Proteomics and mathematical modeling of longitudinal CSF differentiates fast versus slow ALS progression.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with a complex etiology that lacks biomarkers predicting disease progression. The objective of this study was to use longitudinal cerebrospinal fluid (CSF) samples to identify biomarkers that distinguish fast progression (FP) from slow progression (SP) and assess their temporal response. METHODS: We utilized mass spectrometry (MS)-based proteomics to identify candidate biomarkers using longitudinal CSF from a discovery cohort of SP and FP ALS patients. Immunoassays were used to quantify and validate levels of the top biomarkers. A state-transition mathematical model was created using the longitudinal MS data that also predicted FP versus SP. RESULTS: We identified a total of 1148 proteins in the CSF of all ALS patients. Pathway analysis determined enrichment of pathways related to complement and coagulation cascades in FPs and synaptogenesis and glucose metabolism in SPs. Longitudinal analysis revealed a panel of 59 candidate markers that could segregate FP and SP ALS. Based on multivariate analysis, we identified three biomarkers (F12, RBP4, and SERPINA4) as top candidates that segregate ALS based on rate of disease progression. These proteins were validated in the discovery and a separate validation cohort. Our state-transition model determined that the overall variance of the proteome over time was predictive of the disease progression rate. INTERPRETATION: We identified pathways and protein biomarkers that distinguish rate of ALS disease progression. A mathematical model of the CSF proteome determined that the change in entropy of the proteome over time was predictive of FP versus SP.

Defining the Caprin-1 Interactome in Unstressed and Stressed Conditions.

Cytoplasmic stress granules (SGs) are dynamic foci containing translationally arrested mRNA and RNA-binding proteins (RBPs) that form in response to a variety of cellular stressors. It has been debated that SGs may evolve into cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1. However, it is widely accepted that multiple baits are required to capture the full SG proteome. To gain further insight into the SG proteome, we employed immunoprecipitation coupled with mass spectrometry of endogenous Caprin-1, an RBP implicated in mRNP granules. Overall, we identified 1543 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, TALIN-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and further determined that SNRNP200 also associated with osmotic- and thermal-induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patient neurons. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.

Cross-sectional and longitudinal measures of chitinase proteins in amyotrophic lateral sclerosis and expression of CHI3L1 in activated astrocytes.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a complex disease with numerous pathological mechanisms resulting in a heterogeneous patient population. Using biomarkers for particular disease mechanisms may enrich a homogeneous subset of patients. In this study, we quantified chitotriosidase (Chit-1) and chitinase-3-like protein 1 (CHI3L1), markers of glial activation, in cerebrospinal fluid (CSF) and plasma and determined the cell types that express CHI3L1 in ALS. METHODS: Immunoassays were used to quantify Chit-1, CHI3L1 and phosphorylated neurofilament heavy chain levels in longitudinal CSF and matching plasma samples from 118 patients with ALS, 17 disease controls (DCs), and 24 healthy controls (HCs). Immunostaining was performed to identify and quantify CHI3L1-positive cells in tissue sections from ALS, DCs and non-neurological DCs. RESULTS: CSF Chit-1 exhibited increased levels in ALS as compared with DCs and HCs. CSF CHI3L1 levels were increased in ALS and DCs compared with HCs. No quantitative differences were noted in plasma for either chitinase. Patients with ALS with fast-progressing disease exhibited higher levels of CSF Chit-1 and CHI3L1 than patients with slow-progressing disease. Increased numbers of CHI3L1-positive cells were observed in postmortem ALS motor cortex as compared with controls, and these cells were identified as a subset of activated astrocytes located predominately in the white matter of the motor cortex and the spinal cord. CONCLUSIONS: CSF Chit-1 and CHI3L1 are significantly increased in ALS, and CSF Chit-1 and CHI3L1 levels correlate to the rate of disease progression. CHI3L1 is expressed by a subset of activated astrocytes predominately located in white matter.

Fasting differentially alters the hypothalamic proteome of chickens from lines with the propensity to be anorexic or obese.

