Peter Pytel

Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice

Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprins luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1alpha, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.

Interferon-γ Induced Medulloblastoma in the Developing Cerebellum

We have generated a mouse model system with a high incidence of medulloblastoma, a malignant neoplasm believed to arise from immature precursors of cerebellar granule neurons. These animals ectopically express interferon-γ (IFN-γ) in astrocytes in the CNS in a controlled manner, exploiting the tetracycline-controllable system. More than 80% of these mice display severe ataxia and develop cerebellar tumors that express synaptophysin, the mouse atonal homolog MATH1, sonic hedgehog (SHH), and Gli1. IFN-γ-induced tumorigenesis in these mice is associated with increased expression of SHH, and SHH induction and tumorigenesis are dependent on signal transducer and activator of transcription 1 (STAT1). When IFN-γ expression is shut down with doxycycline at postnatal day 12 (P12), the clinical symptoms dissipate and the mice do not develop tumors, whereas if transgene expression is shut down at P16, the clinical symptoms and tumors progress to lethality, indicating that IFN-γ is required for tumor induction but not progression. The tumors that occur in the continued presence of IFN-γ display extensive necrosis and apoptosis as well as macrophage and lymphocytic infiltration, whereas the tumors that develop in mice in which IFN-γ expression is shut down at P16 do not. Thus, IFN-γ expression in the perinatal period can induce SHH expression and medulloblastoma in the cerebellum by a STAT1-dependent mechanism, and its continued presence appears to promote a host response to the tumor.

Nesprin-1 mutations in human and murine cardiomyopathy

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.

Undifferentiated intimal sarcoma of large systemic blood vessels : Report of 14 cases with immunohistochemical profile and review of the literature

Intimal sarcoma (IS) is defined as a malignant tumor arising in the tunica intima of large blood vessels. In systemic circulation, the majority of IS develop in the aorta, where close to three fourths of published cases lack specific differentiation and are called undifferentiated intimal sarcomas (UIS). The remaining cases are intima-associated sarcomas of recognized types, also called differentiated intimal sarcomas (DIS). In this report, we further characterize UIS, including its immunohistochemical profile and results of comparative genomic hybridization. A total of 14 cases of UIS were collected from 17 medical institutions, including slides, blocks, electron photomicrographs, clinical abstracts, and reports of surgical pathology specimens and autopsies. The patients, 7 women and 7 men, were 41 to 85 years of age (median, 65.6 years). Twelve tumors arose from the aorta, one from the left external iliac and femoral arteries, and one in a large systemic vein (the venous tumor was included due to histologic similarity with the arterial lesions). Tumors ranged from 1 cm to over 10 cm in diameter. Histopathology was that of a largely necrotic, poorly differentiated epithelioid and pleomorphic malignant neoplasm relating to the tunica intima. Usually there was only a thin layer of viable tumor cells overlying a large thrombus. All tumors stained at least focally with the endothelial markers CD31 and Fli-1; however, there was otherwise considerable variability in immunophenotype. The distinctive histopathologic appearance of the primary luminal lesion was lost whenever tumor invaded outside the vessel wall (into adventitia and beyond) or in metastatic sites. Such extravascular tumors assumed a variety of patterns reminiscent of undifferentiated pleomorphic sarcoma (UPS; in older literature also known as pleomorphic malignant fibrous histiocytoma, MFH) or other distinct types of sarcomas, including osteosarcoma, angiosarcoma, and rhabdomyosarcoma. The results of comparative genomic hybridization were nonspecific. Eleven patients died of the disease, in an average of 11 months after diagnosis. Three patients are still alive and free of disease at 4, 16, and 27 years. UIS of large systemic vessels represents a distinct clinical entity where intraluminal sarcoma presents with thrombosis and occlusion of large vessels. It is associated with a highly characteristic, although not entirely specific, histology and immunohistochemical phenotype. The histogenesis of UIS is not certain; however, it seems that the cell of origin must leave the confines of the vessel wall to show altered morphology. Although there are rare long-term survivors, UIS behaves as a fully malignant neoplasm that is almost uniformly associated with metastases and tumor-related death.

