Faculty Bibliography

Amyloid β (Aβ) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of Aβ heterogeneity likely resulting from the action of multiple proteolytic enzymes. This chapter describes a sequential extraction protocol allowing the differential fractionation of soluble and deposited Aβ species taking advantage of their differential solubility properties. Soluble Aβ is extracted by water-based buffers like phosphate-buffered saline-PBS-whereas pre-fibrillar and fibrillar deposits, usually poorly soluble in PBS, are extractable in detergent containing solutions or more stringent conditions as formic acid. The extraction procedure is followed by the biochemical identification of the extracted Aβ species using Western blot and a targeted proteomic analysis which combines immunoprecipitation with MALDI-ToF mass spectrometry. This approach revealed the presence of numerous C- and N-terminal truncated Aβ species in addition to Aβ1-40/42. Notably, the more soluble C-terminal cleaved fragments constitute a main part of PBS homogenates. On the contrary, N-terminal truncated species typically require more stringent conditions for the extraction in agreement with their lower solubility and enhanced aggregability. Detailed assessment of the molecular diversity of Aβ species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of Aβ truncations to AD pathogenesis and their potential as novel therapeutic targets.

Quantitative proteome analysis allows comparisons of protein or phosphoprotein levels across multiple cell types or conditions. A number of experimental approaches have been described toward quantitative proteomics. In this chapter, we focus on Tandem Mass Tag (TMT) isobaric labeling of peptides for global, relative quantitation of proteins and phosphopeptides. To date, there has been no published protocol describing chemical labeling of small amounts of peptides specifically extracted from small tumor samples, for which rigorous sample preparation is necessary to ensure reproducible TMT labeling.

Mass spectrometry (MS)-based techniques have been finding utility as sensitive, high throughput metabolite analysis tools for complex biological samples. We describe here a nanoflow liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system we developed and applied to metabolic profiling of human cells. Metabolites are extracted from cells using methanol, and filtered through a C18 StageTip to remove large particles. Metabolite samples are separated by HPLC at a flow rate of 400-500 nl/min, then analyzed in both positive and negative ion modes in an LTQ-Orbitrap MS. Metabolite identification and differential analysis are performed using commercial or open source software. Protocols outlined in this chapter describe how nano-LC-MS can be applied to investigate metabolic profiling with limited biomass amount.

Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein alpha-synuclein. alpha-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that alpha-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different alpha-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa alpha-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific alpha-synuclein features in neurodegeneration will allow for the identification of neurotoxic alpha-synuclein species, which represent prime targets to reduce alpha-synuclein pathogenicity.

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1DeltaHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1DeltaHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.