Dissection of astrocyte-mediated cerebral homeostasis in synucleinopathies

Multiple system atrophy (MSA) represents a rare atypical parkinsonian disorder characterized by a rapid and fatal course currently lacking causal therapies. Clinical features of MSA com- prise classical parkinsonism, cerebellar ataxia and pronounced autonomic failure occurring in the mean of the 6th decade of life. So far, the disease is classified as a sporadic disease with rare cases of familial MSA. Data concerning environmental factors causing the disease is still controversial. Dependent on the clinical manifestation, MSA is subdivided into a parkinsonian or a cerebellar type. Due to the low frequency of the disease and the fact that a definitive diagnosis is based on post mortem, animal models have been developed to mirror the molec- ular and motor phenotype of the disease. The MBP29-haSyn mouse model expresses human α-synuclein (aSyn) under the myelin basic protein (MBP) promotor specifically in oligodendro- cytes thus resembling MSA-related molecular pathology, such as glial cytoplasmic aSyn inclu- sions, severe demyelination, and microgliosis. Moreover, MBP29-haSyn mice display motor impairments, gait instability and a reduced lifespan. So far, comprehensive molecular profiling of astrocytes in MSA is lacking. Astrocytic expres- sion of glial fibrillary acidic protein (GFAP) was assessed as an indicator for astrocytic re- sponse to alterations in the microenvironment of MBP29-haSyn mice. Reactive astrogliosis was observed in the cortex and striatum on protein and RNA level analyzing expression levels of GFAP and vimentin. Moreover, focusing on cortex and striatum, an impaired capacity of upregulation of homeostasis-associated aquaporin-4, growth-associated protein 43, and glu- tamine synthetase was observed in the striatum of MBP29-haSyn-Syn mice compared to cor- tex. Additionally, expression of glutamate reuptake transporters was downregulated in the stri- atum of MBP29-haSyn mice, but not altered in the cortex. Based on these differences, a magnetic-activated cell sorting protocol for astrocyte isolation was adapted to ensure a high yield of pure astrocytes and bulk RNA sequencing was per- formed subsequently. Using these datasets, a transcriptomic landscape was generated high- lighting altered gene expression in astrocytes in the cortex and the striatum of MBP29-haSyn mice. Comprehensive analysis of both astrocyte populations revealed a dichotomous profile of astrocytes in the regions analyzed. Striatal astrocytes exhibit a pronounced immuno-active profile with upregulated pro-inflammatory transcripts involved in cytokine-cytokine interaction, toll-like receptor signaling, and phagocytosis. Conversely, analysis of the cortical astrocyte population demonstrated a decreased expression of transcripts associated with the inflamma- tory response, such as cytokine-cytokine interaction, and chemokine signaling. Since an increased presence of oligodendrocyte-associated transcripts was found upregulat-ed, the cellular components were further analyzed using gene set enrichment. Notably, the results indicated an enrichment of upregulated transcripts predominantly in the myelin sheath and the maintenance of axon implying a supportive role of astrocytes in the cortex of MBP29-haSyn mice. To examine the presence of oligodendroglial transcripts in astrocytes, in situ RNA hybridization was performed targeting Sry-related HMG-box 10 (SOX10) and myelin regulatory factor (MYRF) as prototypical oligodendroglial transcription factor. SOX10 and MYRF were identified in the proximity of astrocytic processes suggesting a potential oligoden-droglia-astroglia interaction in the cortex of MBP29-haSyn mice. Finally, reactive astrogliosis was investigated in human MSA-P patients post mortem. MSA-P patients display a strong reactive astrogliosis in the cortex, the striatum, and the substantia nigra. Moreover, expression of excitatory amino acid transporter 2 (EAAT2) was investigated expand the analysis of astrocytic characteristics. While no general decrease in expression of EAAT2 was detected, astrocytes in MSA-P patients show a re-distribution of EAAT2 from the branches towards the soma. The re-distribution of EAAT2 implies altered astrocyte functions in MSA-P patients, with EAAT2 as a potential target for a more detailed investigation of the glutamate metabolism in MSA. In conclusion, the findings of this thesis strongly demonstrate the heterogeneity of astrocytes within the CNS dependent on the region and the predominant pathology. The MBP29-haSyn mouse model provided a powerful tool to generate a transcriptomic landscape, thus highlight-ing the dynamic characteristic of astrocytes, displaying distinguished molecular profiles de-spite showing a similar upregulation pattern of GFAP. This work highlights the necessity of a comprehensive profiling of astrocytes to unravel the molecular complexity of this cell type to identify dysregulated pathways. Identification of potential interventional targets may be a promising approach to modulate the astrocytic phenotype in MSA-related pathology and thus slowing disease progression

