Neuronal Glycogen Breakdown Mitigates Tauopathy via Pentose Phosphate Pathway-Mediated Oxidative Stress Reduction.

Tauopathies encompass a range of neurodegenerative disorders, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD). Unfortunately, current treatment approaches for tauopathies have yielded limited success, underscoring the pressing need for novel therapeutic strategies. We observed distinct signatures of impaired glycogen metabolism in the Drosophila brain of the tauopathy model and the brain of AD patients, indicating a link between tauopathies and glycogen metabolism. We demonstrate that the breakdown of neuronal glycogen by activating glycogen phosphorylase (GlyP) ameliorates the tauopathy phenotypes in flies and induced pluripotent stem cell (iPSC) derived neurons from FTD patients. We observed that glycogen breakdown redirects the glucose flux to the pentose phosphate pathway to alleviate oxidative stress. Our findings uncover a critical role for increased GlyP activity in mediating the neuroprotection benefit of dietary restriction (DR) through the cAMP-mediated protein kinase A (PKA) activation. Our studies identify impaired glycogen metabolism as a key hallmark for tauopathies and offer a promising therapeutic target in tauopathy treatment.

Dual Blockade of EP2 and EP4 Signaling is Required for Optimal Immune Activation and Antitumor Activity Against Prostaglandin-Expressing Tumors.

While the role of prostaglandin E2 (PGE2) in promoting malignant progression is well established, how to optimally block the activity of PGE2 signaling remains to be demonstrated. Clinical trials with prostaglandin pathway targeted agents have shown activity but without sufficient significance or dose-limiting toxicities that have prevented approval. PGE2 signals through four receptors (EP1-4) to modulate tumor progression. EP2 and EP4 signaling exacerbates tumor pathology and is immunosuppressive through potentiating cAMP production. EP1 and EP3 signaling has the opposite effect through increasing IP3 and decreasing cAMP. Using available small-molecule antagonists of single EP receptors, the cyclooxygenase-2 (COX-2) inhibitor celecoxib, or a novel dual EP2/EP4 antagonist generated in this investigation, we tested which approach to block PGE2 signaling optimally restored immunologic activity in mouse and human immune cells and antitumor activity in syngeneic, spontaneous, and xenograft tumor models. We found that dual antagonism of EP2 and EP4 together significantly enhanced the activation of PGE2-suppressed mouse and human monocytes and CD8+ T cells in vitro as compared with single EP antagonists. CD8+ T-cell activation was dampened by single EP1 and EP3 antagonists. Dual EP2/EP4 PGE2 receptor antagonists increased tumor microenvironment lymphocyte infiltration and significantly reduced disease burden in multiple tumor models, including in the adenomatous polyposis coli (APC)min+/- spontaneous colorectal tumor model, compared with celecoxib. These results support a hypothesis that redundancy of EP2 and EP4 receptor signaling necessitates a therapeutic strategy of dual blockade of EP2 and EP4. Here we describe TPST-1495, a first-in-class orally available small-molecule dual EP2/EP4 antagonist. Significance: Prostaglandin (PGE2) drives tumor progression but the pathway has not been effectively drugged. We demonstrate significantly enhanced immunologic potency and antitumor activity through blockade of EP2 and EP4 PGE2 receptor signaling together with a single molecule.

Proteomic Analysis of Huntington's Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium-spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72-induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis. Using data-dependent acquisitions with FAIMS for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6323 proteins with at least two unique peptides. Of these, 901 proteins were altered significantly more in the HD72-MSNs than in isogenic controls. Functional enrichment analysis of upregulated proteins demonstrated extracellular matrix and DNA signaling (DNA replication pathway, double-strand break repair, G1/S transition) with the highest significance. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A:EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism, and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., septin protein members) were dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and using quantitative image analysis, we found that lipid droplets accumulated in the HD72-MSN, suggesting a deficit in the turnover of lipids possibly through lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.

Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection.

Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).

Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches.

Post-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.

Lengthening Neurogenic Period during Neocortical Development Causes a Hallmark of Neocortex Expansion.

A hallmark of the evolutionary expansion of the neocortex is a specific increase in the number of neurons generated for the upper neocortical layers during development. The cause underlying this increase is unknown. Here, we show that lengthening the neurogenic period during neocortical development is sufficient to specifically increase upper-layer neuron generation. Thus, embryos of mouse strains with longer gestation exhibited a longer neurogenic period and generated more upper-layer, but not more deep-layer, neurons than embryos with shorter gestation. Accordingly, long-gestation embryos showed a greater abundance of neurogenic progenitors in the subventricular zone than short-gestation embryos at late stages of cortical neurogenesis. Analysis of a mouse-rat chimeric embryo, developing inside a rat mother, pointed to factors in the rat environment that influenced the upper-layer neuron generation by the mouse progenitors. Exploring a potential maternal source of such factors, short-gestation strain mouse embryos transferred to long-gestation strain mothers exhibited an increase in the length of the neurogenic period and upper-layer neuron generation. The opposite was the case for long-gestation strain mouse embryos transferred to short-gestation strain mothers, indicating a dominant maternal influence on the length of the neurogenic period and hence upper-layer neuron generation. In summary, our study uncovers a hitherto unknown link between embryonic cortical neurogenesis and the maternal gestational environment and provides experimental evidence that lengthening the neurogenic period during neocortical development underlies a key aspect of neocortical expansion.

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The "one-pot" affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

A proteomic atlas of senescence-associated secretomes for aging biomarker development.

The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo.

Accumulation of "Old Proteins" and the Critical Need for MS-based Protein Turnover Measurements in Aging and Longevity.

Aging and age-related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin-mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half-lives and protein turnover at the level of individual proteins in vivo. For large-scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long-lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age-related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.

Sirtuin 5 Regulates Proximal Tubule Fatty Acid Oxidation to Protect against AKI.

BACKGROUND: The primary site of damage during AKI, proximal tubular epithelial cells, are highly metabolically active, relying on fatty acids to meet their energy demands. These cells are rich in mitochondria and peroxisomes, the two organelles that mediate fatty acid oxidation. Emerging evidence shows that both fatty acid pathways are regulated by reversible posttranslational modifications, particularly by lysine acylation. Sirtuin 5 (Sirt5), which localizes to both mitochondria and peroxisomes, reverses post-translational lysine acylation on several enzymes involved in fatty acid oxidation. However, the role of the Sirt5 in regulating kidney energy metabolism has yet to be determined. METHODS: We subjected male Sirt5-deficient mice (either +/- or -/-) and wild-type controls, as well as isolated proximal tubule cells, to two different AKI models (ischemia-induced or cisplatin-induced AKI). We assessed kidney function and injury with standard techniques and measured fatty acid oxidation by the catabolism of 14C-labeled palmitate to 14CO2. RESULTS: Sirt5 was highly expressed in proximal tubular epithelial cells. At baseline, Sirt5 knockout (Sirt5-/- ) mice had modestly decreased mitochondrial function but significantly increased fatty acid oxidation, which was localized to the peroxisome. Although no overt kidney phenotype was observed in Sirt5-/- mice, Sirt5-/- mice had significantly improved kidney function and less tissue damage compared with controls after either ischemia-induced or cisplatin-induced AKI. This coincided with higher peroxisomal fatty acid oxidation compared with mitochondria fatty acid oxidation in the Sirt5-/- proximal tubular epithelial cells. CONCLUSIONS: Our findings indicate that Sirt5 regulates the balance of mitochondrial versus peroxisomal fatty acid oxidation in proximal tubular epithelial cells to protect against injury in AKI. This novel mechanism might be leveraged for developing AKI therapies.

Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation.

Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.