Proteomics analysis of periplaque and chronic inactive multiple sclerosis lesions.

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by increased inflammation and immune responses, oxidative injury, mitochondrial dysfunction, and iron dyshomeostasis leading to demyelination and axonal damage. In MS, incomplete remyelination results in chronically demyelinated axons and degeneration coinciding with disability. This suggests a failure in the ability to remyelinate in MS, however, the precise underlying mechanisms remain unclear. We aimed to identify proteins whose expression was altered in chronic inactive white matter lesions and periplaque white matter in MS tissue to reveal potential pathophysiological mechanisms. Methods: Laser capture microdissection coupled to proteomics was used to interrogate spatially altered changes in formalin-fixed paraffin-embedded brain tissue from three chronic MS individuals and three controls with no apparent neurological complications. Histopathological maps guided the capture of inactive lesions, periplaque white matter, and cortex from chronic MS individuals along with corresponding white matter and cortex from control tissue. Label free quantitation by liquid chromatography tandem mass spectrometry was used to discover differentially expressed proteins between the various brain regions. Results: In addition to confirming loss of several myelin-associated proteins known to be affected in MS, proteomics analysis of chronic inactive MS lesions revealed alterations in myelin assembly, metabolism, and cytoskeletal organization. The top altered proteins in MS inactive lesions compared to control white matter consisted of PPP1R14A, ERMN, SIRT2, CARNS1, and MBLAC2. Conclusion: Our findings highlight proteome changes in chronic inactive MS white matter lesions and periplaque white matter, which may be crucial for proper myelinogenesis, bioenergetics, focal adhesions, and cellular function. This study highlights the importance and feasibility of spatial approaches such as laser capture microdissection-based proteomics analysis of pathologically distinct regions of MS brain tissue. Identification of spatially resolved changes in the proteome of MS brain tissue should aid in the understanding of pathophysiological mechanisms and the development of novel therapies.

Infrared spectral profiling of demyelinating activity in multiple sclerosis brain tissue.

Multiple sclerosis (MS) is a leading cause of non-traumatic disability in young adults. The highly dynamic nature of MS lesions has made them difficult to study using traditional histopathology due to the specificity of current stains. This requires numerous stains to track and study demyelinating activity in MS. Thus, we utilized Fourier transform infrared (FTIR) spectroscopy to generate holistic biomolecular profiles of demyelinating activities in MS brain tissue. Multivariate analysis can differentiate MS tissue from controls. Analysis of the absorbance spectra shows profound reductions of lipids, proteins, and phosphate in white matter lesions. Changes in unsaturated lipids and lipid chain length indicate oxidative damage in MS brain tissue. Altered lipid and protein structures suggest changes in MS membrane structure and organization. Unique carbohydrate signatures are seen in MS tissue compared to controls, indicating altered metabolic activities. Cortical lesions had increased olefinic lipid content and abnormal membrane structure in normal appearing MS cortex compared to controls. Our results suggest that FTIR spectroscopy can further our understanding of lesion evolution and disease mechanisms in MS paving the way towards improved diagnosis, prognosis, and development of novel therapeutics.

Biomolecular alterations detected in multiple sclerosis skin fibroblasts using Fourier transform infrared spectroscopy.

Multiple sclerosis (MS) is the leading cause of non-traumatic disability in young adults. New avenues are needed to help predict individuals at risk for developing MS and aid in diagnosis, prognosis, and outcome of therapeutic treatments. Previously, we showed that skin fibroblasts derived from patients with MS have altered signatures of cell stress and bioenergetics, which likely reflects changes in their protein, lipid, and biochemical profiles. Here, we used Fourier transform infrared (FTIR) spectroscopy to determine if the biochemical landscape of MS skin fibroblasts were altered when compared to age- and sex-matched controls (CTRL). More so, we sought to determine if FTIR spectroscopic signatures detected in MS skin fibroblasts are disease specific by comparing them to amyotrophic lateral sclerosis (ALS) skin fibroblasts. Spectral profiling of skin fibroblasts from MS individuals suggests significant alterations in lipid and protein organization and homeostasis, which may be affecting metabolic processes, cellular organization, and oxidation status. Sparse partial least squares-discriminant analysis of spectral profiles show that CTRL skin fibroblasts segregate well from diseased cells and that changes in MS and ALS may be unique. Differential changes in the spectral profile of CTRL, MS, and ALS cells support the development of FTIR spectroscopy to detect biomolecular modifications in patient-derived skin fibroblasts, which may eventually help establish novel peripheral biomarkers.

Partial inhibition of mitochondrial complex I ameliorates Alzheimer's disease pathology and cognition in APP/PS1 female mice.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

Signatures of cell stress and altered bioenergetics in skin fibroblasts from patients with multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system inflammatory demyelinating disease and the most common cause of non-traumatic disability in young adults. Despite progress in the treatment of the active relapsing disease, therapeutic options targeting irreversible progressive decline remain limited. Studies using skin fibroblasts derived from patients with neurodegenerative disorders demonstrate that cell stress pathways and bioenergetics are altered when compared to healthy individuals. However, findings in MS skin fibroblasts are limited. Here, we collected skin fibroblasts from 24 healthy control individuals, 30 patients with MS, and ten with amyotrophic lateral sclerosis (ALS) to investigate altered cell stress profiles. We observed endoplasmic reticulum swelling in MS skin fibroblasts, and increased gene expression of cell stress markers including BIP, ATF4, CHOP, GRP94, P53, and P21. When challenged against hydrogen peroxide, MS skin fibroblasts had reduced resiliency compared to ALS and controls. Mitochondrial and glycolytic functions were perturbed in MS skin fibroblasts while exhibiting a significant increase in lactate production over ALS and controls. Our results suggest that MS skin fibroblasts have an underlying stress phenotype, which may be disease specific. Interrogating MS skin fibroblasts may provide patient specific molecular insights and aid in prognosis, diagnosis, and therapeutic testing enhancing individualized medicine.