Generation of a homozygous and a heterozygous SNCA gene knockout human-induced pluripotent stem cell line by CRISPR/Cas9 mediated allele-specific tuning of SNCA expression

Aggregation of alpha-synuclein (aSyn) is closely linked to Parkinson's disease, probably due to the loss of physiological functions and/or gain of toxic functions of aggregated aSyn. Significant efforts have been made elucidating the physiological structure and function of aSyn, however, with limited success thus far in human-derived cells, partly because of restricted resources. Here, we developed two human-induced pluripotent stem cell lines using CRISPR/Cas9-mediated allele-specific frame-shift deletion of the aSyn encoding gene SNCA, resulting in homo- and heterozygous SNCA knockout. The generated cell lines are promising cellular tools for studying aSyn dosage-dependent functions and structural alterations in human neural cells

Interaction of Alpha Synuclein and Microtubule Organization Is Linked to Impaired Neuritic Integrity in Parkinson’s Patient-Derived Neuronal Cells

Parkinson’s disease (PD) is neuropathologically characterized by the loss of dopaminergic neurons and the deposition of aggregated alpha synuclein (aSyn). Mounting evidence suggests that neuritic degeneration precedes neuronal loss in PD. A possible underlying mechanism could be the interference of aSyn with microtubule organization in the neuritic development, as implied by several studies using cell-free model systems. In this study, we investigate the impact of aSyn on microtubule organization in aSyn overexpressing H4 neuroglioma cells and midbrain dopaminergic neuronal cells (mDANs) generated from PD patient-derived human induced pluripotent stem cells (hiPSCs) carrying an aSyn gene duplication (SNCADupl). An unbiased mass spectrometric analysis reveals a preferential binding of aggregated aSyn conformers to a number of microtubule elements. We confirm the interaction of aSyn with beta tubulin III in H4 and hiPSC-derived mDAN cell model systems, and demonstrate a remarkable redistribution of tubulin isoforms from the soluble to insoluble fraction, accompanied by a significantly increased insoluble aSyn level. Concordantly, SNCADupl mDANs show impaired neuritic phenotypes characterized by perturbations in neurite initiation and outgrowth. In summary, our findings suggest a mechanistic pathway, through which aSyn aggregation interferes with microtubule organization and induces neurite impairments

Atrioventricular Node Dysfunction and Ion Channel Transcriptome in Pulmonary Hypertension

Background: Heart block is associated with pulmonary hypertension, and the aim of the study was to test the hypothesis that the heart block is the result of a change in the ion channel transcriptome of the atrioventricular (AV) node. Methods and Results: The most commonly used animal model of pulmonary hypertension, the monocrotaline-injected rat, was used. The functional consequences of monocrotaline injection were determined by echocardiography, ECG recording, and electrophysiological experiments on the Langendorff-perfused heart and isolated AV node. The ion channel transcriptome was measured by quantitative PCR, and biophysically detailed computer modeling was used to explore the changes observed. After monocrotaline injection, echocardiography revealed the pattern of pulmonary artery blood flow characteristic of pulmonary hypertension and right-sided hypertrophy and failure; the Langendorff-perfused heart and isolated AV node revealed dysfunction of the AV node (eg, 50% incidence of heart block in isolated AV node); and quantitative PCR revealed a widespread downregulation of ion channel and related genes in the AV node (eg, >50% downregulation of Cav1.2/3 and HCN1/2/4 channels). Computer modeling predicted that the changes in the transcriptome if translated into protein and function would result in heart block. Conclusions: Pulmonary hypertension results in a derangement of the ion channel transcriptome in the AV node, and this is the likely cause of AV node dysfunction in this disease

Reciprocal effects of alpha-synuclein aggregation and lysosomal homeostasis in synucleinopathy models

Abstract Background Lysosomal dysfunction has been implicated in a number of neurodegenerative diseases such as Parkinson’s disease (PD). Various molecular, clinical and genetic studies have highlighted a central role of lysosomal pathways and proteins in the pathogenesis of PD. Within PD pathology the synaptic protein alpha-synuclein (αSyn) converts from a soluble monomer to oligomeric structures and insoluble amyloid fibrils. The aim of this study was to unravel the effect of αSyn aggregates on lysosomal turnover, particularly focusing on lysosomal homeostasis and cathepsins. Since these enzymes have been shown to be directly involved in the lysosomal degradation of αSyn, impairment of their enzymatic capacity has extensive consequences. Methods We used patient-derived induced pluripotent stem cells and a transgenic mouse model of PD to examine the effect of intracellular αSyn conformers on cell homeostasis and lysosomal function in dopaminergic (DA) neurons by biochemical analyses. Results We found impaired lysosomal trafficking of cathepsins in patient-derived DA neurons and mouse models with αSyn aggregation, resulting in reduced proteolytic activity of cathepsins in the lysosome. Using a farnesyltransferase inhibitor, which boosts hydrolase transport via activation of the SNARE protein ykt6, we enhanced the maturation and proteolytic activity of cathepsins and thereby decreased αSyn protein levels. Conclusions Our findings demonstrate a strong interplay between αSyn aggregation pathways and function of lysosomal cathepsins. It appears that αSyn directly interferes with the enzymatic function of cathepsins, which might lead to a vicious cycle of impaired αSyn degradation. Graphical abstract Lysosomal trafficking of cathepsin D (CTSD), CTSL and CTSB is disrupted when alpha-synuclein (αSyn) is aggregated. This results in a decreased proteolytic activity of cathepsins, which directly mediate αSyn clearance. Boosting the transport of the cathepsins to the lysosome increases their activity and thus contributes to efficient αSyn degradation. </jats:sec

