Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000–80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1–21% and 2–20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.     

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging

Mouse models of neurodegenerative diseases such as Alzheimer’s disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.     

Spastic Paraplegia Type 7 Is Associated with Multiple Mitochondrial DNA Deletions

Spastic paraplegia 7 is an autosomal recessive disorder caused by mutations in the gene encoding paraplegin, a protein located at the inner mitochondrial membrane and involved in the processing of other mitochondrial proteins. The mechanism whereby paraplegin mutations cause disease is unknown. We studied two female and two male adult patients from two Norwegian families with a combination of progressive external ophthalmoplegia and spastic paraplegia. Sequencing of SPG7 revealed a novel missense mutation, c.2102A>C, p.H 701P, which was homozygous in one family and compound heterozygous in trans with a known pathogenic mutation c.1454_1462del in the other. Muscle was examined from an additional, unrelated adult female patient with a similar phenotype caused by a homozygous c.1047insC mutation in SPG7. Immunohistochemical studies in skeletal muscle showed mosaic deficiency predominantly affecting respiratory complex I, but also complexes III and IV. Molecular studies in single, microdissected fibres showed multiple mitochondrial DNA deletions segregating at high levels (38–97%) in respiratory deficient fibres. Our findings demonstrate for the first time that paraplegin mutations cause accumulation of mitochondrial DNA damage and multiple respiratory chain deficiencies. While paraplegin is not known to be directly associated with the mitochondrial nucleoid, it is known to process other mitochondrial proteins and it is possible therefore that paraplegin mutations lead to mitochondrial DNA deletions by impairing proteins involved in the homeostasis of the mitochondrial genome. These studies increase our understanding of the molecular pathogenesis of SPG7 mutations and suggest that SPG7 testing should be included in the diagnostic workup of autosomal recessive, progressive external ophthalmoplegia, especially if spasticity is present.     

Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during data analysis.     

Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection

The action of type I IFN (IFN-I) on APCs is well studied, but their direct effect on CD4 T cells is unclear. To address this, we transferred IFN-I receptor-deficient (IFN-IR(0)) and -sufficient (wild-type, WT) TCR-transgenic CD4 T cells into WT mice and analyzed their response to immunization. In response to lymphocytic choriomeningitis virus immunization, WT CD4 T cells expanded approximately 100-fold, whereas IFN-IR(0) CD4 T cells expanded <10-fold. However, both WT and IFN-IR(0) CD4 T cells expanded approximately 10-fold after Listeria monocytogenes immunization. Poor expansion of IFN-IR(0) CD4 T cells after lymphocytic choriomeningitis virus immunization was not due to a defect in proliferation or initial activation but to poor survival of the daughter cells. Thus, direct IFN-I signals can play either a critical or minimal role in CD4 T cell clonal expansion depending on the specific pathogen.     

Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.