Excess membrane cholesterol is an early contributing reversible aspect of skeletal muscle insulin resistance in C57BL/6NJ mice fed a Western-style high-fat diet.

Skeletal muscle insulin resistance manifests shortly after high-fat feeding, yet mechanisms are not known. Here we set out to determine whether excess skeletal muscle membrane cholesterol and cytoskeletal derangement known to compromise glucose transporter (GLUT)4 regulation occurs early after high-fat feeding. We fed 6-wk-old male C57BL/6NJ mice either a low-fat (LF, 10% kcal) or a high-fat (HF, 45% kcal) diet for 1 wk. This HF feeding challenge was associated with an increase, albeit slight, in body mass, glucose intolerance, and hyperinsulinemia. Liver analyses did not reveal signs of hepatic insulin resistance; however, skeletal muscle immunoblots of triad-enriched regions containing transverse tubule membrane showed a marked loss of stimulated GLUT4 recruitment. An increase in cholesterol was also found in these fractions from HF-fed mice. These derangements were associated with a marked loss of cortical filamentous actin (F-actin) that is essential for GLUT4 regulation and known to be compromised by increases in membrane cholesterol. Both the withdrawal of the HF diet and two subcutaneous injections of the cholesterol-lowering agent methyl-ß-cyclodextrin at 3 and 6 days during the 1-wk HF feeding intervention completely mitigated cholesterol accumulation, cortical F-actin loss, and GLUT4 dysregulation. Moreover, these beneficial membrane/cytoskeletal changes occurred concomitant with a full restoration of metabolic responses. These results identify skeletal muscle membrane cholesterol accumulation as an early, reversible, feature of insulin resistance and suggest cortical F-actin loss as an early derangement of skeletal muscle insulin resistance.

Adapting the Patient-Centered Medical Home to Address Psychosocial Adversity: Results of a Qualitative Study.

OBJECTIVE: The patient-centered medical home (PCMH) seeks to improve population health. However, PCMH models often focus on improving treatment of chronic diseases rather than on addressing psychosocial adversity. We sought to gather key stakeholder input about how PCMHs might feasibly and sustainably address psychosocial adversity within their patient populations. METHODS: We conducted 25 semistructured interviews with key stakeholders, such as physicians, nurses, medical assistants, and patients. The audiorecorded interviews focused on participants' perceptions of the best ways to modify the PCMH to address patients' psychosocial adversity. To facilitate information gathering, a fictional patient case was presented. Analyses were conducted using a 3-stage content-analysis process. RESULTS: Participants identified provider-related and systems-level changes necessary for addressing these psychosocial adversities effectively. On the provider level, participants thought that practitioners should foster trusting relationships with patients and should be emotionally present as patients describe their life experiences. Participants also emphasized that providers need to have sensitive conversations about adversity and resilience. On a systems level, participants discussed that documentation must balance privacy and include relevant information in the medical record. In addition, care should be delivered not by a single provider but by a team that has a longitudinal relationship with the patient; this care team should include behavioral health support. CONCLUSIONS: Participants provided practical strategies and highlighted provider and systems level changes to adequately address patients' prior psychosocial adversity. Future studies need to assess the degree to which such a trauma-informed approach improves patient access, outcomes, and care quality, and reduces cost.

Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG. METHODS: A collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo. RESULTS: In vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice. CONCLUSION: Our collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.

A Novel Mouse Model of Diffuse Intrinsic Pontine Glioma Initiated in Pax3-Expressing Cells.

