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1.  RNA-Seq Profiling of Spinal Cord Motor Neurons from a Presymptomatic SOD1 ALS Mouse 
PLoS ONE  2013;8(1):e53575.
Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ∼10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5–6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.
doi:10.1371/journal.pone.0053575
PMCID: PMC3536741  PMID: 23301088
2.  Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis 
Autophagy  2011;7(7):788-789.
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 therapeutic strategy for AD.
doi:10.4161/auto.7.7.15596
PMCID: PMC3359468  PMID: 21464620
autophagy; lysosome; cathepsin; cystatin B; proteolysis; Alzheimer disease; transgenic
3.  Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits 
Brain  2010;134(1):258-277.
Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer’s disease brain contributes to Alzheimer’s 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 Alzheimer’s disease mouse model TgCRND8 similar to that previously described in Alzheimer’s 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 Alzheimer’s disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer’s disease.
doi:10.1093/brain/awq341
PMCID: PMC3009842  PMID: 21186265
autophagy; lysosome; cystatin B; cathepsin; Alzheimer’s disease
4.  NOVEL REGULATORS OF CHAPERONE-MEDIATED AUTOPHAGY 
Molecular cell  2010;39(4):535-547.
Summary
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 EF1α, 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 EF1α 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 negative regulator of CMA.
doi:10.1016/j.molcel.2010.08.004
PMCID: PMC2945256  PMID: 20797626
5.  Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines 
PLoS ONE  2010;5(3):e9819.
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, Alzheimer's disease, and other neurodegenerative disorders.
doi:10.1371/journal.pone.0009819
PMCID: PMC2843718  PMID: 20352108
6.  Entering the lysosome through a transient gate by chaperone-mediated autophagy 
Autophagy  2008;4(8):1101-1103.
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.
PMCID: PMC2832301  PMID: 18927485
autophagy; chaperones; membrane dynamics; membrane proteins; protein translocation
7.  The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane ▿ †  
Molecular and Cellular Biology  2008;28(18):5747-5763.
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.
doi:10.1128/MCB.02070-07
PMCID: PMC2546938  PMID: 18644871

Results 1-7 (7)