Regulation of the MAD1 promoter by G-CSF

MAD family proteins are transcriptional repressors that antagonize the functions of MYC oncoproteins. In particular, MAD1 has been demonstrated to interfere with MYC-induced proliferation, transformation and apoptosis. The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence. We observed that MAD1 is directly activated by G-CSF in promyelocytic cell lines. To investigate the transcriptional regulation of the human MAD1 gene, we have cloned and characterized its promoter. A region of high homology between the MAD1 orthologs of human, mouse and rat contains the core promoter, marked by open chromatin, high GC content and the lack of a TATA box. Using deletion constructs we identified two CCAAT-boxes occupied by C/EBPα and β in the homology region that mediate responsiveness to G-CSF receptor signaling. The necessary signals include the activation of STAT3 and the RAS/RAF/ERK pathway. STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPβ. In summary, our studies provide a first analysis of the MAD1 promoter and suggest STAT3 functions as a C/EBPβ cofactor in the regulation of the MAD1 gene. Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1

Glutamatergic and Resting-State Functional Connectivity Correlates of Severity in Major Depression – The Role of Pregenual Anterior Cingulate Cortex and Anterior Insula

Glutamatergic mechanisms and resting-state functional connectivity alterations have been recently described as factors contributing to major depressive disorder (MDD). Furthermore, the pregenual anterior cingulate cortex (pgACC) seems to play an important role for major depressive symptoms such as anhedonia and impaired emotion processing. We investigated 22 MDD patients and 22 healthy subjects using a combined magnetic resonance spectroscopy (MRS) and resting-state functional magnetic resonance imaging (fMRI) approach. Severity of depression was rated using the 21-item Hamilton depression scale (HAMD) and patients were divided into severely and mildly depressed subgroups according to HAMD scores. Because of their hypothesized role in depression we investigated the functional connectivity between pgACC and left anterior insular cortex (AI). The sum of Glutamate and Glutamine (Glx) in the pgACC, but not in left AI, predicted the resting-state functional connectivity between the two regions exclusively in depressed patients. Furthermore, functional connectivity between these regions was significantly altered in the subgroup of severely depressed patients (HAMD > 15) compared to healthy subjects and mildly depressed patients. Similarly the Glx ratios, relative to Creatine, in the pgACC were lowest in severely depressed patients. These findings support the involvement of glutamatergic mechanisms in severe MDD which are related to the functional connectivity between pgACC and AI and depression severity

