Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway.

Autophagy allows the degradation of cytosolic endogenous and exogenous material in the lysosome. Substrates are engulfed by double-membrane vesicles, coined autophagosomes, which subsequently fuse with lysosomes. Depending on the involvement of specific receptor proteins, autophagy occurs in a selective or nonselective manner. While this process is well understood at the level of bulky cargo such as mitochondria and bacteria, we know very little about individual proteins and protein complexes that are engulfed and degraded by autophagy. In contrast to the critical role of autophagy in balancing proteostasis, our current knowledge of the autophagic degradome is very limited. Here, we combined proximity labeling with quantitative proteomics to systematically map the protein inventory of autophagosomes. Using this strategy, we uncovered a basal, housekeeping mitophagy pathway that involves piecemeal degradation of mitochondrial proteins in a LC3C- and p62-dependent manner and contributes to mitochondrial homeostasis maintenance when cells rely on oxidative phosphorylation.

Digital PCR cluster predictor: a universal R-package and shiny app for the automated analysis of multiplex digital PCR data.

Digital polymerase chain reaction (dPCR) is an emerging technology that enables accurate and sensitive quantification of nucleic acids. Most available dPCR systems have two channel optics, with ad hoc software limited to the analysis of single and duplex assays. Although multiplexing strategies were developed, variable assay designs, dPCR systems, and the analysis of low DNA input data restricted the ability for a universal automated clustering approach. To overcome these issues, we developed dPCR Cluster Predictor (dPCP), an R package and a Shiny app for automated analysis of up to 4-plex dPCR data. dPCP can analyse and visualize data generated by multiple dPCR systems carrying out accurate and fast clustering not influenced by the amount and integrity of input of nucleic acids. With the companion Shiny app, the functionalities of dPCP can be accessed through a web browser.

Computational Assessment of Spectral Heterogeneity within Fresh Glioblastoma Tissue Using Raman Spectroscopy and Machine Learning Algorithms.

Understanding and classifying inherent tumor heterogeneity is a multimodal approach, which can be undertaken at the genetic, biochemical, or morphological level, among others. Optical spectral methods such as Raman spectroscopy aim at rapid and non-destructive tissue analysis, where each spectrum generated reflects the individual molecular composition of an examined spot within a (heterogenous) tissue sample. Using a combination of supervised and unsupervised machine learning methods as well as a solid database of Raman spectra of native glioblastoma samples, we succeed not only in distinguishing explicit tumor areas-vital tumor tissue and necrotic tumor tissue can correctly be predicted with an accuracy of 76%-but also in determining and classifying different spectral entities within the histomorphologically distinct class of vital tumor tissue. Measurements of non-pathological, autoptic brain tissue hereby serve as a healthy control since their respective spectroscopic properties form an individual and reproducible cluster within the spectral heterogeneity of a vital tumor sample. The demonstrated decipherment of a spectral glioblastoma heterogeneity will be valuable, especially in the field of spectroscopically guided surgery to delineate tumor margins and to assist resection control.

Impact of Formalin- and Cryofixation on Raman Spectra of Human Tissues and Strategies for Tumor Bank Inclusion.

Reliable training of Raman spectra-based tumor classifiers relies on a substantial sample pool. This study explores the impact of cryofixation (CF) and formalin fixation (FF) on Raman spectra using samples from surgery sites and a tumor bank. A robotic Raman spectrometer scans samples prior to the neuropathological analysis. CF samples showed no significant spectral deviations, appearance, or disappearance of peaks, but an intensity reduction during freezing and subsequent recovery during the thawing process. In contrast, FF induces sustained spectral alterations depending on molecular composition, albeit with good signal-to-noise ratio preservation. These observations are also reflected in the varying dual-class classifier performance, initially trained on native, unfixed samples: The Matthews correlation coefficient is 81.0% for CF and 58.6% for FF meningioma and dura mater. Training on spectral differences between original FF and pure formalin spectra substantially improves FF samples' classifier performance (74.2%). CF is suitable for training global multiclass classifiers due to its consistent spectrum shape despite intensity reduction. FF introduces changes in peak relationships while preserving the signal-to-noise ratio, making it more suitable for dual-class classification, such as distinguishing between healthy and malignant tissues. Pure formalin spectrum subtraction represents a possible method for mathematical elimination of the FF influence. These findings enable retrospective analysis of processed samples, enhancing pathological work and expanding machine learning techniques.

