Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation.

The intricate protein-chaperone network is vital for cellular function. Recent discoveries have unveiled the existence of specialized chaperone complexes called epichaperomes, protein assemblies orchestrating the reconfiguration of protein-protein interaction networks, enhancing cellular adaptability and proliferation. This study delves into the structural and regulatory aspects of epichaperomes, with a particular emphasis on the significance of post-translational modifications in shaping their formation and function. A central finding of this investigation is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 situated within an intrinsically disordered region, as critical determinants in epichaperome assembly. Our data demonstrate that the phosphorylation of these serine residues enhances HSP90's interaction with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Furthermore, this study establishes a direct link between epichaperome function and cellular physiology, especially in contexts where robust proliferation and adaptive behavior are essential, such as cancer and stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone complexes in diseases characterized by epichaperome dysregulation, bridging the gap between fundamental research and precision medicine.

Over-expression of N-acetylaspartate synthase exacerbates pathological energetic deficit and accelerates cognitive decline in the 5xFAD mouse.

N-acetylaspartate (NAA) is an abundant central nervous system amino acid derivative that is tightly coupled to mitochondria and energy metabolism in neurons. A reduced NAA signature is a prominent early pathological biomarker in multiple neurodegenerative diseases and becomes progressively more pronounced as disease advances. Because NAA synthesis requires aspartate drawn directly from mitochondria, we argued that this process is in direct competition with oxidative phosphorylation for substrate and that sustained high levels of NAA synthesis would be incompatible with pathological energy crisis. We show here that over-expression of the rate-limiting NAA synthetic enzyme in the hippocampus of the 5x familial Alzheimer's disease (5xFAD) mouse results in an exaggerated pathological ATP deficit and accelerated cognitive decline. Over-expression of NAA synthase did not increase amyloid burden or result in cell loss but did significantly deplete mitochondrial aspartate and impair the ability of mitochondria to oxidize glutamate for adenosine triphosphate (ATP) synthesis. These results define NAA as a sink for energetic substrate and suggest initial pathological reductions in NAA are part of a response to energetic crisis designed to preserve substrate bioavailability for mitochondrial ATP synthesis.

Combination Therapy With MDM2 and MEK Inhibitors Is Effective in Patient-Derived Models of Lung Adenocarcinoma With Concurrent Oncogenic Drivers and MDM2 Amplification.

INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.

Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease.

Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease.

N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylase.

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.

Transcriptional regulation of N-acetylaspartate metabolism in the 5xFAD model of Alzheimer's disease: evidence for neuron-glia communication during energetic crisis.

N-acetylaspartate (NAA) provides a non-invasive clinical index of neuronal metabolic integrity across the entire neurodegenerative spectrum. While NAA function is not comprehensively defined, reductions in the brain are associated with compromised mitochondrial metabolism and are tightly linked to ATP. We have undertaken an analysis of abnormalities in NAA during early stage pathology in the 5xFAD mouse model of familial Alzheimer's disease and show here that dysregulated expression of the gene encoding for the rate-limiting NAA synthetic enzyme (Nat8L) is associated with deficits in mitochondrial oxidative phosphorylation in this model system. Downreguation of Nat8L is particularly pronounced in the 5xFAD hippocampus, and is preceded by a significant upregulation of oligodendrocytic aspartoacylase (aspa), which encodes for the sole known NAA-catabolizing enzyme in the brain. Reductions in 5xFAD NAA and Nat8L cannot be accounted for by discrepancies in either neuron content or activity of the substrate-providing malate-aspartate shuttle, thereby implicating transcriptional regulation in a coordinated response to pathological energetic crisis. A central role for ASPA in this response is supported by a parallel developmental analysis showing highly significant increases in Nat8L expression in an ASPA-null mouse model during a period of early postnatal development normally punctuated by the transcriptional upregulation of aspa. These results provide preliminary evidence of a signaling mechanism in Alzheimer's disease that involves cross talk between neurons and oligodendrocytes, and suggest that ASPA acts to negatively regulate Nat8L expression. This mechanism is proposed to be a fundamental means by which the brain conserves available substrate during energy crises.

Dietary triheptanoin rescues oligodendrocyte loss, dysmyelination and motor function in the nur7 mouse model of Canavan disease.

The inherited pediatric leukodystrophy Canavan disease is characterized by dysmyelination and severe spongiform degeneration, and is currently refractory to treatment. A definitive understanding of core disease mechanisms is lacking, but pathology is believed to result at least in part compromised fatty acid synthesis during myelination. Recent evidence generated in an animal model suggests that the breakdown of N-acetylaspartate metabolism in CD results in a heightened coupling of fatty acid synthesis to oligodendrocyte oxidative metabolism during the early stages of myelination, thereby causing acute oxidative stress. We present here the results of a dietary intervention designed to support oxidative integrity during developmental myelination in the nur7 mouse model of Canavan disease. Provision of the odd carbon triglyceride triheptanoin to neonatal nur7 mice reduced oxidative stress, promoted long-term oligodendrocyte survival, and increased myelin in the brain. Improvements in oligodendrocyte survival and myelination were associated with a highly significant reduction in spongiform degeneration and improved motor function in triheptanoin treated mice. Initiation of triheptanoin treatment in older animals resulted in markedly more modest effects on these same pathological indices, indicating a window of therapeutic intervention that corresponds with developmental myelination. These results support the targeting of oxidative integrity at early stages of Canavan disease, and provide a foundation for the clinical development of a non-invasive dietary triheptanoin treatment regimen.

