Unraveling dystonia circuitry in rodent models using novel neuromodulation techniques

Dystonia is a network disorder presumed to result from abnormalities in multiple brain regions and in multiple cell populations. The specific pathomechanisms affecting the motor circuits in dystonia are, however, still largely unclear. Animal models for dystonia have long been used to advance our understanding on how specific brain regions and cell populations are involved in dystonia symptomatogenesis. Lesioning, pharmacological modulation and electrical stimulation paradigms were able to highlight that both the basal ganglia and the cerebellum are pathologically altered in these animal models for dystonia. Techniques such as optogenetics and chemogenetics now offer the opportunity for targeted modulation of brain regions and most importantly cell populations and circuits. This could not only allow for a better understanding of the dystonic brain, but potentially improve and expand treatment options. In hopes that the insights from these neuromodulation techniques will eventually translate into therapies, we aim to summarize and critically discuss the findings from different in vivo approaches used to dissect the network dysfunctions underlying dystonia

Strukturelle und funktionelle Charakterisierung von Exosomen aus Prostatakarzinomzellen

Die Umgestaltung der Tumor-Mikroumgebung, die Modulation nicht-maligner Zellen sowie die Induktion des metastatischen Potentials sind essentielle Vorgänge im Rahmen der Tumorprogression. Exosomen sind extrazelluläre Vesikel eines Durchmessers von 50-150 nm, welche im Rahmen des interzellulären Informationsaustauschs eine möglicherweise maßgebliche Rolle in Tumorwachstum und Malignitätssteigerung innehaben. Ein genaueres Verständnis ihrer Funktion könnte zu neuen diagnostischen und therapeutischen Möglichkeiten in der Onkologie verhelfen. Im Rahmen dieser Arbeit erfolgte die Charakterisierung von Exosomen aus Prostatakarzinom (PCa)-Zellen auf molekularer und funktioneller Ebene. Exosomen aus den PCa-Zelllinien LNCaP, PC-3 und PC3-HSP27 wurden anhand Ultrazentrifugation angereichert. Es erfolgte eine Validierung der Präparationsmethode mittels dynamischer Lichtstreuung und Transmissionselektronenmikroskopie. Das exosomale Transkriptom wurde anhand DNA-Microarray, das exosomale Proteom anhand Massenspektrometrie analysiert. Der Auswirkung von PCa-Exosomen auf ihre Umgebung wurde mittels MTT-Vitalitätstest und LDH-Zytotoxizitätstest nachgegangen. Anhand der SILAC-Methodik erfolgte der Nachweis einer Aufnahme exosomaler Proteine in canine Zellen. Die Analyse der exosomalen Nukleinsäuren- und Protein-Zusammensetzung zeigte, dass Exosomen einerseits einen Fingerabdruck des Zytoplasmas der Ursprungszelle darstellen und, dass andererseits eine spezifische Anreicherung von Membranproteinen stattfindet. Dabei erschien vor allem die Inkorporation kleiner Proteine keinem bestimmten Sortierungsmechanismus zu unterliegen. Die Zellkultur-Experimente ließen auf einen Wachstums-inhibierenden Effekt der Exosomen kanzerösen Ursprungs auf nicht-maligne Zellen schließen. Des Weiteren konnte eine Aufnahme exosomaler, humaner Proteine in canine Zellen nach einer Inkubationszeit von 6 h nachgewiesen werden. In der Zusammenschau weisen die Ergebnisse auf eine bedeutende Funktion der Exosomen im Rahmen der Modulation der PCa-Mikroumgebung hin. Exosomen ist die Induktion multipler Effekte in Tumor-assoziierten Zielzellen zuzuschreiben, unter anderem die Regulation des Zellwachstums.Exosomes are small vesicles with a diameter between 50–150nm, which are secreted into the extracellular space. Emerging evidence shows that they may serve as vehicles for signal molecules in order to modulate target cells. As such, exosomes may play a pivotal role in tumor progression by influencing the remodeling of the tumor environment and modulating tumor associated non-malignant cells. A better understanding of composition and function of these nanovesicles could lead to diagnostic and therapeutic advances in the treatment of cancer. Protein and mRNA composition as well as functions of prostate cancer cell-derived exosomes were analyzed within this work. Exosomes were prepared from prostate cancer cell lines LNCaP, PC-3 and PC3-HSP27 via ultracentrifugation. Validation of exosomal enrichment and characterization of morphology were conducted by means of dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis as well as mass spectrometry. Effects of tumor-exosomes on non-malignant target cells were studied by means of cell survival assays and cytotxicity assays. Using the SILAC method, the potential uptake of exosomal human proteins into canine cells and de novo synthesis of proteins through transferred exosomal mRNA was analyzed in MDCK cells via mass spectrometry. Analysis of exosomal composition showed that these vesicles are highly enriched in membrane proteins, the packaging of small proteins was however unspecific, as shown with the example of two model proteins. In cell culture assays exosomes from prostate cancer cells had an anti-proliferative effect on non-malignant cells. Mass spectrometry revealed the uptake of exosomal proteins into canine cells after 6 hr of incubation. The results reveal a distinct function of exosomal particles in the modulation of prostate tumor environment, including impact on cellular growth and incorporation of exosomal proteins into adjacent target cells

Second hit hypothesis in dystonia: Dysfunctional cross talk between neuroplasticity and environment?

