of 24S-OHC induces formation of atypical lipid droplet-like structures, leading to neuronal cell death

“…It should be noted, however, with regard to physiological relevance, that it is not clear that the changes observed in our present study would necessarily occur in accompaniment to the increase in CYP46A1 expression (14)(15)(16) or the increase in brain 24S-OHC level that has been observed in early-and middlestage AD (11)(12)(13). Whereas LD structures have been observed in brain tissue from AD patients and in AD mouse models (46)(47)(48), there remains a need to elucidate whether an increase in 24S-OHC esters and formation of atypical LD-like structures, as reported in our present study, occur in vivo as a consequence of the normal progression of AD.…”