MR imaging profile and histopathological characteristics of tumour vasculature, cell density and proliferation rate define two distinct growth patterns of human brain metastases from lung cancer.

PURPOSE: Non-invasive prediction of the tumour of origin giving rise to brain metastases (BMs) using MRI measurements obtained in radiological routine and elucidating the biological basis by matched histopathological analysis. METHODS: Preoperative MRI and histological parameters of 95 BM patients (female, 50; mean age 59.6 ± 11.5 years) suffering from different primary tumours were retrospectively analysed. MR features were assessed by region of interest (ROI) measurements of signal intensities on unenhanced T1-, T2-, diffusion-weighted imaging and apparent diffusion coefficient (ADC) normalised to an internal reference ROI. Furthermore, we assessed BM size and oedema as well as cell density, proliferation rate, microvessel density and vessel area as histopathological parameters. RESULTS: Applying recursive partitioning conditional inference trees, only histopathological parameters could stratify the primary tumour entities. We identified two distinct BM growth patterns depending on their proliferative status: Ki67high BMs were larger (p = 0.02), showed less peritumoural oedema (p = 0.02) and showed a trend towards higher cell density (p = 0.05). Furthermore, Ki67high BMs were associated with higher DWI signals (p = 0.03) and reduced ADC values (p = 0.004). Vessel density was strongly reduced in Ki67high BM (p < 0.001). These features differentiated between lung cancer BM entities (p ≤ 0.03 for all features) with SCLCs representing predominantly the Ki67high group, while NSCLCs rather matching with Ki67low features. CONCLUSION: Interpretable and easy to obtain MRI features may not be sufficient to predict directly the primary tumour entity of BM but seem to have the potential to aid differentiating high- and low-proliferative BMs, such as SCLC and NSCLC.

Neurodegeneration and neuroinflammation are linked, but independent of alpha-synuclein inclusions, in a seeding/spreading mouse model of Parkinson's disease.

A key pathological process in Parkinson's disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein (α-syn) is a presynaptic protein that, in PD, forms pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-syn particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used a model of spreading of α-syn induced by striatal injection of α-syn preformed fibrils into the mouse striatum to address this question. We performed quantitative analysis for α-syn inclusions, neurodegeneration, and microgliosis in different brain regions, and generated gene expression profiles of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-syn inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. Using longitudinal gene expression profiling, we observed early gene expression changes, linked to neuroinflammation, that preceded neurodegeneration, indicating an active role of microglia in this process. Altered gene pathways overlapped with those typical of PD. Our observations indicate that α-syn inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that microglia, activated by diffusible, oligomeric α-syn, may play a key role in this process. Our findings uncover new features of α-syn induced pathologies, in particular microgliosis, and point to the necessity for a broader view of the process of α-syn spreading.

Parkin Deficiency Impairs Mitochondrial DNA Dynamics and Propagates Inflammation.

BACKGROUND: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patient-derived neurons. OBJECTIVES: We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. METHODS: We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. RESULTS: Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkin-deficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. CONCLUSIONS: Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Apomorphine Reduces A53T α-Synuclein-Induced Microglial Reactivity Through Activation of NRF2 Signalling Pathway.

The chiral molecule, apomorphine, is currently used for the treatment of Parkinson's disease (PD). As a potent dopamine receptor agonist, this lipophilic compound is especially effective for treating motor fluctuations in advanced PD patients. In addition to its receptor-mediated actions, apomorphine has also antioxidant and free radical scavenger activities. Neuroinflammation, oxidative stress, and microglia reactivity have emerged as central players in PD. Thus, modulating microglia activation in PD may be a valid therapeutic strategy. We previously reported that murine microglia are strongly activated upon exposure to A53T mutant α-synuclein. The present study was designed to investigate whether apomorphine enantiomers could modulate this A53T-induced microglial activation. Taken together, the results provided evidence that apomorphine enantiomers decrease A53T-induced microgliosis, through the activation of the NRF2 signalling pathway, leading to a lower pro-inflammatory state and restoring the phagocytic activity. Suppressing NRF2 recruitment (trigonelline exposure) or silencing specifically Nfe2l2 gene (siRNA treatment) abolished or strongly decreased the anti-inflammatory activity of apomorphine. In conclusion, apomorphine, which is already used in PD patients to mimic dopamine activity, may also be suitable to decrease α-synuclein-induced microglial reactivity.