Charakterisierung und Regulation des humanen mad1-Promotors

The transcriptionfactor Mad1 is a potential tumorsuppressor. As a member of the Myc/Max/Madnetwork it antagonizes the transforming ability of the protooncogene c-myc. It was shown, that Mad1 can inhibit the transformation of REFs by c-myc/Ras through blockade of the G1/S-transition. Moreover it was shown, that Mad1 is an inhibitor of apoptosis. The members of the Myc/Max/Madnetwork are transcriptonfactors and regulate gene-expression after binding to specific DNA-sequences and then recruiting chromatin-modifying factors. In principle c-myc is described as an activator of transcription while mad1 is described as an inhibitor of transcription. Mad1 is an essential factor in the development of myeloid cells and loss of Mad1-function results in delayed differentiation of granulocytes and monocytes. Accordingly to this it fits, that mad1 gets upregulated after TPA- or GCSF-stimulation of U937-cells. Therefore it is interesting to know how Mad1 itself is transcriptionally regulated, to get more insight into crucial cellular processes like proliferation and differentiation. What are essential extracellular factors and what different intracellular signaltransduction-pathways are involved? In this study a human DNA-fragment was isolated which has mad1-promoter-function and contains a set of regulatory elements like binding sites for transcriptionfactors. This promoter-fragment is located only a few basepairs upstream of the translation-start-site. Alignement of this human sequence with DNA-sequences of mouse and rat reveales a box with nearly 70% homology and binding sites for transcriptionfactors important for myeloid development and differentiation. The mad1-promoter is very GC-rich and contains no TATA-box. In this study one transcription-start-site of the mad1-gene was identified. It is located at the 3´-end of the homologous promoter region. The influence of chromatin and its modifications on transcription is known. So it is important to investigate the nucleosomal structure of cis-acting DNA-elements like promoters and/or enhancers. In this study nucleosomal mapping of the mad1-promoter in U937-cells was done. It reveales that exactly the previous identified homologous region is nucleosome-free. This fits to other results, which show, that free access to regulatory DNA is a prereqiusite for transcription initiation. Unfortunately no GCSF- or TPA-effect on the nucleosomal structure was observed. The homology-box of the mad1- promoter is nucleosome-free in proliferating and/or differentiating U937-cells. To investigate the influence of different transcriptionfactors on Mad1-expression in detail, the Mad1- promoter was cloned as a reportergene-construct and several deletion-mutants were constructed. In following reportergene-assays c-myb, PU.1 and C/EBP Alpha, Beta and Epsilon were identified as positive regulators of mad1-transcription in RK13-cells in vitro. In the case of c-myb, cotransfection of GCSF/R and stimulation with GCSF results in a twofold increase of luciferase-activity. STAT1 and STAT3 exhibit only low but GCSF-dependent transcriptional potential. The ChIP-method (Chromatin-Immunpräzipitation) is very sensitive and specific in investigating protein/DNA-interactions in vivo. Using ChIP binding of RNA-Pol II and PU.1 to the human mad1- promoter was detected. This confirms some in vitro results. Additionally with the ChIP-method also histone-acetylation at the 3´-side of the homology-box was observed. But for other transcriptionfactors like STATs or C/EBPs no DNA-binding to the mad1-promoter was detected. Also no GCSFdependent recruitment of factors or histone-modification. In conclusion the results obtained in this study prove the essential role of the identified DNA-region in regulation of mad1-transcription and reveales the nucleosomal structure of this mad1-promoter. Also potential transcriptionfactors involved in upregulating mad1-expression were detected. But to completely understand the regulation of mad1-expression further investigation like timedependent and cofactor-dependent ChIP- and Reportergene-assays are needed

Characterization of Extracranial Giant Cell Arteritis with Intracranial Involvement and its Rapidly Progressive Subtype.

OBJECTIVE The objective of this study was to characterize patients with extracranial giant cell arteritis with intracranial involvement. METHODS In a multicenter retrospective study, we included 31 patients with systemic giant cell arteritis (GCA) with intracranial involvement. Clinical characteristics, pattern of arterial involvement, and cytokine profiles were assessed. Patients with GCA without intracranial involvement (n = 17), and with intracranial atherosclerosis (n = 25) served as controls. RESULTS Erythrocyte sedimentation rate (ESR) was elevated in 18 patients (69.2%) with and in 16 patients (100%) without intracranial involvement (p = 0.02). Headache was complained by 15 patients (50.0%) with and 13 patients (76.5%) without intracranial involvement (p = 0.03). Posterior circulation arteries were affected in 26 patients (83.9%), anterior circulation arteries in 17 patients (54.8%), and both territories in 12 patients (38.7%). Patients with GCA had vertebral artery stenosis proximal and, in contrast, patients with atherosclerosis distal to the origin of posterior inferior cerebellar artery (PICA). Among patients with GCA with intracranial involvement, 11 patients (37.9%) had a rapid progressive disease course characterized by short-term recurrent ischemic events. The median modified Rankin Scale (mRS) at follow-up in these patients was 4 (interquartile range [IQR] = 2.0-6.0) and 4 patients (36.4%) died. Vessel wall expression of IL-6 and IL-17 was significantly increased in patients with rapid progressive course. INTERPRETATION Typical characteristics of GCA, headache, and an elevated ESR, are frequently absent in patients with intracranial involvement. However, differentiation of intracranial GCA from atherosclerosis can be facilitated by the typical pattern of vertebral artery stenosis. About one-third of patients with intracranial GCA had a rapid progressive course with poor outcome. IL-17 and IL-6 may represent potential future treatment targets. ANN NEUROL 2021;90:118-129