BACKGROUND: The hypothalamus is the ultimate modulator of appetite and energy balance and therefore sensitive to changes in nutritional state. Chicks from lines selected for low (LWS) and high (HWS) body weight are hypophagic and compulsive eaters, respectively, and differ in their propensity to become obese and in their hypothalamic mRNA response to fasting. METHODS: As fasting-induced changes in hypothalamic proteins are unknown, we investigated the hypothalamic proteomes of 5-day old LWS and HWS chicks in the fed and fasted states using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. RESULTS: A total of 744 proteins were identified in the chicken hypothalamus, and 268 differentially abundant proteins were identified among four pairwise comparisons. Ninety-five proteins were associated with the response to fasting in HWS chicks, and 23 proteins were associated with the response to fasting in LWS chicks. Fasting-responsive proteins in HWS chicks were significantly enriched in ATP metabolic processes, glyoxylate/dicarboxylate metabolism, and ribosome function. There was no enrichment for any pathways in LWS chicks in response to fasting. In the fasted and fed states, 159 and 119 proteins differed between HWS and LWS, respectively. Oxidative phosphorylation, citric acid cycle, and carbon metabolism were the main pathways associated with differences between the two lines of chicks. Enzymes associated with metabolic pathways differed between HWS and LWS in both nutritional states, including fumarase, aspartate aminotransferase, mitochondrial GOT2, 3-hydroxyisobutyrate dehydrogenase, chondrogenesis associated lipocalin, sialic acid synthase, arylamine N-acetyltransferase, pineal gland isozyme NAT-3, and succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial. CONCLUSIONS: These results provide insights into the hypothalamic metabolic pathways that are affected by nutritional status and the regulation of appetite and eating behavior.

Transcriptomic Analysis of Hepatic Cells in Multicellular Organotypic Liver Models.

Liver homeostasis requires the presence of both parenchymal and non-parenchymal cells (NPCs). However, systems biology studies of the liver have primarily focused on hepatocytes. Using an organotypic three-dimensional (3D) hepatic culture, we report the first transcriptomic study of liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) cultured with hepatocytes. Through computational pathway and interaction network analyses, we demonstrate that hepatocytes, LSECs and KCs have distinct expression profiles and functional characteristics. Our results show that LSECs in the presence of KCs exhibit decreased expression of focal adhesion kinase (FAK) signaling, a pathway linked to LSEC dedifferentiation. We report the novel result that peroxisome proliferator-activated receptor alpha (PPARα) is transcribed in LSECs. The expression of downstream processes corroborates active PPARα signaling in LSECs. We uncover transcriptional evidence in LSECs for a feedback mechanism between PPARα and farnesoid X-activated receptor (FXR) that maintains bile acid homeostasis; previously, this feedback was known occur only in HepG2 cells. We demonstrate that KCs in 3D liver models display expression patterns consistent with an anti-inflammatory phenotype when compared to monocultures. These results highlight the distinct roles of LSECs and KCs in maintaining liver function and emphasize the need for additional mechanistic studies of NPCs in addition to hepatocytes in liver-mimetic microenvironments.

Fluid-Based Biomarkers for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a highly heterogeneous disease with no effective treatment. Drug development has been hampered by the lack of biomarkers that aid in early diagnosis, demonstrate target engagement, monitor disease progression, and can serve as surrogate endpoints to assess the efficacy of treatments. Fluid-based biomarkers may potentially address these issues. An ideal biomarker should exhibit high specificity and sensitivity for distinguishing ALS from control (appropriate disease mimics and other neurologic diseases) populations and monitor disease progression within individual patients. Significant progress has been made using cerebrospinal fluid, serum, and plasma in the search for ALS biomarkers, with urine and saliva biomarkers still in earlier stages of development. A few of these biomarker candidates have demonstrated use in patient stratification, predicting disease course (fast vs slow progression) and severity, or have been used in preclinical and clinical applications. However, while ALS biomarker discovery has seen tremendous advancements in the last decade, validating biomarkers and moving them towards the clinic remains more elusive. In this review, we highlight biomarkers that are moving towards clinical utility and the challenges that remain in order to implement biomarkers at all stages of the ALS drug development process.

The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironment.

BACKGROUND: Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. METHODS: To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. RESULTS: All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based ß-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. CONCLUSIONS: Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.

Cell migration on planar and three-dimensional matrices: a hydrogel-based perspective.