A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts.

INTRODUCTION: Patient-derived skin fibroblasts offer a unique translational model to study molecular mechanisms of multiple human diseases. Metabolomics profiling allows to track changes in a broad range of metabolites and interconnected metabolic pathways that could inform on molecular mechanisms involved in disease development and progression, and on the efficacy of therapeutic interventions. Therefore, it is important to establish standardized protocols for metabolomics analysis in human skin fibroblasts for rigorous and reliable metabolic assessment. OBJECTIVES: We aimed to develop an optimized protocol for concurrent measure of the concentration of amino acids, acylcarnitines, and components of the tricarboxylic acid (TCA) cycle in human skin fibroblasts using gas (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS). METHODS: The suitability of four different methods of cell harvesting on the recovery of amino acids, acylcarnitines, and TCA cycle metabolites was established using GC/MS and LC/MS analytical platforms. For each method, metabolite stability was determined after 48 h, 2 weeks and 1 month of storage at - 80 °C. RESULTS: Harvesting cells in 80% methanol solution allowed the best recovery and preservation of metabolites. Storage of samples in 80% methanol up to 1  month at - 80 °C did not significantly impact metabolite concentrations. CONCLUSION: We developed a robust workflow for metabolomics analysis in human skin fibroblasts suitable for a high-throughput multiplatform analysis. This method allows a direct side-by-side comparison of metabolic changes in samples collected at different time that could be used for studies in large patient cohorts.

Application of Metabolomics in Alzheimer's Disease.

Progress toward the development of efficacious therapies for Alzheimer's disease (AD) is halted by a lack of understanding early underlying pathological mechanisms. Systems biology encompasses several techniques including genomics, epigenomics, transcriptomics, proteomics, and metabolomics. Metabolomics is the newest omics platform that offers great potential for the diagnosis and prognosis of neurodegenerative diseases as an individual's metabolome reflects alterations in genetic, transcript, and protein profiles and influences from the environment. Advancements in the field of metabolomics have demonstrated the complexity of dynamic changes associated with AD progression underscoring challenges with the development of efficacious therapeutic interventions. Defining systems-level alterations in AD could provide insights into disease mechanisms, reveal sex-specific changes, advance the development of biomarker panels, and aid in monitoring therapeutic efficacy, which should advance individualized medicine. Since metabolic pathways are largely conserved between species, metabolomics could improve the translation of preclinical research conducted in animal models of AD into humans. A summary of recent developments in the application of metabolomics to advance the AD field is provided below.

Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV.

Interferon-induced transmembrane (IFITM) proteins are potent antiviral factors shown to restrict the infection of many enveloped viruses, including HIV. Here we report cloning and characterization of a panel of nonhuman primate IFITMs. We show that, similar to human IFITM, nonhuman primate IFITM proteins inhibit HIV and other primate lentiviruses. While some nonhuman primate IFITM proteins are more potent than human counterparts to inhibit HIV-1, they are generally not effective against HIV-2 similar to that of human IFITMs. Notably, depending on SIV strains and also IFITM species tested, nonhuman primate IFITM proteins exhibit distinct activities against SIVs; no correlation was found to support the notion that IFITM proteins are most active in non-natural primate hosts. Consistent with our recent findings for human IFITMs, nonhuman primate IFITM proteins interact with HIV-1 Env and strongly act in viral producer cells to impair viral infectivity and block cell-to-cell transmission. Accordingly, knockdown of primate IFITM3 increases HIV-1 replication in nohuman primate cells. Interestingly, analysis of DNA sequences of human and nonhuman primate IFITMs suggest that IFITM proteins have been undergoing purifying selection, rather than positive selection typical for cellular restriction factors. Overall, our study reveals some new and unexpected features of IFITMs in restricting primate lentiviruses, which enhances our understanding of virus-host interaction and AIDS pathogenesis.

IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein.

The interferon-induced transmembrane (IFITM) proteins have been recently shown to restrict HIV-1 and other viruses. Here, we provide evidence that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env), thereby inhibiting viral infection. IFITM proteins interact with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions does not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes leads to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appears to be virus-strain dependent. Overall, our study uncovers a mechanism by which IFITM proteins specifically antagonize HIV-1 Env to restrict HIV-1 infection and provides insight into the specialized role of IFITMs in HIV infection.

From Gigabyte to Kilobyte: A Bioinformatics Protocol for Mining Large RNA-Seq Transcriptomics Data.

RNA-Seq techniques generate hundreds of millions of short RNA reads using next-generation sequencing (NGS). These RNA reads can be mapped to reference genomes to investigate changes of gene expression but improved procedures for mining large RNA-Seq datasets to extract valuable biological knowledge are needed. RNAMiner--a multi-level bioinformatics protocol and pipeline--has been developed for such datasets. It includes five steps: Mapping RNA-Seq reads to a reference genome, calculating gene expression values, identifying differentially expressed genes, predicting gene functions, and constructing gene regulatory networks. To demonstrate its utility, we applied RNAMiner to datasets generated from Human, Mouse, Arabidopsis thaliana, and Drosophila melanogaster cells, and successfully identified differentially expressed genes, clustered them into cohesive functional groups, and constructed novel gene regulatory networks. The RNAMiner web service is available at http://calla.rnet.missouri.edu/rnaminer/index.html.

A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5.

Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5.