Faculty Bibliography
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130
The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space
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.
PMCID:3522305
PMID: 23129776
ISSN: 1083-351x
CID: 3629662
Guidelines for the use and interpretation of assays for monitoring autophagy [Guideline]
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
PMCID:3404883
PMID: 22966490
ISSN: 1554-8627
CID: 181862
Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain
The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker alpha-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.
PMID: 22528096
ISSN: 1064-3745
CID: 165625
In Vitro Assays Measuring Protection by Proteins such as Cystatin C of Primary Cortical Neuronal and Smooth Muscle Cells
Neuronal cell culture models have been used to demonstrate the protective effects of cystatin C against a variety of insults, including the toxicity induced by oligomeric and fibrillar amyloid beta (Abeta). Here, we describe assays quantifying cystatin C protective effects against cytotoxicity induced by nutrient deprivation, oxidative stress, or cytotoxic forms of Abeta. Three methods for the evaluation of either cell death or cell survival are described: measurement of metabolic activity, cell death, and cell division. The cell culture models used are murine primary cortical neurons and murine primary cerebral smooth muscle cells. The effects of exogenously applied cystatin C are studied by comparing the viability of nonstressed control, stressed control, and cystatin C-treated stressed cells. The effect of endogenous level of cystatin C expression is studied by comparing stressed primary cells isolated from brains of cystatin C transgenic, cystatin C knockout, and wild-type mice.
PMID: 22528097
ISSN: 1064-3745
CID: 165626
Cystatin C in Alzheimer's disease
Changes in expression and secretion levels of cystatin C (CysC) in the brain in various neurological disorders and in animal models of neurodegeneration underscore a role for CysC in these conditions. A polymorphism in the CysC gene (CST3) is linked to increased risk for Alzheimer's disease (AD). AD pathology is characterized by deposition of oligomeric and fibrillar forms of amyloid beta (Abeta) in the neuropil and cerebral vessel walls, neurofibrillary tangles composed mainly of hyperphosphorylated tau, and neurodegeneration. The implication of CysC in AD was initially suggested by its co-localization with Abeta in amyloid-laden vascular walls, and in senile plaque cores of amyloid in the brains of patients with AD, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), and cerebral infarction. CysC also co-localizes with Abeta amyloid deposits in the brains of non-demented aged individuals. Multiple lines of research show that CysC plays protective roles in AD. In vitro studies have shown that CysC binds Abeta and inhibits Abeta oligomerization and fibril formation. In vivo results from the brains and plasma of Abeta-depositing transgenic mice confirmed the association of CysC with the soluble, non-pathological form of Abeta and the inhibition of Abeta plaques formation. The association of CysC with Abeta was also found in brain and in cerebrospinal fluid (CSF) from AD patients and non-demented control individuals. Moreover, in vitro results showed that CysC protects neuronal cells from a variety of insults that may cause cell death, including cell death induced by oligomeric and fibrillar Abeta. These data suggest that the reduced levels of CysC manifested in AD contribute to increased neuronal vulnerability and impaired neuronal ability to prevent neurodegeneration. This review elaborates on the neuroprotective roles of CysC in AD and the clinical relevance of this protein as a therapeutic agent.
PMCID:3390601
PMID: 22783166
ISSN: 1662-5099
CID: 175779
Sensory Network Dysfunction, Behavioral Impairments, and Their Reversibility in an Alzheimer's beta-Amyloidosis Mouse Model
The unique vulnerability of the olfactory system to Alzheimer's 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 beta-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-beta (Abeta) 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 Abeta-dependent, because liver-X receptor agonist treatment to promote Abeta 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
PMCID:3417321
PMID: 22049439
ISSN: 1529-2401
CID: 145504
Network dysfunction, olfactory behavior impairments, and their reversibility in an Alzheimer's b-amyloidosis mouse model [Meeting Abstract]
The vulnerability of the olfactory system to Alzheimer's disease (AD) pathology and the high incidence of olfactory perceptual dysfunction in early stages of the disease makes the olfactory system a unique model for understanding mechanisms of synaptic and neural network dysfunction in AD. Here we demonstrate aberrant neural oscillations within the olfactory bulb (OB) and piriform cortex (PCX) of mice overexpressing human mutations of amyloid precursor protein (APP). Network dysfunction was evident starting at 3 months of age in APP mice, prior to the onset of significant behavioral impairments or comparable hippocampal network dysfunction. Coinciding with the onset of behavioral impairments, we found hyperactivity of odor-evoked responses in the PCX and enhanced coherence between the OB and PCX. In contrast, older APP mice with established disease-related pathology were characterized by hyporesponsive PCX odor-evoked activity and impaired behavior which were both recovered by treatment with a Liver-X Receptor (LXR) agonist. These results complement recent findings in other neural networks and suggest that disease-relevant network dysfunction can be transient and region specific, yet with lasting effects on cognition and behavior
EMBASE:71027912
ISSN: 0379-864x
CID: 288242
Cystatin C is released in association with exosomes: a new tool of neuronal communication which is unbalanced in Alzheimer's disease
It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. Multiple lines of research support a neuroprotective role for cystatin C in Alzheimer's disease (AD). Herein we demonstrate that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, is also secreted by mouse primary neurons in association with exosomes. Immunoproteomic analysis using SELDI-TOF MS revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familial AD-associated presenilin 2 mutations (PS2 M239I and PS2 T122R) resulted in reduced levels of all cystatin C forms (native and glycosylated) and of amyloid-β precursor protein (APP) metabolites within exosomes. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against AD and other neurodegenerative diseases.
PMCID:2891183
PMID: 19773092
ISSN: 1558-1497
CID: 3629642
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-beta peptide (Abeta) accumulation, extracellular beta-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 Abeta, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Abeta40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Abeta 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
PMCID:3359468
PMID: 21464620
ISSN: 1554-8635
CID: 134440
Mechanisms of neural and behavioral dysfunction in Alzheimer's disease
This review critically examines progress in understanding the link between Alzheimer's disease (AD) molecular pathogenesis and behavior, with an emphasis on the impact of amyloid-beta. We present the argument that the AD research field requires more multifaceted analyses into the impacts of Alzheimer's pathogenesis which combine simultaneous molecular-, circuit-, and behavior-level approaches. Supporting this argument is a review of particular research utilizing similar, 'systems-level' methods in mouse models of AD. Related to this, a critique of common physiological and behavioral models is made-highlighting the likely usefulness of more refined and specific tools in understanding the relationship between candidate molecular pathologies and behavioral dysfunction. Finally, we propose challenges for future research which, if met, may greatly extend our current understanding of how AD molecular pathology impacts neural network function and behavior and possibly may lead to refinements in disease therapeutics
PMCID:3090690
PMID: 21424679
ISSN: 1559-1182
CID: 131960
