Oliver Oehlke

Transforming Growth Factor β Cooperates with Persephin for Dopaminergic Phenotype Induction

The aim of the present study was to investigate the putative cooperative effects of transforming growth factor β (TGF‐β) and glial cell line‐derived neurotrophic factor (GDNF) family ligands in the differentiation of midbrain progenitors toward a dopaminergic phenotype. Therefore, a mouse midbrain embryonic day (E) 12 neurospheres culture was used as an experimental model. We show that neurturin and persephin (PSPN), but not GDNF, are capable of transient induction of dopaminergic neurons in vitro. This process, however, requires the presence of endogenous TGF‐β. In contrast, after 8 days in vitro GDNF rescued the TGF‐β neutralization‐dependent loss of the TH‐positive cells. In vivo, at E14.5, no apparent phenotype concerning dopaminergic neurons was observed in Tgf‐β2−/−/gdnf−/− double mutant mice. In vitro, combined TGF‐β/PSPN treatment achieved a yield of approximately 20% TH‐positive cells that were less vulnerable against 1‐methyl‐4‐phenyl pyridinium ion toxicity. The underlying TGF‐β/PSPN differentiation signaling is receptor‐mediated, involving p38 mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase pathways. These results indicate that phenotype induction and survival of fully differentiated neurons are accomplished through distinct pathways and individual factor requirement. TGF‐β is required for the induction of dopaminergic neurons, whereas GDNF is required for regulating and/or maintaining a differentiated neuronal phenotype. Moreover, this study suggests that the combination of TGF‐β with PSPN is a potent inductive cocktail for the generation of dopaminergic neurons that should be considered in tissue engineering and cell replacement therapies for Parkinsons disease.

Rab11b and its effector Rip11 regulate the acidosis-induced traffic of V-ATPase in salivary ducts.

Redistribution of acid‐base transporters is a crucial regulatory mechanism for many types of cells to cope with extracellular pH changes. In epithelial cells, however, translocation of acid‐base transporters ultimately leads to changes in vectorial transport of H+ and HCO  3− . We have previously shown that the bicarbonate‐secreting epithelium of salivary ducts responds to changes of systemic acid‐base balance by adaptive redistribution of H+ and HCO  3− transporters, thereby influencing the ionic composition and buffering capacity of saliva. However, the specific proteins involved in regulated vesicular traffic of acid‐base transporters are largely unknown. In the present study we have investigated the impact of Rab11 family members on the acidosis‐induced trafficking of the vacuolar‐type H+‐ATPase (V‐ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. The results show that Rab11b is expressed in salivary ducts and exhibits a significantly higher co‐localization with V‐ATPase than Rab11a and Rab25. We also show that Rab11 but not Rab25 interacts with the ε subunit of V‐ATPase. Extracellular acidosis up‐regulates Rab11b expression and protein abundance in HSG cells and causes translocation of the V‐ATPase from intracellular pools toward the plasma membrane. Loss‐of‐function experiments using specific siRNA either against Rab11b or against its effector Rip11 prevent acidosis‐induced V‐ATPase translocation. These data introduce Rab11b as a crucial regulator and Rip11 as mediator of acidosis‐induced V‐ATPase traffic in duct cells of submandibular gland. J. Cell. Physiol. 226: 638–651, 2011.

Stereotactic fractionated radiotherapy of the resection cavity in patients with one to three brain metastases

