Hannes Vogel

p63 is a p53 homologue required for limb and epidermal morphogenesis

The p53 tumour suppressor is a transcription factor that regulates the progression of the cell through its cycle and cell death (apoptosis) in response to environmental stimuli such as DNA damage and hypoxia. Even though p53 modulates these critical cellular processes, mice that lack p53 are developmentally normal, suggesting that p53-related proteins might compensate for the functions of p53 during embryogenesis. Two p53 homologues, p63 and p73, are known and here we describe the function of p63 in vivo. Mice lacking p63 are born alive but have striking developmental defects. Their limbs are absent or truncated, defects that are caused by a failure of the apical ectodermal ridge to differentiate. The skin of p63-deficient mice does not progress past an early developmental stage: it lacks stratification and does not express differentiation markers. Structures dependent upon epidermal–mesenchymal interactions during embryonic development, such as hair follicles, teeth and mammary glands, are absent in p63-deficient mice. Thus, in contrast to p53, p63 is essential for several aspects of ectodermal differentiation during embryogenesis.

Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice.

Despite the high doses of radiation delivered in the treatment of patients with glioblastoma multiforme (GBM), the tumors invariably recur within the irradiation field, resulting in a low cure rate. Understanding the mechanism of such recurrence is therefore important. Here we have shown in an intracranial GBM xenograft model that irradiation induces recruitment of bone marrow-derived cells (BMDCs) into the tumors, restoring the radiation-damaged vasculature by vasculogenesis and thereby allowing the growth of surviving tumor cells. BMDC influx was initiated by induction of HIF-1 in the irradiated tumors, and blocking this influx prevented tumor recurrence. Previous studies have indicated that BMDCs are recruited to tumors in part through the interaction between the HIF-1-dependent stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4. Pharmacologic inhibition of HIF-1 or of the SDF-1/CXCR4 interaction prevented the influx of BMDCs, primarily CD11b+ myelomonocytes, and the postirradiation development of functional tumor vasculature, resulting in abrogation of tumor regrowth. Similar results were found using neutralizing antibodies against CXCR4. Our data therefore suggest a novel approach for the treatment of GBM: in addition to radiotherapy, the vasculogenesis pathway needs to be blocked, and this can be accomplished using the clinically approved drug AMD3100, a small molecule inhibitor of SDF-1/CXCR4 interactions.

Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery

Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinsons disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in ∼50% of human sporadic cancers and in an inherited cancer predisposition (Li–Fraumeni syndrome). We have analyzed the status of the wild‐type p53 allele in tumors taken from p53‐deficient heterozygous (p53+/−) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild‐type p53. We present evidence that a high proportion of the tumors from the p53+/− mice retain an intact, functional, wild‐type p53 allele. Unlike p53+/− tumors which lose their wild‐type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following γ‐irradiation, activates p21WAF1/CIP1 and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild‐type p53), shows high levels of binding to oligonucleotides containing a wild‐type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.

Neuronal Activity Promotes Oligodendrogenesis and Adaptive Myelination in the Mammalian Brain

Introduction Myelin is formed by mature oligodendrocytes to facilitate fast propagation of action potentials in axons. Small changes in myelin thickness can confer substantial changes in conduction speed and may thus alter neural circuit function. The idea that active neurons may modulate myelination is supported by in vitro studies and correlations between experience and myelin microstructure, but direct in vivo evidence demonstrating that neuronal activity regulates oligodendrocyte precursor cell (OPC) proliferation, differentiation, or changes in myelin microstructure has been lacking. We use in vivo optogenetic techniques in awake, behaving mice to provide direct evidence that neuronal activity regulates changes in myelin-forming cells within an active circuit. Neuronal activity promotes OPC proliferation, oligodendrogenesis, and myelin remodeling. Optogenetic stimulation of unilateral premotor cortex layer V projection neurons in awake, behaving Thy1::ChR2 mice promotes OPC proliferation (light green cells, red EdU+ nuclei), oligodendrogenesis (newly generated, EdU-marked oligodendrocyte; dark green cell), and an increase in myelin sheath thickness (viewed in cross section, gray). Together with these adaptive myelin changes, motor performance of the correlate limb is improved during normal gait 4 weeks after premotor cortex stimulation. Rationale Demonstrating the direct effects of behaviorally relevant neuronal activity on oligodendroglial lineage cells in vivo has been a challenge because traditional methods of directly promoting neuronal activity involve placement of an electrode, and the resultant tissue injury and subsequent inflammation affects OPC dynamics. Optogenetic technology allows for in vivo control of neuronal firing with millisecond precision using light delivered at a distance from the target and, thus, avoids extensive electrode-related tissue damage. We used an optogenetic (Thy1::ChR2) mouse model in which 470-nm light delivered near the brain surface stimulates the excitatory opsin channelrhodopsin expressed by cortical layer V projection neurons. Wild-type littermate controls lacking channelrhodopsin were identically manipulated to control for effects of surgery, optical fiber placement, and light exposure. In Thy1::ChR2 mice, light stimulation delivered unilaterally to the premotor cortex elicits complex motor behavior (unidirectional ambulation). The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) was administered at the time of optogenetic stimulation to mark actively dividing cells. Animals were evaluated at various time points to examine the effects of neuronal activity on myelin-forming cells and myelin microstructure, as well as the functional consequences of neuronal activity–regulated myelin changes. Results Optogenetic stimulation of cortical layer V projection neurons resulted in robust proliferation of OPCs within the premotor circuit, from the deep layers of the premotor cortex to the subcortical projections through the corpus callosum. Four weeks later, an increase in newly generated oligodendrocytes and increased myelin sheath thickness were found within the stimulated premotor circuit. Behavioral testing revealed increased swing speed of the correlate forelimb. Pharmacological blockade of OPC differentiation prevented activity-regulated oligodendrogenesis and myelin changes, as well as the associated behavioral change. Conclusion Neuronal activity regulates OPC proliferation, differentiation, and myelin remodeling in the murine brain with accompanying changes in behavioral function. Taken together, these findings suggest that adaptive changes in myelin-forming cells represent a type of behaviorally relevant neural plasticity, raising numerous conceptual and mechanistic questions. Mechanisms regulating myelin plasticity may be important for adaptive neural function and could be leveraged for interventions in diseases of myelin. Conversely, dysregulated myelin plasticity could conceivably contribute to disease. On-Demand Activity Oligodendroglia ensheath axons in the brain with myelin, which provides the insulation that speeds up transmission of neuronal electrical impulses. The process of myelination in the human brain goes on for decades, concurrent with all manner of brain development and cognitive activity. Gibson et al. (p. 10.1126/science.1252304, published online 10 April; see the Perspective by Bechler and ffrench-Constant) used optogenetics to study myelination in response to neural activity. Electrical activity in the motor cortex of the brain of awake mice led to proliferation and differentiation of oligodendrocytes and consequently increased myelination and alterations in motor response. Optogenetic stimulation of the mouse motor cortex incites proliferation of myelin-producing cells and axonal myelination. [Also see Perspective by Bechler and ffrench-Constant] Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPCs). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. In this work, we use optogenetic stimulation of the premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis, and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity–regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