One of the great mysteries in dystonia pathophysiology is the role of environmental factors in disease onset and development. Progress has been made in defining the genetic components of dystonic syndromes, still the mechanisms behind the discrepant relationship between dystonic genotype and phenotype remain largely unclear. Within this review, the preclinical and clinical evidence for environmental stressors as disease modifiers in dystonia pathogenesis are summarized and critically evaluated. The potential role of extragenetic factors is discussed in monogenic as well as adult-onset isolated dystonia. The available clinical evidence for a "second hit" is analyzed in light of the reduced penetrance of monogenic dystonic syndromes and put into context with evidence from animal and cellular models. The contradictory studies on adult-onset dystonia are discussed in detail and backed up by evidence from animal models. Taken together, there is clear evidence of a gene-environment interaction in dystonia, which should be considered in the continued quest to unravel dystonia pathophysiology

Age-dependent neurodegeneration and neuroinflammation in a genetic A30P/A53T double-mutated α-synuclein mouse model of Parkinson's disease

The pathogenesis of Parkinson's disease (PD) is closely interwoven with the process of aging. Moreover, increasing evidence from human postmortem studies and from animal models for PD point towards inflammation as an additional factor in disease development. We here assessed the impact of aging and inflammation on dopaminergic neurodegeneration in the hm2α-SYN-39 mouse model of PD that carries the human, A30P/A53T double-mutated α-synuclein gene. At 2–3 months of age, no significant differences were observed comparing dopaminergic neuron numbers of the substantia nigra (SN) pars compacta of hm2α-SYN-39 mice with wildtype controls. At an age of 16–17 months, however, hm2α-SYN-39 mice revealed a significant loss of dopaminergic SN neurons, of dopaminergic terminals in the striatum as well as a reduction of striatal dopamine levels compared to young, 2–3 months transgenic mice and compared to 16–17 months old wildtype littermates. A significant age-related correlation of infiltrating CD4+ and CD8+ T cell numbers with dopaminergic terminal loss of the striatum was found in hm2α-SYN-39 mice, but not in wildtype controls. In the striatum of 16–17 months old wildtype mice a slightly elevated CD8+ T cell count and CD11b+ microglia cell count was observed compared to younger aged mice. Additional analyses of neuroinflammation in the nigrostriatal tract of wildtype mice did not yield any significant age-dependent changes of CD4+, CD8+ T cell and B220+ B cell numbers, respectively. In contrast, a significant age-dependent increase of CD8+ T cells, GFAP+ astrocytes as well as a pronounced increase of CD11b+ microglia numbers were observed in the SN of hm2α-SYN-39 mice pointing towards a neuroinflammatory processes in this genetic mouse model for PD. The findings in the hm2α-SYN-39 mouse model strengthen the evidence that T cell and glial cell responses are involved in the age-related neurodegeneration in PD. The slow and age-dependent progression of neurodegeneration and neuroinflammation in the hm2α-SYN-39 PD rodent model underlines its translational value and makes it suitable for studying anti-inflammatory therapies

The evolution of dystonia-like movements in TOR1A rats after transient nerve injury is accompanied by dopaminergic dysregulation and abnormal oscillatory activity of a central motor network

TOR1A is the most common inherited form of dystonia with still unclear pathophysiology and reduced penetrance of 30–40%. ∆ETorA rats mimic the TOR1A disease by expression of the human TOR1A mutation without presenting a dystonic phenotype. We aimed to induce dystonia-like symptoms in male ∆ETorA rats by peripheral nerve injury and to identify central mechanism of dystonia development. Dystonia-like movements (DLM) were assessed using the tail suspension test and implementing a pipeline of deep learning applications. Neuron numbers of striatal parvalbumin+, nNOS+, calretinin+, ChAT+ interneurons and Nissl+ cells were estimated by unbiased stereology. Striatal dopaminergic metabolism was analyzed via in vivo microdialysis, qPCR and western blot. Local field potentials (LFP) were recorded from the central motor network. Deep brain stimulation (DBS) of the entopeduncular nucleus (EP) was performed. Nerve-injured ∆ETorA rats developed long-lasting DLM over 12 weeks. No changes in striatal structure were observed. Dystonic-like ∆ETorA rats presented a higher striatal dopaminergic turnover and stimulus-induced elevation of dopamine efflux compared to the control groups. Higher LFP theta power in the EP of dystonic-like ∆ETorA compared to wt rats was recorded. Chronic EP-DBS over 3 weeks led to improvement of DLM. Our data emphasizes the role of environmental factors in TOR1A symptomatogenesis. LFP analyses indicate that the pathologically enhanced theta power is a physiomarker of DLM. This TOR1A model replicates key features of the human TOR1A pathology on multiple biological levels and is therefore suited for further analysis of dystonia pathomechanism