Introgressive hybridisation between domestic pigs (Sus scrofa domesticus) and endemic Corsican wild boars (S. s. meridionalis): effects of human-mediated interventions.

Owing to the intensified domestication process with artificial trait selection, introgressive hybridisation between domestic and wild species poses a management problem. Traditional free-range livestock husbandry, as practiced in Corsica and Sardinia, is known to facilitate hybridisation between wild boars and domestic pigs (Sus scrofa). Here, we assessed the genetic distinctness and genome-wide domestic pig ancestry levels of the Corsican wild boar subspecies S. s. meridionalis, with reference to its Sardinian conspecifics, employing a genome-wide single nucleotide polymorphism (SNP) assay and mitochondrial control region (mtCR) haplotypes. We also assessed the reliance of morphological criteria and the melanocortin-1 receptor (MC1R) coat colour gene to identify individuals with domestic introgression. While Corsican wild boars showed closest affinity to Sardinian and Italian wild boars compared to other European populations based on principal component analysis, the observation of previously undescribed mtCR haplotypes and high levels of nuclear divergence (Weir's θ > 0.14) highlighted the genetic distinctness of Corsican S. s. meridionalis. Across three complementary analyses of mixed ancestry (i.e., STRUCTURE, PCADMIX, and ELAI), proportions of domestic pig ancestry were estimated at 9.5% in Corsican wild boars, which was significantly higher than in wild boars in Sardinia, where free-range pig keeping was banned in 2012. Comparison of morphologically pure- and hybrid-looking Corsican wild boars suggested a weak correlation between morphological criteria and genome-wide domestic pig ancestry. The study highlights the usefulness of molecular markers to assess the direct impacts of management practices on gene flow between domestic and wild species.

Immune profile and radiological characteristics of progressive multifocal leukoencephalopathy.

BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) constitutes a severe disease with increasing incidence, mostly in the context of immunosuppressive therapies. A detailed understanding of immune response in PML appears critical for the treatment strategy. The aim was a comprehensive immunoprofiling and radiological characterization of magnetic resonance imaging (MRI) defined PML variants. METHODS: All biopsy-confirmed PML patients (n = 15) treated in our department between January 2004 and July 2019 were retrospectively analysed. Data from MRI, histology as well as detailed clinical and outcome data were collected. The MRI-defined variants of classical (cPML) and inflammatory (iPML) PML were discriminated based on the intensity of gadolinium enhancement. In these PML variants, intensity and localization (perivascular vs. parenchymal) of inflammation in MRI and histology as well as the cellular composition by immunohistochemistry were assessed. The size of the demyelinating lesions was correlated with immune cell infiltration. RESULTS: Patients with MRI-defined iPML showed a stronger intensity of inflammation with an increased lymphocyte infiltration on histological level. Also, iPML was characterized by a predominantly perivascular inflammation. However, cPML patients also demonstrated certain inflammatory tissue alterations. Infiltration of CD163-positive microglia and macrophage (M/M) subtypes correlated with PML lesion size. CONCLUSIONS: The non-invasive MRI-based discrimination of PML variants allows for an estimation of inflammatory tissue alterations, although exhibiting limitations in MRI-defined cPML. The association of a distinct phagocytic M/M subtype with the extent of demyelination might reflect disease progression.

Effects of Low Intensity Pulsed Ultrasound Stimulation on the Temporal Dynamics of Irradiated Bone Tissue Healing: A Histomorphometric Study in Rabbits.

The present study evaluated the influence of Low-Intensity Pulsed Ultrasound (LIPUS) on the regeneration processes of non-critical-size bone defects in irradiated and non-irradiated rabbit tibias. Bone defects were surgically created on both tibiae of six rabbits. The control group had no additional treatment. In one intervention group, one tibia was irradiated with 15 Gy in a single dose. A second group was treated with LIPUS, and a third with a combination of both treatments. The control samples showed 83.10% ± 17.79% of bone repair after 9 weeks, while the irradiated bone had regenerated significantly less during the same period (66.42% ± 29.36%). The LIPUS treatment on irradiated bones performed a 79.21% ± 21.07% bone fill and could not significantly improve the response compared to the non-treated irradiated specimens. However, LIPUS treatment on non-irradiated bone showed bone formations beyond the size defect (115.91% ± 33.69%), which was a highly significant increase when compared to the control group or any irradiated group. The application of ultrasound to healthy bone produced highly significant and enhanced bone formations with 36.70% more regenerated bone when compared to the same application on irradiated bone. LIPUS vibration stimuli may be considered as a promising complementary treatment approach in non-irradiated bone regeneration procedures to shorten the treatment and enhance bone healing. In irradiated bones, the effect of ultrasound application is less clear, and further studies are needed to refine the dynamics of the present results.

