Claudia A. Doege

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

A SNARE required for retrograde transport to the endoplasmic reticulum.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1). Slt1 is an essential protein localized in the endoplasmic reticulum (ER). It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1. Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER. We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER. Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin.

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

Perfusion and diffusion magnetic resonance imaging in human cerebral venous thrombosis

Background Diagnosis of cerebral venous thrombosis (CVT) is usually achieved by digital subtraction angiography or magnetic resonance angiography, while structural brain tissue damage can be assessed by computed tomography or magnetic resonance imaging (MRI). Using perfusion and diffusion weighted imaging (PWI, DWI) we aimed in this study to identify pathophysiological patterns corresponding to only functional and hence reversible tissue involvement. Methods PWI, DWI, and conventional MRI were performed in six CVT patients acutely and after 16–26 days when their clinical condition had improved. All patients were treated with partial thromboplastin time-effective intravenous heparin. After intravenous administration of a paramagnetic contrast agent, bolus track PWI allows pixel based determination of mean transit time (MTT) and cerebral blood volume (CBV). DWI was performed with two different b values (0, 1000 s/mm2) for calculation of apparent diffusion coefficient (ADC) maps. Results In five of six cases increased MTT values were observed initially, whereas the CBV was normal, indicating a reduction of cerebral blood flow. ADC values were normal. On follow up after clinical recovery MTT prolongations had resolved. Areas with prolonged MTT did not evolve into structural lesions. Conclusion In patients with CVT, prolongations of MTT in the absence of changes in CBV and ADC seem to indicate reversible involvement of brain tissue, a situation corresponding to the ischaemic penumbra.

Progressive change in primary progressive multiple sclerosis normal-appearing white matter: a serial diffusion magnetic resonance imaging study

In spite of marked disability, patients with primary progressive multiple sclerosis (PPMS) display smaller lesion volumes on conventional magnetic resonance imaging (MRI) compared with other forms of multiple sclerosis (MS). Hence, damage to the normal-appear ing brain tissue (NA BT) may play an important role in explaining the patho genesis of disability in PPMS. Diffusion-weighted MRI (DW-MRI) probes water diffusion in vivo that can be altered by patho logic changes. Using DW-MRI we investigated diffusion in the NA BT of 15 patients with PPMS over one year. The average apparent diffusion coefficient (A DC av) was measured in 10 regions of interest located in the normal- appearing thalamus and the normal-appearing white matter (NAWM). Six healthy subjects served as a reference. In contrast to healthy subjects, patients with PPMS showed an increment within 12 months of the A DC av in NAWM which was associated with an increase of the T2- and T1-lesion volumes. The A DC av in frontal NAWM was associated with disability as measured by the MS Functio nal C omposite Measure. Serial DW-MRI depicts progressive changes in the NAWM of patients with PPMS. O ur preliminary findings suggest that the processes causing structural damage in NAWM and lesions in patients with PPMS are partially linked and that changes of water diffusion in NAWM depicted by DW-MRI are clinically relevant.

Cerebral oxygenation changes during motor and somatosensory stimulation in humans as measured by near-infrared spectroscopy

Functional cortical activation is coupled to changes of cerebral hemodynamics and metabolism. These changes have been used in positron emission tomography and recently developed functional magnetic resonance techniques to localize task-related cortical activation. Near-infrared spectroscopy has the potential to monitor changes of intracerebral blood oxygenation during functional activation (Villringer 1993, Hoshi 1993). We proceeded from a rather global, cognitive stimulus to experimental paradigms which entail a more localized cerebral activation and which have been examined by other functional techniques. In establishing a simple motor and a vibratory stimulus we sought a means to further examine the issue of coupling of neuronal function and cerbral hemodynamics. In recent experiments we were able to demonstrate that these stimulation models are useful to retrieve additional information about oxygenation dependent signal changes in functional MRI (Kleinschmidt 1994, Obrig 1994). As near-infrared spectroscopy has a good temporal resolution, we were also interested in the time course of local oxygenation changes.

REPROGRAMMING THERAPEUTICS: IPS CELL PROSPECTS FOR NEURODEGENERATIVE DISEASE

The recent description of somatic cell reprogramming to an embryonic stem (ES) cell-like phenotype, termed induced pluripotent stem (iPS) cell technology, presents an exciting potential venue toward cell-based therapeutics and disease models for neurodegenerative disorders. Two recent studies (Dimos et al. and Ebert et al.) describe the initial characterization of neurodegenerative disease patient-derived iPS cell cultures as proof of concept for the utility of this technology.

Optimized mouse ES cell culture system by suspension growth in a fully defined medium.

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.

MRI of small human stroke shows reversible diffusion changes in subcortical gray matter.

Six patients who had suffered small cerebral ischemia affecting subcortical gray matter were examined with diffusion weighted imaging (DWI) and T2-weighted imaging within the first 8 h, during the next 2 days and after 5–16 days. Areas of apparent diffusion coefficient (ADC) decreases were observed acutely and reached the maximum size during the subsequent 2 days. Noticeably, in all subjects, a large portion of the ADC lesion was reversible as judged from the lesion size on final T2 image. The regular reversibility of the ADC decreases in this setting may indicate a hitherto not understood pathophysiological process occurring in small ischemic stroke.

Dementia in a Dish

Neurodegenerative disorders of aging represent a growing public health concern. In the United States alone, there are now >5 million patients with Alzheimers disease (AD), the most common form of dementia. No therapeutic approaches are available that alter the relentless course of AD or other dementias of aging. A major hurdle to the development of effective therapeutics has been the lack of predictive model systems in which to develop and validate candidate therapies. Animal model studies based on the analysis of transgenic mice that overexpress rare familial AD-associated mutant genes have been informative about mechanisms of familial disease, but they have not proven predictive for drug development. New approaches to disease modeling are of particular interest. Methods such as epigenetic reprogramming of patient skin fibroblasts to human induced pluripotent stem cells, which can be differentiated into patient-derived neuron subtypes, have generated significant excitement because of their potential to model more accurately aspects of human neurodegeneration. Studies focused on the generation of human neuron models of AD and frontotemporal dementia have pointed to pathologic pathways and potential therapeutic venues. This article discusses the promise and potential pitfalls of modeling of dementia disorders based on somatic cell reprogramming.