A Shot in the Dark: Failing to Recognize the Link Between Physical and Mental Illness

A 74-year-old widowed white man with chronic rheumatoid arthritis presented with nausea and weight loss. He was diagnosed with failure to thrive and admitted for hydration. Misoprostol was determined to be the etiology of his symptoms and he was discharged home. Three days later, he killed himself with a gunshot to the head. Clinicians often fail to recognize those at high risk for suicide. Suicidal risk is increased in both psychiatric and physical illness, and particularly when both are present. Psychiatric illness, particularly depression, often underlies chronic medical illness. The purpose of this case report is to remind health care providers of the strong association between depression and chronic medical illness, and to consider this in all patients, including those who present solely with physical symptoms. Recognizing this association and screening for it, as recommended by the U.S. Preventive Services Task Force, may prevent the unnecessary tragedy of suicide

Relationship between the morphology of the foveal avascular zone, retinal structure, and macular circulation in patients with diabetes mellitus

Diabetic Retinopathy (DR) is an extremely severe and common degenerative disease. The purpose of this study was to quantify the relationship between various parameters including the Foveal Avascular Zone (FAZ) morphology, retinal layer thickness, and retinal hemodynamic properties in healthy controls and patients with diabetes mellitus (DM) with and with no mild DR (MDR) using Spectral-Domain Optical Coherence Tomography (Spectralis SDOCT, Heidelberg Engineering GmbH, Germany) and the Retinal Function Imager (Optical Imaging, Ltd., Rehovot, Israel). Our results showed a higher FAZ area and diameter in MDR patients. Blood flow analysis also showed that there is a significantly smaller venous blood flow velocity in MDR patients. Also, a significant difference in roundness was observed between DM and MDR groups supporting the development of asymmetrical FAZ expansion with worsening DR. Our results suggest a potential anisotropy in the mechanical properties of the diabetic retina with no retinopathy that may trigger the FAZ elongation in a preferred direction resulting in either thinning or thickening of intraretinal layers in the inner and outer segments of the retina as a result of autoregulation. A detailed understanding of these relationships may facilitate earlier detection of DR, allowing for preservation of vision and better clinical outcomes

Postoperative outcomes in oesophagectomy with trainee involvement

BACKGROUND: The complexity of oesophageal surgery and the significant risk of morbidity necessitates that oesophagectomy is predominantly performed by a consultant surgeon, or a senior trainee under their supervision. The aim of this study was to determine the impact of trainee involvement in oesophagectomy on postoperative outcomes in an international multicentre setting. METHODS: Data from the multicentre Oesophago-Gastric Anastomosis Study Group (OGAA) cohort study were analysed, which comprised prospectively collected data from patients undergoing oesophagectomy for oesophageal cancer between April 2018 and December 2018. Procedures were grouped by the level of trainee involvement, and univariable and multivariable analyses were performed to compare patient outcomes across groups. RESULTS: Of 2232 oesophagectomies from 137 centres in 41 countries, trainees were involved in 29.1 per cent of them (n = 650), performing only the abdominal phase in 230, only the chest and/or neck phases in 130, and all phases in 315 procedures. For procedures with a chest anastomosis, those with trainee involvement had similar 90-day mortality, complication and reoperation rates to consultant-performed oesophagectomies (P = 0.451, P = 0.318, and P = 0.382, respectively), while anastomotic leak rates were significantly lower in the trainee groups (P = 0.030). Procedures with a neck anastomosis had equivalent complication, anastomotic leak, and reoperation rates (P = 0.150, P = 0.430, and P = 0.632, respectively) in trainee-involved versus consultant-performed oesophagectomies, with significantly lower 90-day mortality in the trainee groups (P = 0.005). CONCLUSION: Trainee involvement was not found to be associated with significantly inferior postoperative outcomes for selected patients undergoing oesophagectomy. The results support continued supervised trainee involvement in oesophageal cancer surgery