Diffuse intrinsic pontine glioma (DIPG) is a rare and incurable brain tumor that arises predominately in children and involves the pons, a structure that along with the midbrain and medulla makes up the brainstem. We have previously developed genetically engineered mouse models of brainstem glioma using the RCAS/Tv-a system by targeting PDGF-B overexpression, p53 loss, and H3.3K27M mutation to Nestin-expressing brainstem progenitor cells of the neonatal mouse. Here we describe a novel mouse model targeting these same genetic alterations to Pax3-expressing cells, which in the neonatal mouse pons consist of a Pax3+/Nestin+/Sox2+ population lining the fourth ventricle and a Pax3+/NeuN+ parenchymal population. Injection of RCAS-PDGF-B into the brainstem of Pax3-Tv-a mice at postnatal day 3 results in 40% of mice developing asymptomatic low-grade glioma. A mixture of low- and high-grade glioma results from injection of Pax3-Tv-a;p53(fl/fl) mice with RCAS-PDGF-B and RCAS-Cre, with or without RCAS-H3.3K27M. These tumors are Ki67+, Nestin+, Olig2+, and largely GFAP- and can arise anywhere within the brainstem, including the classic DIPG location of the ventral pons. Expression of the H3.3K27M mutation reduces overall H3K27me3 as compared with tumors without the mutation, similar to what has been previously shown in human and mouse tumors. Thus, we have generated a novel genetically engineered mouse model of DIPG, which faithfully recapitulates the human disease and represents a novel platform with which to study the biology and treatment of this deadly disease.

A high-throughput in vitro drug screen in a genetically engineered mouse model of diffuse intrinsic pontine glioma identifies BMS-754807 as a promising therapeutic agent.

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 µM, significant inhibition of proliferation at a concentration of 1.5 µM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.

Pax3 expression enhances PDGF-B-induced brainstem gliomagenesis and characterizes a subset of brainstem glioma.

High-grade Brainstem Glioma (BSG), also known as Diffuse Intrinsic Pontine Glioma (DIPG), is an incurable pediatric brain cancer. Increasing evidence supports the existence of regional differences in gliomagenesis such that BSG is considered a distinct disease from glioma of the cerebral cortex (CG). In an effort to elucidate unique characteristics of BSG, we conducted expression analysis of mouse PDGF-B-driven BSG and CG initiated in Nestin progenitor cells and identified a short list of expression changes specific to the brainstem gliomagenesis process, including abnormal upregulation of paired box 3 (Pax3). In the neonatal mouse brain, Pax3 expression marks a subset of brainstem progenitor cells, while it is absent from the cerebral cortex, mirroring its regional expression in glioma. Ectopic expression of Pax3 in normal brainstem progenitors in vitro shows that Pax3 inhibits apoptosis. Pax3-induced inhibition of apoptosis is p53-dependent, however, and in the absence of p53, Pax3 promotes proliferation of brainstem progenitors. In vivo, Pax3 enhances PDGF-B-driven gliomagenesis by shortening tumor latency and increasing tumor penetrance and grade, in a region-specific manner, while loss of Pax3 function extends survival of PDGF-B-driven;p53-deficient BSG-bearing mice by 33%. Importantly, Pax3 is regionally expressed in human glioma as well, with high PAX3 mRNA characterizing 40% of human BSG, revealing a subset of tumors that significantly associates with PDGFRA alterations, amplifications of cell cycle regulatory genes, and is exclusive of ACVR1 mutations. Collectively, these data suggest that regional Pax3 expression not only marks a novel subset of BSG but also contributes to PDGF-B-induced brainstem gliomagenesis.

PD-0332991, a CDK4/6 inhibitor, significantly prolongs survival in a genetically engineered mouse model of brainstem glioma.

Diffuse intrinsic pontine glioma (DIPG) is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb) pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG) mouse model. We found that PD-0332991 (PD), a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT) followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas.

Targeting sonic hedgehog-associated medulloblastoma through inhibition of Aurora and Polo-like kinases.

