Philip Stavrides

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.

Neuronal Apoptosis and Autophagy Cross Talk in Aging PS/APP Mice, a Model of Alzheimer's Disease

Mechanisms of neuronal loss in Alzheimers disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved alpha-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration.

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.

Specific Calpain Inhibition by Calpastatin Prevents Tauopathy and Neurodegeneration and Restores Normal Lifespan in Tau P301L Mice

Tau pathogenicity in Alzheimers disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

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

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimers 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 Alzheimers disease.

Declining phosphatases underlie aging-related hyperphosphorylation of neurofilaments

Cytoskeletal protein phosphorylation is frequently altered in neuropathologic states but little is known about changes during normal aging. Here we report that declining protein phosphatase activity, rather than activation of kinases, underlies aging-related neurofilament hyperphosphorylation. Purified PP2A or PP2B dephosphorylated the heavy neurofilament (NFH) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting either phosphatase induced NFH phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after NFH expression plateaued at p21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.

Cyclodextrin has conflicting actions on autophagy flux in vivo in brains of normal and Alzheimer model mice.

Abstract 2‐hydroxypropyl‐&bgr;‐cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo‐lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann‐Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild‐type mice and TgCRND8 mice—an Alzheimers Disease (AD) model exhibiting &bgr;‐amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14‐day intracerebroventricular administration of CYCLO to 8‐month‐old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and A&bgr;‐immunoreactivity without detectably altering amyloid precursor protein processing or extracellular A&bgr;/&bgr;‐amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome‐lysosome fusion as evidenced by the accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

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.

Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo

ABSTRACT Autophagy-lysosome pathway (ALP) disruption is considered pathogenic in multiple neurodegenerative diseases; however, current methods are inadequate to investigate macroautophagy/autophagy flux in brain in vivo and its therapeutic modulation. Here, we describe a novel autophagy reporter mouse (TRGL6) stably expressing a dual-fluorescence-tagged LC3 (tfLC3, mRFP-eGFP-LC3) by transgenesis selectively in neurons. The tfLC3 probe distributes widely in the central nervous system, including spinal cord. Expression levels were similar to endogenous LC3 and induced no detectable ALP changes. This ratiometric reporter registers differential pH-dependent changes in color as autophagosomes form, fuse with lysosomes, acidify, and degrade substrates within autolysosomes. We confirmed predicted changes in neuronal autophagy flux following specific experimental ALP perturbations. Furthermore, using a third fluorescence label in TRGL6 brains to identify lysosomes by immunocytochemistry, we validated a novel procedure to detect defective autolysosomal acidification in vivo. Thus, TRGL6 mice represent a unique tool to investigate in vivo ALP dynamics in specific neuron populations in relation to neurological diseases, aging, and disease modifying agents. Abbreviations: ACTB: actin, beta; AD: Alzheimer disease; AL: autolysosomes; ALP: autophagy-lysosome pathway; AP: autophagosome; APP: amyloid beta (Abeta) precursor protein; ATG5: autophagy related 5; ATG7: autophagy related 7; AV: autophagic vacuoles; CNS: central nervous system; CTSD: cathepsin D; CQ: chloroquine; DMEM: Dulbecco’s modified Eagle’s medium; GFP: green fluorescent protein; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2/GATE16: gamma-aminobutyric acid (GABA) receptor-associated protein-like 2; ICC: immunocytochemistry; ICV: intra-cerebroventricular; LAMP2: lysosomal-associated membrane protein 2; Leup: leupeptin; LY: lysosomes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RFP: red fluorescent protein; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; tfLC3: mRFP-eGFP-LC3; TRGL6: Thy1 mRFP eGFP LC3-line 6; PCR: polymerase chain reaction; PD: Parkinson disease