Faculty Bibliography
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136
MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease
Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.
PMCID:4496044
PMID: 26154133
ISSN: 1553-7374
CID: 1663192
Early hyperactivity in lateral entorhinal cortex is associated with elevated levels of AbetaPP metabolites in the Tg2576 mouse model of Alzheimer's disease
Alzheimer's disease (AD) is a neurodegenerative disorder which is the most common cause of dementia in the elderly today. One of the earliest symptoms of AD is olfactory dysfunction. The present study investigated the effects of amyloid beta precursor protein (AbetaPP) metabolites, including amyloid-beta (Abeta) and AbetaPP C-terminal fragments (CTF), on olfactory processing in the lateral entorhinal cortex (LEC) using the Tg2576 mouse model of human AbetaPP over-expression. The entorhinal cortex is an early target of AD related neuropathology, and the LEC plays an important role in fine odor discrimination and memory. Cohorts of transgenic and age-matched wild-type (WT) mice at 3, 6, and 16months of age (MO) were anesthetized and acute, single-unit electrophysiology was performed in the LEC. Results showed that Tg2576 exhibited early LEC hyperactivity at 3 and 6MO compared to WT mice in both local field potential and single-unit spontaneous activity. However, LEC single-unit odor responses and odor receptive fields showed no detectable difference compared to WT at any age. Finally, the very early emergence of olfactory system hyper-excitability corresponded not to detectable Abeta deposition in the olfactory system, but rather to high levels of intracellular AbetaPP-CTF and soluble Abeta in the anterior piriform cortex (aPCX), a major afferent input to the LEC, by 3MO. The present results add to the growing evidence of AbetaPP-related hyper-excitability, and further implicate both soluble Abeta and non-Abeta AbetaPP metabolites in its early emergence.
PMCID:4324092
PMID: 25500142
ISSN: 0014-4886
CID: 1453232
Glutamatergic Transmission Aberration: A Major Cause of Behavioral Deficits in a Murine Model of Down's Syndrome
Trisomy 21, or Down's syndrome (DS), is the most common genetic cause of intellectual disability. Altered neurotransmission in the brains of DS patients leads to hippocampus-dependent learning and memory deficiency. Although genetic mouse models have provided important insights into the genes and mechanisms responsible for DS-specific changes, the molecular mechanisms leading to memory deficits are not clear. We investigated whether the segmental trisomy model of DS, Ts[Rb(12.1716)]2Cje (Ts2), exhibits hippocampal glutamatergic transmission abnormalities and whether these alterations cause behavioral deficits. Behavioral assays demonstrated that Ts2 mice display a deficit in nest building behavior, a measure of hippocampus-dependent nonlearned behavior, as well as dysfunctional hippocampus-dependent spatial memory tested in the object-placement and the Y-maze spontaneous alternation tasks. Magnetic resonance spectra measured in the hippocampi revealed a significantly lower glutamate concentration in Ts2 as compared with normal disomic (2N) littermates. The glutamate deficit accompanied hippocampal NMDA receptor1 (NMDA-R1) mRNA and protein expression level downregulation in Ts2 compared with 2N mice. In concert with these alterations, paired-pulse analyses suggested enhanced synaptic inhibition and/or lack of facilitation in the dentate gyrus of Ts2 compared with 2N mice. Ts2 mice also exhibited disrupted synaptic plasticity in slice recordings of the hippocampal CA1 region. Collectively, these findings imply that deficits in glutamate and NMDA-R1 may be responsible for impairments in synaptic plasticity in the hippocampus associated with behavioral dysfunctions in Ts2 mice. Thus, these findings suggest that glutamatergic deficits have a significant role in causing intellectual disabilities in DS.