TGF-ß activates pericytes via induction of the epithelial-to-mesenchymal transition protein SLUG in glioblastoma.

AIMS: In primary central nervous system tumours, epithelial-to-mesenchymal transition (EMT) gene expression is associated with increased malignancy. However, it has also been shown that EMT factors in gliomas are almost exclusively expressed by glioma vessel-associated pericytes (GA-Peris). In this study, we aimed to identify the mechanism of EMT in GA-Peris and its impact on angiogenic processes. METHODS: In glioma patients, vascular density and the expression of the pericytic markers platelet derived growth factor receptor (PDGFR)-ß and smooth muscle actin (αSMA) were examined in relation to the expression of the EMT transcription factor SLUG and were correlated with survival of patients with glioblastoma (GBM). Functional mechanisms of SLUG regulation and the effects on primary human brain vascular pericytes (HBVP) were studied in vitro by measuring proliferation, cell motility and growth characteristics. RESULTS: The number of PDGFR-ß- and αSMA-positive pericytes did not change with increased malignancy nor showed an association with the survival of GBM patients. However, SLUG-expressing pericytes displayed considerable morphological changes in GBM-associated vessels, and TGF-ß induced SLUG upregulation led to enhanced proliferation, motility and altered growth patterns in HBVP. Downregulation of SLUG or addition of a TGF-ß antagonising antibody abolished these effects. CONCLUSIONS: We provide evidence that in GA-Peris, elevated SLUG expression is mediated by TGF-ß, a cytokine secreted by most glioma cells, indicating that the latter actively modulate neovascularisation not only by modulating endothelial cells, but also by influencing pericytes. This process might be responsible for the formation of an unstructured tumour vasculature as well as for the breakdown of the blood-brain barrier in GBM.

Linking epigenetic signature and metabolic phenotype in IDH mutant and IDH wildtype diffuse glioma.

AIMS: Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. METHODS: We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n = 57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n = 22) for mitochondrial copy number and validated these results in a large glioma cohort (n = 293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n = 29). RESULTS: DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. CONCLUSION: A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.

HIF-1α is involved in blood-brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

Delay in diagnosing patients with right-sided glioblastoma induced by hemispheric-specific clinical presentation.

PURPOSE: Cognitive functions are differentially represented in brain hemispheres. Aphasia is an "easy to recognize" symptom of diseases affecting the left side. In contrast, lesions in the right hemisphere cause subtle neuropsychological deficits such as neglect and anosognosia. We evaluated whether right-sided malignant brain tumors are on average larger at the time of first diagnosis as compared to left-sided tumors, and extrapolated the delay in diagnosing right-sided tumors compared to the left side. METHODS: All first-ever diagnosed glioblastoma (GBM) patients between 2005 and 2012 were identified using our hospital-based prospective research registry. Baseline data, information on initial clinical presentation and imaging findings (including tumor volume) were collected. Extrapolation of time since tumor initiation was based on an established gompertzian growth model. RESULTS: We included 173 patients. Mean age of the study population was 58 ± 13 years. Tumors located in the right hemisphere (n = 96) were larger as compared to tumors located in the left hemisphere (n = 77) (median 36.4 mL [interquartile range 13.0-56.0; minimum 0.2, maximum 140.0] vs. 17.2 mL [7.7-45.1 mL; 0.4, 105.2]; p = 0.011). Right-sided tumors grew longer than left-sided tumors (378 ± 95 days vs. 341 ± 74 days; p = 0.006). Initial neuropsychological symptoms differed depending on the affected hemisphere. CONCLUSION: Right-hemispheric symptoms appear to be less clinically conspicuous resulting in a delayed diagnosis of GBM, which might be improved by raising awareness for the corresponding neuropsychological deficits. Whether our findings have prognostic implications needs to be evaluated in future studies.

Cholinergic innervation and ganglion cell distribution in Hirschsprung's disease.

