Urmi Bandyopadhyay

Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimers disease brain contributes to Alzheimers disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimers disease mouse model TgCRND8 similar to that previously described in Alzheimers disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimers disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimers disease.

The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane

ABSTRACT Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane.

Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age

Rates of autophagy, the mechanism responsible for lysosomal clearance of cellular components, decrease with age. We have previously described an age-related decline in chaperone-mediated autophagy (CMA), a selective form of autophagy, by which particular cytosolic proteins are delivered to lysosomes after binding to the lysosome-associated membrane protein type 2A (LAMP-2A), a receptor for this pathway. Rates of CMA decrease with age because of a decrease in the levels of LAMP-2A. In this work we have investigated the reasons for the reduced levels of LAMP-2A with age. While transcriptional rates of LAMP-2A remain unchanged with age, the dynamics and stability of the receptor in the lysosomal compartment are altered. The mobilization of the lysosomal lumenal LAMP-2A to the membrane when CMA is activated is altered in lysosomes from old animals, leading to the presence of an unstable pool of lumenal LAMP-2A. By contrast, the regulated cleavage of LAMP-2A at the lysosomal membrane is reduced owing to altered association of the receptor and the protease responsible for its cleavage to particular membrane microdomain regions. We conclude that age-related changes at the lysosomal membrane are responsible for the altered turnover of the CMA receptor in old organisms and the consequent decline in this pathway.

Chaperone-mediated autophagy at a glance.

Chaperone-mediated autophagy (CMA) is an intracellular catabolic pathway that mediates the degradation of a selective subset of cytosolic proteins in lysosomes ([Dice, 2007][1]; [Cuervo, 2010][2]; [Kon and Cuervo, 2010][3]; [Orenstein and Cuervo, 2010][4]). The term autophagy (or self-eating) is

Identification of Regulators of Chaperone-Mediated Autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of cytosolic proteins in lysosomes that contributes to cellular quality control and becomes an additional source of amino acids when nutrients are scarce. A chaperone complex delivers CMA substrates to a receptor protein at the lysosomal membrane that assembles into multimeric translocation complexes. However, the mechanisms regulating this process remain, for the most part, unknown. In this work, we have identified two regulatory proteins, GFAP and EF1alpha, that mediate a previously unknown inhibitory effect of GTP on CMA. GFAP stabilizes the multimeric translocation complex against chaperone-mediated disassembly, whereas GTP-mediated release of EF1alpha from the lysosomal membrane promotes self-association of GFAP, disassembly of the CMA translocation complex, and the consequent decrease in CMA. The dynamic interactions of these two proteins at the lysosomal membrane unveil now a role for GTP as a negative regulator of CMA.

Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimers disease, and other neurodegenerative disorders.

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

Entering the lysosome through a transient gate by chaperone-mediated autophagy.

A subset of cytosolic proteins can be selectively degraded in lysosomes through chaperone-mediated autophagy. The lysosomal-membrane protein type 2A (LAMP-2A) acts as the receptor for the substrates of chaperone-mediated autophagy (CMA), which should undergo unfolding before crossing the lysosomal membrane and reaching the lumen for degradation. Translocation of substrates is assisted by chaperones on both sides of the membrane, but the actual steps involved in this process and the characteristics of the translocation complex were, for the most part, unknown. We have now found that rather than a stable translocon at the lysosomal membrane, CMA substrates bind to monomers of LAMP-2A driving the organization of this protein into a high molecular weight multimeric complex that mediates translocation. Assembly and disassembly of LAMP-2A into and from this complex is dynamic and it is regulated by hsc70 and hsp90, the two lysosomal chaperones related to CMA. This work thus unveils a unique mechanism of protein translocation across the lysosomal membrane, which involves only transient discontinuity of the membrane. The possible advantages of this transitory lysosomal translocon are discussed in light of the unique properties of the lysosomal compartment. Addendum to: Bandyopadhyay U, Kaushik S, Vartikovski L, Cuervo AM. Dynamic organization of the receptor for chaperone-mediated autophagy at the lysosomal membrane. Mol Cell Biol 2008; 28:5747-63; DOI: 10.1128/MCB.02070-07.

P1-059: Cystatin B deletion in a mouse model of Alzheimer's disease, TgCRND8, ameliorates both autophagic-lysosomal and amyloid pathologies

(11-12 months) and elderly (16-18 months) offspring. Following an assessment of neuromuscular function using the SHIRPA protocol, the spatial reference memories of these mice were tested using the Morris water maze. The protocol consisted of 3 days visible platform training, followed by 9 days hidden platform training. Probe trials, where the platform was removed and the mice were left to swim for 1min, were conducted on days 4, 7 and 10 of the hidden platform training. Mouse performance was recorded and assessed using the Ethovision analysis package. Results: X11 overexpression did not alter cognitive function as single transgenic X11 mice performed similarly to wild-type littermates in all age groups. In contrast, and consistent with previous reports, elderly APPswe mice performed significantly worse than their wild-type littermates, displaying increased latency to platform (p 0.026), and reduced memory retention as assessed using probe trials (p 0.950 target vs. opposite quadrant, day 10). Interestingly, APPswe/X11 double transgenic mice performed significantly better than their APPswe single transgenic littermates, displaying latencies (p 0.031, APPswe/X11 vs. APPswe) and memory retention (p 0.001, target vs. opposite quadrant, day 10) similar to those seen in their wild-type littermates. A similar result was observed in the middleaged cohort: APPswe mice displayed reduced memory retention (p 0.177 target vs. opposite quadrant, day 10), while their APPswe/X11 double transgenic littermates displayed retention (p 0.003, target vs. opposite quadrant, day 10) comparable to wild-type mice (p 0.001 target vs. opposite quadrant, day 10). Young mice displayed no memory deficiencies, regardless of genotype (p 0.001 target vs opposite quadrant, day 10). Conclusions: These data suggest that overexpression of X11 , in addition to decreasing the cerebral A load, reduces cognitive impairment in middle-aged and elderly Tg2576 mice.