PMCID:3983795
PMID: 24719089
ISSN: 0270-6474
CID: 881932
Spared piriform cortical single-unit odor processing and odor discrimination in the tg2576 mouse model of Alzheimer's disease
Alzheimer's disease is a neurodegenerative disorder that is the most common cause of dementia in the elderly today. One of the earliest reported signs of Alzheimer's disease is olfactory dysfunction, which may manifest in a variety of ways. The present study sought to address this issue by investigating odor coding in the anterior piriform cortex, the primary cortical region involved in higher order olfactory function, and how it relates to performance on olfactory behavioral tasks. An olfactory habituation task was performed on cohorts of transgenic and age-matched wild-type mice at 3, 6 and 12 months of age. These animals were then anesthetized and acute, single-unit electrophysiology was performed in the anterior piriform cortex. In addition, in a separate group of animals, a longitudinal odor discrimination task was conducted from 3-12 months of age. Results showed that while odor habituation was impaired at all ages, Tg2576 performed comparably to age-matched wild-type mice on the olfactory discrimination task. The behavioral data mirrored intact anterior piriform cortex single-unit odor responses and receptive fields in Tg2576, which were comparable to wild-type at all age groups. The present results suggest that odor processing in the olfactory cortex and basic odor discrimination is especially robust in the face of amyloid beta precursor protein (AbetaPP) over-expression and advancing amyloid beta (Abeta) pathology. Odor identification deficits known to emerge early in Alzheimer's disease progression, therefore, may reflect impairments in linking the odor percept to associated labels in cortical regions upstream of the primary olfactory pathway, rather than in the basic odor processing itself.
PMCID:4152226
PMID: 25181487
ISSN: 1932-6203
CID: 1173742
Immunization targeting a minor plaque constituent clears beta-amyloid and rescues behavioral deficits in an Alzheimer's disease mouse model
Although anti-human beta-amyloid (Abeta) immunotherapy clears brain beta-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Abeta is a minor Abeta plaque component in amyloid precursor protein (APP) transgenic AD models, which we show is approximately 3%-8% of the total accumulated Abeta in various human APP transgenic mice. Murine Abeta codeposits and colocalizes with human Abeta in amyloid plaques, and the two Abeta species coimmunoprecipitate together from brain extracts. In the human APP transgenic mouse model Tg2576, passive immunization for 8 weeks with a murine-Abeta-specific antibody reduced beta-amyloid plaque pathology, robustly decreasing both murine and human Abeta levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine Abeta immunization clears Abeta plaque pathology-including the major human Abeta component-and decreases behavioral deficits, arguing that targeting minor endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics.
PMCID:3426627
PMID: 22608241
ISSN: 0197-4580
CID: 180342
Early Endosomal Abnormalities and Cholinergic Neuron Degeneration in Amyloid-beta Protein Precursor Transgenic Mice
Early endosomal changes, a prominent pathology in neurons early in Alzheimer's disease, also occur in neurons and peripheral tissues in Down syndrome. While in Down syndrome models increased amyloid-beta protein precursor (AbetaPP) expression is known to be a necessary contributor on the trisomic background to this early endosomal pathology, increased AbetaPP alone has yet to be shown to be sufficient to drive early endosomal alterations in neurons. Comparing two AbetaPP transgenic mouse models, one that contains the AbetaPP Swedish K670N/M671L double mutation at the beta-cleavage site (APP23) and one that has the AbetaPP London V717I mutation near the gamma-cleavage site (APPLd2), we show significantly altered early endosome morphology in fronto-parietal neurons as well as enlargement of early endosomes in basal forebrain cholinergic neurons of the medial septal nucleus in the APP23 model, which has the higher levels of AbetaPP beta-C-terminal fragment (betaCTF) accumulation. Early endosomal changes correlate with a marked loss of the cholinergic population, which is consistent with the known dependence of the large projection cholinergic cells on endosome-mediated retrograde neurotrophic transport. Our findings support the idea that increased expression of AbetaPP and AbetaPP metabolites in neurons is sufficient to drive early endosomal abnormalities in vivo, and that disruption of the endocytic system is likely to contribute to basal forebrain cholinergic vulnerability.
PMCID:3616896
PMID: 23254640
ISSN: 1387-2877
CID: 231162
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
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
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