Pathogenesis of septic encephalopathy.

Purpose of reviewSeptic encephalopathy is a frequent complication in severe sepsis, the pathogenesis and mechanisms of which are not fully understood. Here, we review recent advances in our understanding of septic encephalopathy, from molecular mechanisms to behavioral alterations, from diagnostic tools to potential therapeutic agents. Recent findingsRecent insights into septic encephalopathy include: microcirculatory failure precedes changes in evoked potential responses; blood–brain barrier alteration is prevented by reducing intercellular adhesion molecule expression and pericyte detachment; reducing infiltration of CD68 macrophages and inhibiting complement activation alleviates neuroinflammation in septic encephalopathy; and reducing mitochondrial dysfunction and inducible nitric oxide synthase expression can restore altered brain function. In addition, other factors such as the circulating levels of growth hormone are independent predictors for mortality and correlate with the severity of sepsis. Similar to humans, septic rats present recognition memory impairment and depressive-like symptoms but not anxiety-like behavior and will serve as efficient models to study the underlying mechanisms of septic encephalopathy. SummarySeptic encephalopathy is a dynamic disease caused by a complex network of systems and pathways going awry. More insights into the pathogenesis of septic encephalopathy are expected to lead to new cellular and molecular targets, which in turn will permit design of specific septic encephalopathy-alleviating drugs and prevent its negative influence on survival.

Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.

Background Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. Methods/Findings To investigate the hypothesis that mutant lamin A/C changes the laminas ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. Conclusions These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.

Sarcomere Mutations in Cardiomyopathy with Left Ventricular Hypertrabeculation

Background— Mutations in the genes encoding sarcomere proteins have been associated with both hypertrophic and dilated cardiomyopathy. Recently, mutations in myosin heavy chain (MYH7), cardiac actin (ACTC), and troponin T (TNNT2) were associated with left ventricular noncompaction, a form of cardiomyopathy characterized with hypertrabeculation that may also include reduced function of the left ventricle. Methods and Results— We used clinically available genetic testing on 3 cases referred for evaluation of left ventricular dysfunction and noncompaction of the left ventricle and found that all 3 individuals carried sarcomere mutations. The first patient presented with neonatal heart failure and was referred for left ventricular noncompaction cardiomyopathy. Genetic testing found 2 different mutations in MYBPC3 in trans. The first mutation, 3776delA, Q1259fs, rendered a frame shift at 1259 of cardiac myosin-binding protein C and the second mutation was L1200P. The frameshift mutation was also found in this mother who displayed mild echocardiographic features of cardiomyopathy, with only subtle increase in trabeculation and an absence of hypertrophy. A second pediatric patient presented with heart failure and was found to carry a de novo MYH7 R369Q mutation. The third case was an adult patient with dilated cardiomyopathy referred for ventricular hypertrabeculation. This patient had a family history of congestive heart failure, including pediatric onset cardiomyopathy where 3 individuals in the family were found to have the MYH7 mutation R1250W. Conclusion— Genetic testing should be considered for cardiomyopathy with hypertrabeculation.

The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.

CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

Molecular Heterogeneity in Glioblastoma: Therapeutic Opportunities and Challenges

Glioblastoma (GBM) has been recognized as a clinical and pathologic entity for more than a century. Throughout its history, its cells of origin have been in question. Its behavior is aggressive and despite decades of effort, median survival is just beginning to improve. Surgical techniques and radiotherapy schemas continue to be refined, but the most recent progress has been achieved through improved medical therapies. These are the result of both pharmacological advances and a deeper understanding of the biological characteristics of GBM. Due to a combination of its complex phenotype and organ-specific clinical manifestations, efforts to refine GBM treatment with targeted therapies largely have been frustrated. In this review, we discuss recent attempts to exploit new molecular insights, consider the reasons for slow progress in developing better treatments, and examine future therapeutic options.