BACKGROUND: The diagnostic gold standard of Hirschsprung's disease (HD) is based on the histopathological assessment of colorectal biopsies. Although data on cholinergic innervation and ganglion cell (GC) distribution exist, only few studies have examined these two key features together. We assessed the pattern of cholinergic innervation and the amount of GCs in colorectal specimens of 14 HD patients. METHODS: We established a semi-quantitative score for cholinergic innervation using acetylcholinesterase (AChE) enzyme histochemistry and quantitatively analyzed the number of GCs via NADH tetrazolium reductase (NADH) enzyme histochemistry. We examined both the entire length of the resected specimens as well as defined areas of the transition zone of both pathological and healthy appearing segment. RESULTS: High AChE score values were associated with absence of GCs, and AChE scores were inversely correlated with the number of GCs. Nevertheless, we observed several cases in which one of the two features revealed a normal distribution pattern, whereas the other still displayed pathological features. CONCLUSIONS: Our data support the need for transmural colon biopsies, to enable the best evaluation of both cholinergic innervation and GCs for a reliable assessment of HD.

Generation of an Atxn2-CAG100 knock-in mouse reveals N-acetylaspartate production deficit due to early Nat8l dysregulation.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion mutations in the ATXN2 gene, mainly affecting motor neurons in the spinal cord and Purkinje neurons in the cerebellum. While the large expansions were shown to cause SCA2, the intermediate length expansions lead to increased risk for several atrophic processes including amyotrophic lateral sclerosis and Parkinson variants, e.g. progressive supranuclear palsy. Intense efforts to pioneer a neuroprotective therapy for SCA2 require longitudinal monitoring of patients and identification of crucial molecular pathways. The ataxin-2 (ATXN2) protein is mainly involved in RNA translation control and regulation of nutrient metabolism during stress periods. The preferential mRNA targets of ATXN2 are yet to be determined. In order to understand the molecular disease mechanism throughout different prognostic stages, we generated an Atxn2-CAG100-knock-in (KIN) mouse model of SCA2 with intact murine ATXN2 expression regulation. Its characterization revealed somatic mosaicism of the expansion, with shortened lifespan, a progressive spatio-temporal pattern of pathology with subsequent phenotypes, and anomalies of brain metabolites such as N-acetylaspartate (NAA), all of which mirror faithfully the findings in SCA2 patients. Novel molecular analyses from stages before the onset of motor deficits revealed a strong selective effect of ATXN2 on Nat8l mRNA which encodes the enzyme responsible for NAA synthesis. This metabolite is a prominent energy store of the brain and a well-established marker for neuronal health. Overall, we present a novel authentic rodent model of SCA2, where in vivo magnetic resonance imaging was feasible to monitor progression and where the definition of earliest transcriptional abnormalities was possible. We believe that this model will not only reveal crucial insights regarding the pathomechanism of SCA2 and other ATXN2-associated disorders, but will also aid in developing gene-targeted therapies and disease prevention.

The physiological mTOR complex 1 inhibitor DDIT4 mediates therapy resistance in glioblastoma.

BACKGROUND: Despite significant advances in the understanding of glioblastoma genetics and biology, survival is still poor. Hypoxia and nutrient depletion in the tumour microenvironment induce adaptive signalling and metabolic responses, which can influence sensitivity to therapeutic regimens. DNA damage-inducible transcript 4 (DDIT4) is a protein induced by hypoxia and in response to DNA stress. Mechanistically, DDIT4 inhibits mammalian target of rapamycin complex 1 (mTORC1) signalling by activation of the tuberous sclerosis 1/2 (TSC1/2) complex. METHODS: Using short hairpin RNA-mediated gene suppression as well as doxycycline-regulated gene induction, we developed a glioblastoma cell model to study effects of DDIT4 under conditions of the glioblastoma microenvironment and therapy. RESULTS: We found an intact DDIT4-mTORC1 signalling axis in human glioblastoma cells that was inducible by hypoxia. Temozolomide and radiotherapy also induced DDIT4 and repressed mTORC1 activity in some glioblastoma cell lines. DDIT4 gene suppression sensitised glioma cells towards hypoxia-induced cell death, while DDIT4 overexpression protected them. Additionally, in clonogenic survival analyses, DDIT4 induction conferred protection from radiotherapy and temozolomide, while DDIT4 gene suppression sensitised cells. CONCLUSIONS: We identified DDIT4 as a cell-intrinsic regulator for adaptive responses and therapy resistance in glioblastoma cells which may interfere with cell death induction by temozolomide, radiotherapy or hypoxia by inhibiting mTORC1 activity.