Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.

https://www.jneurosci.org/content/jneuro/28/27/6926.full.pdf

Autophagy Induction and Autophagosome Clearance in Neurons: Relationship to Autophagic Pathology in Alzheimer's Disease

Professional phagocytes have a vast and sophisticated arsenal of microbicidal features. They are capable of ingesting and destroying invading organisms, and can present microbial antigens on their surface, eliciting acquired immune responses. To survive this hostile response, certain bacterial species have developed evasive strategies that often involve the secretion of effectors to co-opt the cellular machinery of the host. In this Review, we present an overview of the antimicrobial defences of the host cell, with emphasis on macrophages, for which phagocytosis has been studied most extensively. In addition, using Mycobacterium tuberculosis, Listeria monocytogenes, Legionella pneumophila and Coxiella burnetii as examples, we describe some of the evasive strategies used by bacteria.

/pdf/antimicrobial-mechanisms-of-phagocytes-and-bacterial-evasion-238mnfz1n0.pdf

Antimicrobial mechanisms of phagocytes and bacterial evasion strategies

Unraveling the role of proteases in cancer.

https://corpus.ulaval.ca/jspui/bitstream/20.500.11794/70644/1/JBiolChem_2003.pdf

Studies on the Internalization Mechanism of Cationic Cell-penetrating Peptides *

Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design.

The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H(+)-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

/pdf/endolysosomal-proteolysis-and-its-regulation-1vec35xwpk.pdf

Endolysosomal proteolysis and its regulation.

Alterations in trafficking of cathepsins B and D have been reported in human and animal tumors. In MCF10 human breast epithelial cells, altered trafficking of cathepsin B occurs during their progression from a preneoplastic to neoplastic state. We now show that this is also the case for altered trafficking of cathepsin D. Nevertheless, the two cathepsins are not necessarily trafficked to the same vesicles. Perinuclear vesicles of immortal MCF10A cells label for both cathepsins B and D, yet the peripheral vesicles found in ras-transfected MCF10AneoT cells label for cathepsin B, cathepsin D or both enzymes. Studies at the electron microscopic level confirm these findings and show in addition surface labeling for both enzymes in the transfected cells. By immunofluorescence staining, cathepsin B can be localized on the outer surface of the cells. Similar patterns of peripheral intracellular and surface staining for cathepsin B are seen in the human breast carcinoma lines MCF7 and BT20. We suggest that the altered trafficking of cathepsins B and D may be of functional significance in malignant progression of human breast epithelial cells. Translocation of vesicles containing cathepsins B and D toward the cell periphery occurs in human breast epithelial cells that are at the point of transition between the pre-neoplastic and neoplastic state and remains part of the malignant phenotype of breast carcinoma cells.

Cathepsin B and D are Localized at the Surface of Human Breast Cancer Cells.

Of the cysteine proteases, cathepsin B has been most often implicated in tumor progression. Accumulating evidence indicates that the regulation of cathepsin B and other cysteine proteases is under both transcriptional and posttranscriptional control. The elucidation of such control mechanisms may be important for therapeutic strategies aimed at reducing cathepsin B overexpression in tumors and other pathologies. Transcription initiation at more than one initiation site and/or the induction of alternative splicing of mRNA may lead to the production of transcripts that differ in their rate of translation or their stability. Hence, the amount of protein produced may not be proportional to the amount of mRNA observed in tissues. Transcripts lacking exon 3 would encode a truncated procathepsin B, a form predicted to be trafficked to the cytoplasm due to the absence of its signal peptide. Evidence of alterations in trafficking of cathepsin B and distribution of organelles labeling for cathepsin B to more peripheral, basolateral or cell membrane localizations is seen in bladder, breast, colon, prostate and thyroid carcinomas and in gliomas. These may reflect alterations in the cytoskeleton as similar changes in localization of cathepsin B accompany the rearrangement of cytoskeletal elements and the acquisition of an invasive phenotype in MCF-10A breast epithelial cell lines after transfection with oncogenic c-Ha-ras. Alterations in trafficking of cathepsin B and other cysteine proteases may thus be brought about by changes in signal transduction, induced by transfection with an activated oncogene. Signal-transduction pathways as well as the enzymes directly involved in the invasive degradation of basement membranes may therefore both be targets for therapeutic intervention. The results of recent studies suggest that cathepsins B and L are more stable in the extracellular environment than previously believed and so could play a significant extracellular role in invasive cancer.

Edith Elliott

Papers

Endolysosomal proteolysis and its regulation.

Cathepsin B and D are Localized at the Surface of Human Breast Cancer Cells.

The cysteine protease cathepsin B in cancer

Neutrophil tissue inhibitor of matrix metalloproteinases-1 occurs in novel vesicles that do not fuse with the phagosome

Anti-peptide antibodies to cathepsins B, L and D and type IV collagenase. Specific recognition and inhibition of the enzymes.