Faculty Bibliography
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school:SOM
Department/Unit:Cell Biology
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14086
Lysosomal dysfunction in a mouse model of Sandhoff disease leads to accumulation of ganglioside-bound amyloid-beta peptide
Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-beta peptide (Abeta)-like, alpha-synuclein-like, and phospho-tau-like immunoreactivity in the brains of beta-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Abeta-like immunoreactivity (iAbeta-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Abeta. In addition, we observed increased levels of Abeta40 and Abeta42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Abeta (GAbeta) immunoreactivity in a brain region-specific manner. Furthermore, alpha-synuclein and APP-CTFs and/or Abeta were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAbeta-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAbeta-LIR may be associated with the lysosomal-autophagic turnover of Abeta and fragments of APP-containing Abeta epitopes. Importantly, intraneuronal GAbeta immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.
PMID: 22496568
ISSN: 0270-6474
CID: 832862
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2012:109(15):E869-78.DOI: 10.1073/pnas.1115623109
The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule
IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4mu8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.
PMCID:3326519
PMID: 22315414
ISSN: 0027-8424
CID: 165427
A primary neuron culture system for the study of herpes simplex virus latency and reactivation
Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.
PMCID:3466666
PMID: 22491318
ISSN: 1940-087x
CID: 164363
Transcription goes digital
Transcription is a complex process that integrates the state of the cell and its environment to generate adequate responses for cell fitness and survival. Recent microscopy experiments have been able to monitor transcription from single genes in individual cells. These observations have revealed two striking features: transcriptional activity can vary markedly from one cell to another, and is subject to large changes over time, sometimes within minutes. How the chromatin structure, transcription machinery assembly and signalling networks generate such patterns is still unclear. In this review, we present the techniques used to investigate transcription from single genes, introduce quantitative modelling tools, and discuss transcription mechanisms and their implications for gene expression regulation.
PMCID:3321162
PMID: 22410830
ISSN: 1469-3178
CID: 2385252
Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction
BACKGROUND: Barth syndrome is a rare, multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly develop an early-onset cardiomyopathy in infancy or fetal life. METHODS AND RESULTS: Knockdown of tafazzin (TAZKD) in a mouse model was induced from the start of gestation via a doxycycline-inducible shRNA transgenic approach. All liveborn TAZKD mice died within the neonatal period, and in vivo echocardiography revealed prenatal loss of TAZKD embryos at E12.5-14.5. TAZKD E13.5 embryos and newborn mice demonstrated significant tafazzin knockdown, and mass spectrometry analysis of hearts revealed abnormal cardiolipin profiles typical of Barth syndrome. Electron microscopy of TAZKD hearts demonstrated ultrastructural abnormalities in mitochondria at both E13.5 and newborn stages. Newborn TAZKD mice exhibited a significant reduction in total mitochondrial area, smaller size of individual mitochondria, reduced cristae density, and disruption of the normal parallel orientation between mitochondria and sarcomeres. Echocardiography of E13.5 and newborn TAZKD mice showed good systolic function, but early diastolic dysfunction was evident from an abnormal flow pattern in the dorsal aorta. Strikingly, histology of E13.5 and newborn TAZKD hearts showed myocardial thinning, hypertrabeculation and noncompaction, and defective ventricular septation. Altered cellular proliferation occurring within a narrow developmental window accompanied the myocardial hypertrabeculation-noncompaction. CONCLUSIONS: In this murine model, tafazzin deficiency leads to a unique developmental cardiomyopathy characterized by ventricular myocardial hypertrabeculation-noncompaction and early lethality. A central role of cardiolipin and mitochondrial functioning is strongly implicated in cardiomyocyte differentiation and myocardial patterning required for heart development. (J Am Heart Assoc. 2012;1:jah3-e000455 doi: 10.1161/JAHA.111.000455.).
PMCID:3487377
PMID: 23130124
ISSN: 2047-9980
CID: 180952
Is losartan the drug for all seasons?
PMID: 22445547
ISSN: 1471-4892
CID: 169635
Impact of thrombolysis on stroke outcome at 12 months in a population: the Bern stroke project
BACKGROUND AND PURPOSE: Thrombolysis improves outcome of patients with acute ischemic stroke, but it is unknown whether thrombolysis has a measurable effect on long-term outcome in a defined population. METHODS: We prospectively assessed demographic data, management, and outcome of acute ischemic stroke patients admitted within 48 hours to 18 primary care hospitals of the canton of Bern (969 299 inhabitants) during 12 months. Blinded follow-up was obtained at 3 and 12 months. Predictors of mortality and favorable outcome (modified Rankin Scale score
PMID: 22403052
ISSN: 0039-2499
CID: 986902
Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice
Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer (64)Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2-4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with (64)Cu rapidly appearing in milk. Parallel studies in Cp-/- mice (siblings from same colony) gave virtually identical results. However, their milk contained less (64)Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp-/- dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.
PMID: 22080201
ISSN: 0966-0844
CID: 281172
Apoprotein B: quality control early and late in the secretory pathway for this atherogenic protein [Meeting Abstract]
Apoprotein B (apoB100) is required for the formation of VLDL, the precursor of LDL, the plasma level of which is a strong risk factor for atherosclerosis and coronary artery disease (CAD). It is an exceptionally large (>4000 aa) and hydrophobic protein. As the nascent polypeptide is translocating across the ER, if lipids are not transferred to stabilize its conformation, we and others have shown that like other abnormally folded proteins in the ER, its quality control is governed by ER-associated ub-proteasome degradation (ERAD). Using a combination of systems from yeast to genetically modified mice, we and our collaborators have shown apoB100 ERAD to involve chaperone and chaperone-like molecules, such as Hsp70, 90, 110, and p58. The full assembly of VLDL does not occur until the Golgi. We have discovered that nascent particles ("pre-VLDL") that do not complete this maturation process (e.g., under the influence of certain dietary fatty acids), are subject to a late stage quality control process mediated by autophagy. In summary, the production of apoB100-lipoproteins, whose plasma levels correlate with the risk of CAD, is determined in part by quality control processes that recognize features of the protein specific to its stage of assembly and that are separated between the ER and Golgi compartments
EMBASE:70852949
ISSN: 0892-6638
CID: 178107
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