The migration of cells is a complex process that is dependent on the properties of the surrounding environment. In vivo, the extracellular environment is complex with a wide range of physical features, topographies, and protein compositions. There have been a number of approaches to design substrates that can recapitulate the complex architecture in vivo. Two-dimensional (2D) substrates have been widely used to study the effect of material properties on cell migration. However, such substrates do not capture the intricate structure of the extracellular environment. Recent advances in hydrogel assembly and patterning techniques have enabled the design of new three-dimensional (3D) scaffolds and microenvironments. Investigations conducted on these matrices provide growing evidence that several established migratory trends obtained from studies on 2D substrates could be significantly different when conducted in a 3D environment. Since cell migration is closely linked to a wide range of physiological functions, there is a critical need to examine migratory trends on 3D matrices. In this review, our goal is to highlight recent experimental studies on cell migration within engineered 3D hydrogel environments and how they differ from planar substrates. We provide a detailed examination of the changes in cellular characteristics such as morphology, speed, directionality, and protein expression in 3D hydrogel environments. This growing field of research will have a significant impact on tissue engineering, regenerative medicine, and in the design of biomaterials.

The promise of organotypic hepatic and gastrointestinal models.

Advances in the design and assembly of in vitro organotypic liver and gastrointestinal (GI) models can accelerate our understanding of metabolism, nutrient absorption, and the effect of microbial flora. Such models can provide comprehensive information on how of environmental toxins, drugs, and pharmaceuticals interact with and within these organs. Information obtained from such models could elucidate the complicated cascades of signaling mechanisms that occur in vivo. Because experiments on large-scale animal models are expensive and resource intensive, the design of organotypic models has renewed significance. The challenges and approaches to designing liver and GI models are similar. Because these organs are in close proximity and interact continually, we have described recent design considerations to guide future tissue models.

Polymer-enhanced delivery increases adenoviral gene expression in an orthotopic model of bladder cancer.

Gene therapy has garnered significant attention as a therapeutic approach for bladder cancer but efficient delivery and gene expression remain major hurdles. The goal of this study was to determine if cationic polymers can enhance adenoviral gene expression in cells that are difficult to transduce in vitro and to subsequently investigate lead candidates for their capacity to increase adenoviral gene expression in an orthotopic in vivo model of bladder cancer. In vitro screening of linear polyamine-based and aminoglycoside-based polymer libraries identified several candidates that enhanced adenoviral reporter gene expression in vitro. The polyamine-based polymer NPGDE-1,4 Bis significantly enhanced adenoviral gene expression in the orthotopic model of bladder cancer but unfortunately further use of this polymer was limited by toxicity. In contrast, the aminoglycoside-based polymer paromomycin-BGDE, enhanced adenoviral gene expression within the bladder without adverse events. Our study demonstrates for the first time that cationic polymers can enhance adenoviral gene expression in an orthotopic model of bladder cancer, thereby providing the foundation for future studies to determine therapeutic benefits of polymer-adenovirus combination in bladder cancer gene therapy.

Generation of a focused poly(amino ether) library: polymer-mediated transgene delivery and gold-nanorod based theranostic systems.

A focused library of twenty-one cationic poly(amino ethers) was synthesized following ring-opening polymerization of two diglycidyl ethers by different oligoamines. The polymers were screened in parallel for plasmid DNA (pDNA) delivery, and transgene expression efficacies of individual polymers were compared to those of 25 kDa polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Seven lead polymers that demonstrated higher transgene expression than PEI in pancreatic and prostate cancer cells lines were identified from the screen. All seven lead polymers showed highest transgene expression at a polymer:pDNA weight ratio of 5:1 in the MIA PaCa-2 pancreatic cancer cell line. Among the conditions studied, transgene expression efficacy correlated with minimal polymer cytotoxicity but not polyplex sizes. In addition, this study indicated that methylene spacing between amine centers in the monomers, amine content, and molecular weight of the polymers are all significant factors and should be considered when designing polymers for transgene delivery. A lead effective polymer was employed for coating gold nanorods, leading to theranostic nanoassemblies that possess combined transgene delivery and optical imaging capabilities, leading to potential theranostic systems.