OBJECTIVES The goal of this study is to evaluate the role of stereotactic fractionated radiotherapy (SFRT) in patients with one to three brain metastases after surgical resection. METHODS AND MATERIALS We performed a retrospective single-institutional study in patients undergoing SFRT of surgical cavity after resection of ≤3 brain metastases. 60 patients with newly diagnosed brain metastases treated with SFRT following resection were included. The total irradiation dose was 30 Gy (5 Gy/d, BED 45 Gy) after complete macroscopical resection and 35 Gy (5 Gy/d, BED 52.5 Gy) in patients with macroscopic residual tumour after surgery. Macroscopic residual tumour was defined as contrast enhancement next to the resection cavity on the postoperative T1-MRI. The gross tumour volume (GTV) encompassing the residual tumour was delineated on the T1-MRI, the clinical target volume (CTV) encompassed the surgical cavity plus 1mm and the planning target volume (PTV) the CTV plus 2mm. RESULTS Eight of 60 patients had no imaging follow-up due to morbidity/mortality. Two of 52 (3.8%) patients experienced local failures only, 25 of 52 (48.1%) patients experienced distant intracranial failures only and 4 (7.7%) patients experienced both local and distant intracranial failures. In summary, there were 6 (11.5%) local failures and 29 (55.8%) distant failures. Age was significant for local control in the Cox regression test (p=0.046). Thirty-seven of 60 (61.7%) patients died during follow-up. Median follow-up was 8 months. Median overall survival was 15 months. Cox regression for survival was significant for KPS score ≤70% and size of PTV. No severe side effects were seen. Patients undergoing whole brain radiation therapy (WBRT) as salvage therapy in case of progression had no severe side effects either. CONCLUSION In the light of encouraging local control rates, SFRT could be an alternative to WBRT after surgical resection of ≤3 brain metastases. Due to the high rate of distant intracranial failure regular follow-up with MRI is mandatory.

Acidosis-induced V-ATPase trafficking in salivary ducts is initiated by cAMP/PKA/CREB pathway via regulation of Rab11b expression

Changes in systemic acid-base homeostasis cause a series of organ-specific cellular responses, among them changes of acid-base transporter activities, and recruitment or retrieval of these transporters from intracellular pools to the plasma membrane and vice versa. The purpose of this study was to investigate the impact of protein phosphorylation in the acidosis-induced translocation of vacuolar-type H(+)-ATPase (V-ATPase) in salivary ducts and to identify molecular targets. Therefore, the human submandibular gland cell line HSG was exposed to acidosis and V-ATPase trafficking was investigated in the presence or absence of inhibitors and activators of sAC/PKA and Src/ERK signaling pathways. Putative target genes have been identified by RT-PCR and immunoblotting, and validated by loss-of-function experiments. Acidosis caused activation of cAMP/PKA and Src signaling and inhibition of either pathway significantly impaired acidosis-induced V-ATPase redistribution and incorporation into the plasma membrane. Activation of ERK1/2 was Src-independent, whereas activation of PKA caused phosphorylation of cAMP response element-binding (CREB) and activation to regulate Rab11b transcription. Loss-of-function of CREB down-regulated Rab11b transcript and protein and significantly impaired acidosis-induced V-ATPase translocation in HSG cells. These data demonstrate that the cAMP/PKA/CREB signaling pathway initiates acidosis-induced V-ATPase trafficking in salivary ducts via regulation of Rab11b expression and provide first evidence for a molecular mechanism underlying cAMP/PKA-dependent transporter trafficking that could account for accumulation and activity of transporters in other cellular systems as well.

Sim1 Is a Novel Regulator in the Differentiation of Mouse Dorsal Raphe Serotonergic Neurons

Background Mesencephalic dopaminergic neurons (mDA) and serotonergic (5-HT) neurons are clinically important ventral neuronal populations. Degeneration of mDA is associated with Parkinsons disease; defects in the serotonergic system are related to depression, obsessive-compulsive disorder, and schizophrenia. Although these neuronal subpopulations reveal positional and developmental relationships, the developmental cascades that govern specification and differentiation of mDA or 5-HT neurons reveal missing determinants and are not yet understood. Methodology We investigated the impact of the transcription factor Sim1 in the differentiation of mDA and rostral 5-HT neurons in vivo using Sim1-/- mouse embryos and newborn pups, and in vitro by gain- and loss-of-function approaches. Principal Findings We show a selective significant reduction in the number of dorsal raphe nucleus (DRN) 5-HT neurons in Sim1-/- newborn mice. In contrast, 5-HT neurons of other raphe nuclei as well as dopaminergic neurons were not affected. Analysis of the underlying molecular mechanism revealed that tryptophan hydroxylase 2 (Tph2) and the transcription factor Pet1 are regulated by Sim1. Moreover, the transcription factor Lhx8 and the modulator of 5-HT1A-mediated neurotransmitter release, Rgs4, exhibit significant higher expression in ventral hindbrain, compared to midbrain and are target genes of Sim1. Conclusions The results demonstrate for the first time a selective transcription factor dependence of the 5-HT cell groups, and introduce Sim1 as a regulator of DRN specification acting upstream of Pet1 and Tph2. Moreover, Sim1 may act to modulate serotonin release via regulating RGS4. Our study underscores that subpopulations of a common neurotransmitter phenotype use distinct combinations of transcription factors to control the expression of shared properties.