Human umbilical cord blood-derived mesenchymal stem cells in the cultured rabbit intervertebral disc: a novel cell source for disc repair.

OBJECTIVE: Back pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture. DESIGN: This study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant tracer specific for live cells. Human type II collagen gene expression (from the graft) was assessed by polymerase chain reaction. RESULTS: We have shown that hUCB-MSCs cultured in micromass are stained blue with Alcian blue dye, which suggests that proteoglycan-rich extracellular matrix is produced. In the cultured rabbit IVD explants, hUCB-MSCs survived for at least 4 wks and expressed the human type II collagen gene, suggesting that the injected hUCB-MSCs are differentiating into a chondrocyte-like lineage. CONCLUSIONS: This study demonstrates the abiity of hUBC-MSCs to survive and assume a chondrocyte-like phenotype when injected into the rabbit IVD. These data support the potential for hUBC-MSCs as a cell source for disc repair. Further measures of the host response to the injection and studies in animal models are needed before trials in humans.

Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium.

PURPOSE: To test the feasibility of altering the phenotype of umbilical cord blood mesenchymal stem cells (UCB MSCs) toward that of human corneal endothelial cells (HCEC) and to determine whether UCB MSCs can "home" to sites of corneal endothelial cell injury using an ex vivo corneal wound model. METHODS: RNA was isolated and purified from UCB MSCs and HCECs. Baseline information regarding the relative gene expression of UCB MSCs and HCEC was obtained by microarray analysis. Quantitative real-time PCR (q-PCR) verified the microarray findings for a subset of genes. The ability of different culture media to direct UCB MSCs toward a more HCEC-like phenotype was tested in both tissue culture and ex vivo corneal endothelial wound models using three different media: MSC basal medium (MSCBM), a basal medium used to culture lens epithelial cells (LECBM), or lens epithelial cell-conditioned medium (LECCM). Morphology of the MSCs was observed by phase-contrast microscopy or by light microscopic observation of crystal violet-stained cells. Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy. Formation of cell-cell junctions was tested by treatment with the calcium chelator, EGTA. A second microarray analysis compared gene expression between UCB MSCs grown in LECBM and LECCM to identify changes induced by the lens epithelial cell-conditioned culture medium. The ability of UCB MSCs to "home" to areas of endothelial injury was determined using ZO1 immunolocalization patterns in ex vivo corneal endothelial wounds. RESULTS: Baseline microarray analysis provided information regarding relative gene expression in UCB MSCs and HCECs. MSCs attached to damaged, but not intact, corneal endothelium in ex vivo corneal wounds. The morphology of MSCs was consistently altered when cells were grown in the presence of LECCM. In tissue culture and in ex vivo corneal wounds, UCB MSC treated with LECCM were elongated and formed parallel sheets of closely apposed cells. In both tissue culture and ex vivo corneal endothelial wounds, ZO1 and N-cadherin localized mainly to the cytoplasm of UCB MSCs in the presence of MSCBM. However, both proteins localized to cell borders when UCB MSCs were grown in either LECBM or LECCM. This localization was lost when extracellular calcium levels were reduced by treatment with EGTA. A second microarray analysis showed that, when UCB MSCs were grown in LECCM instead of LECBM, the relative expression of a subset of genes markedly differed, suggestive of a more HCEC-like phenotype. CONCLUSIONS: Results indicate that UCB MSCs are able to "home" to areas of injured corneal endothelium and that the phenotype of UCB MSCs can be altered toward that of HCEC-like cells. Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles.

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.

Identification of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model.

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.

Semiflexible amphiphilic polymers: cylindrical-shaped, collagenlike, and toroidal structures.

A coarse-grained model is used to study the conformational properties of semiflexible polymers with amphiphilic monomer units containing both hydrophilic and hydrophobic interaction sites. The hydrophobically driven conformational transitions are studied using molecular dynamics simulations for the chains of varying stiffness, as characterized by intrinsic Kuhn segment lengths that vary over a decade. It is shown that the energy of hydrophobic attraction required for the realization of the coil-to-globule transition increases with increasing chain stiffness. For rather stiff backbone, the coil-to-globule transition corresponds to a first order phase transition. We find that depending on the chain stiffness, a variety of thermodynamically stable anisometric chain morphologies are possible in a solvent selectively poor for hydrophobic sites of amphiphilic monomer units. For flexible chains, the amphiphilic polymer forms a cylindrical globule having blob structure with nearly spherical blobs. With increasing stiffness, the number of blobs composing the globule decreases and the shape of blobs transforms into elongated cylinder. Further increase in stiffness leads to compaction of macromolecules into a collagenlike structure when the chain folds itself several times and different strands wind round each other. In this state, the collagenlike structures coexist with toroidal globules, both conformations having approximately equal energies.