Mosaic Analysis with Double Markers Reveals Tumor Cell of Origin in Glioma

Cancer cell of origin is difficult to identify by analyzing cells within terminal stage tumors, whose identity could be concealed by the acquired plasticity. Thus, an ideal approach to identify the cell of origin is to analyze proliferative abnormalities in distinct lineages prior to malignancy. Here, we use mosaic analysis with double markers (MADM) in mice to model gliomagenesis by initiating concurrent p53/Nf1 mutations sporadically in neural stem cells (NSCs). Surprisingly, MADM-based lineage tracing revealed significant aberrant growth prior to malignancy only in oligodendrocyte precursor cells (OPCs), but not in any other NSC-derived lineages or NSCs themselves. Upon tumor formation, phenotypic and transcriptome analyses of tumor cells revealed salient OPC features. Finally, introducing the same p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis. Our findings suggest OPCs as the cell of origin in this model, even when initial mutations occur in NSCs, and highlight the importance of analyzing premalignant stages to identify the cancer cell of origin.

Clinical Spectrum, Morbidity, and Mortality in 113 Pediatric Patients with Mitochondrial Disease

Objectives. The aim of this study was to elucidate the frequency of major clinical manifestations in children with mitochondrial disease and establish their clinical course, prognosis, and rates of survival depending on their clinical features. Methods. We performed a retrospective review of the medical records of 400 patients who were referred for evaluation of mitochondrial disease. By use of the modified Walker criteria, only patients who were assigned a definite diagnosis were included in the study. Results. A total of 113 pediatric patients with mitochondrial disease were identified. A total of 102 (90%) patients underwent a muscle biopsy as part of the diagnostic workup. A significant respiratory chain (RC) defect, according to the diagnostic criteria, was found in 71% of the patients who were evaluated. In this cohort, complex I deficiency (32%) and combined complex I, III, and IV deficiencies (26%) were the most common causes of RC defects, followed by complex IV (19%), complex III (16%), and complex II deficiencies (7%). Pathogenic mitochondrial DNA abnormalities were found in 11.5% of the patients. A substantial fraction (40%) of patients with mitochondrial disorders exhibited cardiac disease, diagnosed by Doppler echocardiography; however, the majority (60%) of patients had predominant neuromuscular manifestations. No correlation between the type of RC defect and the clinical presentation was observed. Overall, the mean age at presentation was 40 months. However, the mean age at presentation was 33 months in the cardiac group and 44 months in the noncardiac group. Twenty-six (58%) patients in the cardiac group exhibited hypertrophic cardiomyopathy, 29% had dilated cardiomyopathy, and the remainder (13%) had left ventricular noncompaction. Patients with cardiomyopathy had an 18% survival rate at 16 years of age. Patients with neuromuscular features but no cardiomyopathy had a 95% survival at the same age. Conclusions. This study gives strong support to the view that in patients with RC defects, cardiomyopathy is more common than previously thought and tends to follow a different and more severe clinical course. Although with a greater frequency than previously reported, mitochondrial DNA mutations were found in a minority of patients, emphasizing that most mitochondrial disorders of childhood follow a Mendelian pattern of inheritance.

Cancer predisposition caused by elevated mitotic recombination in Bloom mice.

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.

Transcriptional Profiling of Aging in Human Muscle Reveals a Common Aging Signature

We analyzed expression of 81 normal muscle samples from humans of varying ages, and have identified a molecular profile for aging consisting of 250 age-regulated genes. This molecular profile correlates not only with chronological age but also with a measure of physiological age. We compared the transcriptional profile of muscle aging to previous transcriptional profiles of aging in the kidney and the brain, and found a common signature for aging in these diverse human tissues. The common aging signature consists of six genetic pathways; four pathways increase expression with age (genes in the extracellular matrix, genes involved in cell growth, genes encoding factors involved in complement activation, and genes encoding components of the cytosolic ribosome), while two pathways decrease expression with age (genes involved in chloride transport and genes encoding subunits of the mitochondrial electron transport chain). We also compared transcriptional profiles of aging in humans to those of the mouse and fly, and found that the electron transport chain pathway decreases expression with age in all three organisms, suggesting that this may be a public marker for aging across species.

CHD5 Is a Tumor Suppressor at Human 1p36

Cancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.