Variants of the electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse hippocampal neurons are regulated by extracellular pH changes: Evidence for a Rab8a-dependent mechanism

Changes in extracellular pH are common events in both pathological conditions and during normal brain function. In organs other than the brain, cells may respond to pH changes by trafficking of acid-base transporters. However, regulation of neuronal acid-base transporters during pH shifts is not understood. The aim of this study was to investigate regulatory mechanisms of the variants of the electrogenic sodium/bicarbonate cotransporter 1, NBCe1-A and NBCe1-B/C, in neurons following changes of extracellular pH. Therefore, primary mouse hippocampal neurons were exposed to extracellular acidosis or alkalosis. We show that acid-base changes regulated trafficking and membrane expression of neuronal NBCe1 but the underlying molecular cues were distinct for individual NBCe1 variants. Following extracellular acidosis NBCe1-A was recruited from intracellular pools to the plasma membrane, followed by increased membrane expression, whereas NBCe1-B/C was retrieved from the membrane. Extracellular alkalosis had no impact on NBCe1-A, but caused translocation of NBCe1-B/C toward the dendrites. We also show that acidosis-induced NBCe1-A, but not NBCe1-B/C, trafficking is mediated by Rab8a. Rab8a is expressed in hippocampal neurons, co-localizes, and interacts with NBCe1-A. Loss-of-function of Rab8a using specific siRNA prevented acidosis-induced redistribution of NBCe1-A. These data propose opposite recruitment pattern for NBCe1 variants in neurons following extracellular acid-base changes, implicating distinct physiological functions of individual NBCe1 variants, and introduce Rab8a as a novel molecular determinant and crucial mediator of acidosis-induced NBCe1 trafficking in neurons.

PET/MRI and brain tumors: focus on radiation oncology treatment planning

In brain tumors, imaging by magnetic resonance imaging (MRI) can very accurately visualize anatomy and morphology of healthy and malignant tissue, but neither contrast-enhancing areas in T1-weighted sequences, nor hyperintensities in T2/FLAIR sequences are specific for tumor tissue, especially when considering the manifold alterations resulting from previous treatment. Imaging the biology of tumor tissue by positron emission tomography (PET), therefore, is a highly interesting approach to improve the detection of macroscopic tumor which is the prerequisite for high-precision radiotherapy treatment planning. This review will focus on the benefits of amino acid tracers (l-[methyl-11C]methionine (MET) and O-(2-[18F]fluoroethyl)-l-tyrosine (FET)) in neurooncology and their implementation in radiation oncology. Furthermore, a brief overview of the current impact of 2-deoxy-2-(18F)fluoro-d-glucose (FDG), nucleic acid analogs, hypoxia tracers, and Somatostatin receptor (SSTR) analogs on radiotherapy planning in brain tumors is provided. Among advances in multiparametric MRI, Diffusion-weighted imaging (DWI) has attracted particular attention since it can predict prognosis, as well as indicate response to treatment and has already been introduced into target volume definition for radiotherapy of various cancers (e.g., prostate and rectal cancer). Additionally, advances in MR spectroscopy (MRS) are mentioned. Finally, these findings will be discussed concerning their influence on current aspects of integrated PET/MR hybrid imaging.

Multicenter analysis of stereotactic radiotherapy of the resection cavity in patients with brain metastases

Brain metastases show a recurrence rate of about 50% after surgical resection. Adjuvant radiotherapy can prevent progression; however, whole‐brain radiotherapy (WBRT) can be associated with significant side effects. Local hypofractionated stereotactic radiotherapy (HFSRT) is a good alternative to provide local control with minimal toxicity. In this multicenter analysis, we evaluated the treatment outcome of local HFSRT after resection brain metastases in 181 patients. Patients characteristics, treatment data as well as follow‐up data were collected and analyzed with special focus on local control, locoregional control and survival. After a median follow‐up of 12.6 months (range 0.3–80.2 months), the crude rate for local control was 80.5%; 1‐ and 2‐year local recurrence‐free survival rates were 75% and 70% (median not reached). Resection cavity size was a significant predictor for local recurrence (P = 0.033). The median overall survival was 16.0 months. Both graded prognostic assessment score and recursive partitioning analysis were accurate predictors of survival. HFSRT leads to excellent local control and has a high potential to consolidate results after surgery; acute and late toxicity is low. Distant intracerebral metastases occur frequently during follow‐up, and therefore, a close patient monitoring needs to be warranted if whole‐brain radiotherapy is omitted.

