Efrat Levy

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.

A Recessive Mutation in the APP Gene with Dominant-Negative Effect on Amyloidogenesis

β-Amyloid precursor protein (APP) mutations cause familial Alzheimers disease with nearly complete penetrance. We found an APP mutation [alanine-673→valine-673 (A673V)] that causes disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing, resulting in enhanced β-amyloid (Aβ) production and formation of amyloid fibrils in vitro. Co-incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the autosomal recessive pattern of inheritance and have implications for genetic screening and the potential treatment of Alzheimers disease.

Olfactory Dysfunction Correlates with Amyloid-β Burden in an Alzheimer's Disease Mouse Model

Alzheimers disease often results in impaired olfactory perceptual acuity—a potential biomarker of the disorder. However, the usefulness of olfactory screens to serve as informative indicators of Alzheimers is precluded by a lack of knowledge regarding why the disease impacts olfaction. We addressed this question by assaying olfactory perception and amyloid-β (Aβ) deposition throughout the olfactory system in mice that overexpress a mutated form of the human amyloid-β precursor protein. Such mice displayed progressive olfactory deficits that mimic those observed clinically—some evident at 3 months of age. Also, at 3 months of age, we observed nonfibrillar Aβ deposition within the olfactory bulb—earlier than deposition within any other brain region. There was also a correlation between olfactory deficits and the spatial-temporal pattern of Aβ deposition. Therefore, nonfibrillar, versus fibrillar, Aβ-related mechanisms likely contribute to early olfactory perceptual loss in Alzheimers disease. Furthermore, these results present the odor cross-habituation test as a powerful behavioral assay, which reflects Aβ deposition and thus may serve to monitor the efficacy of therapies aimed at reducing Aβ.

Cystatin C modulates cerebral |[beta]|-amyloidosis

The CST3 Thr25 allele of CST3, which encodes cystatin C, leads to reduced cystatin C secretion and conveys susceptibility to Alzheimers disease. Here we show that overexpression of human cystatin C in brains of APP-transgenic mice reduces cerebral amyloid-β deposition and that cystatin C binds amyloid-β and inhibits its fibril formation. Our results suggest that cystatin C concentrations modulate cerebral amyloidosis risk and provide an opportunity for genetic risk assessment and therapeutic interventions.

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.

The exosome-secretory pathway transports amyloid precursor protein carboxyl terminal fragments from the cell into the brain extracellular space

Background: Exosomes isolated in vitro contain full-length amyloid-β precursor protein (flAPP) and APP metabolites. Results: Exosomes secreted in vivo in brains of wild-type and APP-overexpressing mice contain higher levels of APP C-terminal fragments (CTFs) relative to flAPP compared with brain tissue. Conclusion: Brain exosomes are enriched with APP CTFs. Significance: The exosome secretory pathway clears cellular APP CTFs, releasing the toxic fragments into the neuropil. In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain.

Binding of cystatin C to Alzheimer's amyloid β inhibits in vitro amyloid fibril formation

The colocalization of cystatin C, an inhibitor of cysteine proteases, with amyloid beta (Abeta) in parenchymal and vascular amyloid deposits in brains of Alzheimers disease (AD) patients may reflect cystatin C involvement in amyloidogenesis. We therefore sought to determine the association of cystatin C with Abeta. Immunofluorescence analysis of transfected cultured cells demonstrated colocalization of cystatin C and beta amyloid precursor protein (betaAPP) intracellularly and on the cell surface. Western blot analysis of immunoprecipitated cell lysate or medium proteins revealed binding of cystatin C to full-length betaAPP and to secreted betaAPP (sbetaAPP). Deletion mutants of betaAPP localized the cystatin C binding site within betaAPP to the Abeta region. Cystatin C association with betaAPP resulted in increased sbetaAPP but did not affect levels of secreted Abeta. Analysis of the association of cystatin C and Abeta demonstrated a specific, saturable and high affinity binding between cystatin C and both Abeta(1-42) and Abeta(1-40). Notably, cystatin C association with Abeta results in a concentration-dependent inhibition of Abeta fibril formation.

Days-to-criterion as an indicator of toxicity associated with human Alzheimer amyloid-β oligomers

Recent evidence suggests that high molecular weight soluble oligomeric Aβ (oAβ) assemblies (also known as Aβ‐derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAβ/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Aβ‐secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAβ/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes.

Cystatin C protects neuronal cells from amyloid β-induced toxicity

Multiple studies suggest that cystatin C (CysC) has a role in Alzheimers disease (AD) and a decrease in CysC secretion is linked to the disease in patients with a polymorphism in the CysC gene. CysC binds amyloid-beta (Abeta) and inhibits formation of Abeta fibrils and oligomers both in vitro and in mouse models of amyloid deposition. Here we studied the effect of CysC on cultured primary hippocampal neurons and a neuronal cell line exposed to either oligomeric or fibrillar cytotoxic forms of Abeta. The extracellular addition of the secreted human CysC together with preformed either oligomeric or fibrillar Abeta increased cell survival. While CysC inhibits Abeta aggregation, it does not dissolve preformed Abeta fibrils or oligomers. Thus, CysC has multiple protective effects in AD, by preventing the formation of the toxic forms of Abeta and by direct protection of neuronal cells from Abeta toxicity. Therapeutic manipulation of CysC levels, resulting in slightly higher concentrations than physiological could protect neuronal cells from cell death in AD.

Sensory Network Dysfunction, Behavioral Impairments, and Their Reversibility in an Alzheimer's β-Amyloidosis Mouse Model

The unique vulnerability of the olfactory system to Alzheimers disease (AD) provides a quintessential translational tool for understanding mechanisms of synaptic dysfunction and pathological progression in the disease. Using the Tg2576 mouse model of β-amyloidosis, we show that aberrant, hyperactive olfactory network activity begins early in life, before detectable behavioral impairments or comparable hippocampal dysfunction and at a time when amyloid-β (Aβ) deposition is restricted to the olfactory bulb (OB). Hyperactive odor-evoked activity in the piriform cortex (PCX) and increased OB–PCX functional connectivity emerged at a time coinciding with olfactory behavior impairments. This hyperactive activity persisted until later in life when the network converted to a hyporesponsive state. This conversion was Aβ-dependent, because liver-X receptor agonist treatment to promote Aβ degradation rescued the hyporesponsive state and olfactory behavior. These data lend evidence to a novel working model of olfactory dysfunction in AD and, complimentary to other recent works, suggest that disease-relevant network dysfunction is highly dynamic and region specific, yet with lasting effects on cognition and behavior.