Medulloblastoma is the most common malignant brain tumor in children. Although aggressive surgery, radiation, and chemotherapy have improved outcomes, survivors suffer severe long-term side effects, and many patients still succumb to their disease. For patients whose tumors are driven by mutations in the sonic hedgehog (SHH) pathway, SHH antagonists offer some hope. However, many SHH-associated medulloblastomas do not respond to these drugs, and those that do may develop resistance. Therefore, more effective treatment strategies are needed for both SHH and non-SHH-associated medulloblastoma. One such strategy involves targeting the cells that are critical for maintaining tumor growth, known as tumor-propagating cells (TPC). We previously identified a population of TPCs in tumors from patched mutant mice, a model for SHH-dependent medulloblastoma. These cells express the surface antigen CD15/SSEA-1 and have elevated levels of genes associated with the G2-M phases of the cell cycle. Here, we show that CD15(+) cells progress more rapidly through the cell cycle than CD15(-) cells and contain an increased proportion of cells in G2-M, suggesting that they might be vulnerable to inhibitors of this phase. Indeed, exposure of tumor cells to inhibitors of Aurora kinase (Aurk) and Polo-like kinases (Plk), key regulators of G2-M, induces cell-cycle arrest, apoptosis, and enhanced sensitivity to conventional chemotherapy. Moreover, treatment of tumor-bearing mice with these agents significantly inhibits tumor progression. Importantly, cells from human patient-derived medulloblastoma xenografts are also sensitive to Aurk and Plk inhibitors. Our findings suggest that targeting G2-M regulators may represent a novel approach for treatment of human medulloblastoma.

Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra.

Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent ß-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to ß-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ∼2 cm(-1) shift in an apparent ß-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain ß-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible ß-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).

A novel mutation (F71L) in alphaA-crystallin with defective chaperone-like function associated with age-related cataract.

Age-related cataract (ARC) is a multifactorial disease and the leading cause of blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. However, gene mutation studies on ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA (alphaA-crystallin) gene in three unrelated female sporadic cases among 711 ARC patients but not in 265 normal non-cataractous controls by SSCP and RFLP analysis. By comparing human recombinant wild-type and F71L-alphaA-crystallin, we characterized the functional significance of this missense mutation. Chromatography, fluorescence and far- and near-UV CD studies indicated that F71L missense mutation did not significantly affect the apparent molecular mass, secondary and tertiary structures and hydrophobicity of alphaA-crystallin. While the mutant alphaA-crystallin displayed significant (35-90%) loss of chaperone-like activity (CLA) in thermal aggregation of carbonic anhydrase, betaL- and gamma-crystallins, it showed moderate (10-50%) loss in CLA in DTT-induced aggregation of insulin and lysozyme. This is the first report of an alphaA-F71L mutation being associated with ARC and suggests that ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.

The stratified syncytium of the vertebrate lens.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Interactions between small heat shock protein alpha-crystallin and galectin-related interfiber protein (GRIFIN) in the ocular lens.

As a member of the small heat shock protein superfamily, alpha-crystallin has a chaperone-like ability to recognize and bind denatured or unfolded proteins and prevent their aggregation. Recent studies suggest that alpha-crystallin may also interact with a variety of proteins under native conditions in vitro. To identify potential binding partners for alpha-crystallin in the intact ocular lens, we conducted cross-linking studies in transgenic mouse lenses designed for overexpression of His-tagged human alphaA-crystallin. Interacting proteins were copurified with the epitope-tagged crystallin complexes and were identified by tandem mass spectrometry. This approach identified GRIFIN (galectin-related interfiber protein) as a novel binding partner. Consistent with results from cross-linking, GRIFIN subunits copurified with alpha-crystallin complexes during size exclusion chromatography of nontransgenic mouse lens extracts prepared without chemical cross-linking. Equilibrium binding to GRIFIN was studied using native alpha-crystallin isolated from calf lenses as well as oligomeric complexes reconstituted from recombinant alphaA- and alphaB-crystallin subunits. Calf lens alpha-crystallin binds GRIFIN with relatively high affinity (K(d) = 6.5 +/- 0.8 microM) at a stoichiometry of 0.25 +/- 0.01 GRIFIN monomer/alpha-crystallin subunit. The binding interaction between alpha-crystallin and GRIFIN is enhanced up to 5-fold in the presence of 3 mM ATP. These binding data support the hypothesis that GRIFIN is a novel binding partner of alpha-crystallin in the lens.