Disease severity is correlated to tract specific changes of fractional anisotropy in MD and CM thalamus--a DTI study in major depressive disorder

BACKGROUND: Depression is commonly conceptualized as corticolimbic dysregulation. Due to insufficient studies in normal aged populations especially subcortical sources of disconnection are unclear in contrast to potentially general parietal white matter (WM) deficits. This may be due to important influences of variable patient characteristics, most importantly episode severity. Especially thalamic disconnections have been functionally revealed, however, their structural correlates have not been distinctly investigated for its highly diverse subnuclei. METHODS: We compared 20 major depressive disorder (MDD) patients with mixed Hamilton depression rating scale (HAMD) severity to matched controls in fractional anisotropy (FA) derived from diffusion tensor imaging (DTI). Robust acquisition of 4 repetitions restricted to twelve directions, also to match the same parameters used by Eckert et al. (2011) who described a preferential architecture of centromedian (CM) and mediodorsal (MD) thalamic connections. Second to whole brain analysis, we tested for group differences within the preferred structural network of these two nuclei using a tract of interest (TOI) approach. RESULTS: Significant FA deficits in a whole brain analysis were only found in right parietal WM (p<0.05, corrected). Effects of severity were found for increasing thalamic FA. Post hoc analysis revealed this effect to be restricted to CM specific tracts. In contrast, we found MD to dorsolateral prefrontal cortex (DLPFC) tracts to be decreased in FA. Unspecific decreases between MD and CM towards amygdala were paralleled by primary amygdala FA reductions. LIMITATIONS: Specificity of the TOI approach and heterogenous sample. CONCLUSIONS: Robust parietal FA reductions, controlled for age effects were found in MDD. Further we revealed subcortical disease state dependency of FA in thalamic tracts, specific to predescribed preferential connections

Characterization of Extracranial Giant Cell Arteritis with Intracranial Involvement and its Rapidly Progressive Subtype

OBJECTIVE The objective of this study was to characterize patients with extracranial giant cell arteritis with intracranial involvement. METHODS In a multicenter retrospective study, we included 31 patients with systemic giant cell arteritis (GCA) with intracranial involvement. Clinical characteristics, pattern of arterial involvement, and cytokine profiles were assessed. Patients with GCA without intracranial involvement (n = 17), and with intracranial atherosclerosis (n = 25) served as controls. RESULTS Erythrocyte sedimentation rate (ESR) was elevated in 18 patients (69.2%) with and in 16 patients (100%) without intracranial involvement (p = 0.02). Headache was complained by 15 patients (50.0%) with and 13 patients (76.5%) without intracranial involvement (p = 0.03). Posterior circulation arteries were affected in 26 patients (83.9%), anterior circulation arteries in 17 patients (54.8%), and both territories in 12 patients (38.7%). Patients with GCA had vertebral artery stenosis proximal and, in contrast, patients with atherosclerosis distal to the origin of posterior inferior cerebellar artery (PICA). Among patients with GCA with intracranial involvement, 11 patients (37.9%) had a rapid progressive disease course characterized by short-term recurrent ischemic events. The median modified Rankin Scale (mRS) at follow-up in these patients was 4 (interquartile range [IQR] = 2.0-6.0) and 4 patients (36.4%) died. Vessel wall expression of IL-6 and IL-17 was significantly increased in patients with rapid progressive course. INTERPRETATION Typical characteristics of GCA, headache, and an elevated ESR, are frequently absent in patients with intracranial involvement. However, differentiation of intracranial GCA from atherosclerosis can be facilitated by the typical pattern of vertebral artery stenosis. About one-third of patients with intracranial GCA had a rapid progressive course with poor outcome. IL-17 and IL-6 may represent potential future treatment targets. ANN NEUROL 2021;90:118-129