Clinical outcome after high-precision radiotherapy for skull base meningiomas: Pooled data from three large German centers for radiation oncology

PURPOSE To evaluate outcome in patients with base of skull meningiomas treated with modern high precision radiation therapy (RT) techniques. PATIENTS AND METHODS 927 patients from three centers were treated with either radiosurgery or fractionated high-precision RT for meningiomas. Treatment planning was based on CT and MRI following institutional guidelines. For radiosurgery, a median dose of 13 Gy was applied, for fractionated treatments, a median dose of 54 Gy in 1.8 Gy single fractions was prescribed. Follow-up included a clinical examination as well as contrast-enhanced imaging. All patients were followed up prospectively after radiotherapy in the three departments within a strict follow-up regimen. The median follow-up time was 81 months (range 1-348 months). RESULTS Median local control was 79 months (range 1-348 months). Local control (LC) was 98% at 1 year, 94% at 3 years, 92% at 5 years and 86% at 10 years. There was no difference between radiosurgery and fractionated RT. We analyzed the influence of higher doses on LC and could show that dose did not impact LC. Moreover, there was no difference between 54 Gy and 57.6 Gy in the fractionated group. Side effects were below 5% in both groups without any severe treatment-related complications. DISCUSSION Based on the pooled data analysis this manuscript provides a large series of meningiomas of the skull base treated with modern high precision RT demonstrating excellent local control and low rates of side effects. Such data support the recommendation of RT for skull base meningiomas in the interdisciplinary tumor board discussions. The strong role of RT must influence treatment recommendations keeping in mind the individual risk-benefit profile of treatment alternatives.

Diffusion-weighted MRI and ADC versus FET-PET and GdT1w-MRI for gross tumor volume (GTV) delineation in re-irradiation of recurrent glioblastoma

BACKGROUND AND PURPOSE GTV definition for re-irradiation treatment planning in recurrent glioblastoma (rGBM) is usually based on contrast-enhanced MRI (GdT1w-MRI) and, for an increased specificity, on amino acid PET. Diffusion-weighted (DWI) MRI and ADC maps can reveal regions of high cellularity as surrogate for active tumor. The objective of this study was to compare the localization and quality of diffusion restriction foci (GTV-ADClow) with FET-PET (GTV-PET) and GdT1w-MRI (GTV-GdT1w-MRI). MATERIAL AND METHODS We prospectively evaluated 41 patients, who received a fractionated stereotactic re-irradiation for rGBM. GTV-PET was generated automatically (tumor-to-background ratio 1.7-1.8) and manually customized. GTV-ADClow was manually defined based on DWI data (3D diffusion gradients, b = 0, 1000 s/mm2) and parametric ADC maps. The localization of recurrence was correlated with initial GdT1w-MRI and PET data. RESULTS In 30/41 patients, DWI-MRI showed areas with restricted diffusion (mean ADC-value 0.74 ± 0.22 mm2/s). 66% of GTVs-ADClow were located outside the GdT1w-MRI volume and 76% outside increased FET uptake regions. Furthermore, GTVs-ADClow were only partially included in the high dose volume and received in mean 82% of the reference dose. An adjusted volume including GdT1w-MRI, PET-positive and restricted diffusion areas would imply a GTV increase of 48%. GTV-PET and GdT1w-MRI correlated better with the localization of re-recurrence in comparison to GTV-ADClow. CONCLUSION Unexpectedly, GTV-ADClow overlapped only partially with FET-PET and GdT1w-MRI in rGBM. Moreover, GTV-ADClow correlated poorly with later rGBM-recurrences. Seeing as a restricted diffusion is known to correlate with hypercellularity, this imaging discrepancy could only be further explained in histopathological studies.