1,721,075 research outputs found
The Gut-Brain Axis in Inflammatory Bowel Disease—Current and Future Perspectives
Get PDFThe gut–brain axis is a bidirectional communication system driven by neural, hormonal, metabolic, immunological, and microbial signals. Signaling events from the gut can modulate brain function and recent evidence suggests that the gut–brain axis may play a pivotal role in linking gastrointestinal and neurological diseases. Accordingly, accumulating evidence has suggested a link between inflammatory bowel diseases (IBDs) and neurodegenerative, as well as neuroinflammatory diseases. In this context, clinical, epidemiological and experimental data have demonstrated that IBD predisposes a person to pathologies of the central nervous system (CNS). Likewise, a number of neurological disorders are associated with changes in the intestinal environment, which are indicative for disease-mediated gut–brain inter-organ communication. Although this axis was identified more than 20 years ago, the sequence of events and underlying molecular mechanisms are poorly defined. The emergence of precision medicine has uncovered the need to take into account non-intestinal symptoms in the context of IBD that could offer the opportunity to tailor therapies to individual patients. The aim of this review is to highlight recent findings supporting the clinical and biological link between the gut and brain, as well as its clinical significance for IBD as well as neurodegeneration and neuroinflammation. Finally, we focus on novel human-specific preclinical models that will help uncover disease mechanisms to better understand and modulate the function of this complex system
Axon-spezifische mitochondriale Pathologie in Alpha-Motoneuronen von SPG11-Patienten
Get PDFSPG11-HSP is the most common form of complicated autosomal-recessive hereditary spastic paraplegia (HSP). Typical manifestation of HSP is a length-dependant degeneration of axons of corticospinal neurons. Additionally, SPG11-patients are often affected by distal motor neuropathy caused by degeneration of spinal motor neurons. Most notably, altered autophagy and the accumulation of lipids in lysosomes have been studied in SPG11. An affection of mitochondria could be observed in several neurodegenerative diseases like idiopathic Parkinson's disease as well as the HSP-forms SPG15 and SPG48, which are closely related to SPG11 (Denton et al. 2018). Investigations, whether mitochondrial pathology is also present in SPG11 were lacking to this date. The goal of this study was a systematic investigation of mitochondrial morphology as well as axonal mitochondrial transport in SPG11. We used alpha motor neurons that were derived from human induced pluripotent stem cells (iPSC/ in humans hiPSC) as a disease model. Motor neurons derived from human embryonic stem cells and an isogenic SPG11 knockout line were also included in the study.SPG11-HSP ist die häufigste Form der komplizierten autosomal-rezessiven hereditären spastischen Paraparese (HSP). Typisch für die HSP ist eine längenabhängige Degeneration der Axone kortikospinaler Neurone. SPG11-Patienten sind zusätzlich häufig durch eine distal beginnende motorische Neuropathie beeinträchtigt. Dies ist Folge
einer Degeneration spinaler Motoneurone. Bisherige Studien zu SPG11 untersuchten
vor allem die gestörte Autophagie sowie die Akkumulation von Lipiden in Lysosomen.
Eine Beteiligung von Mitochondrien konnte in mehreren neurodegenerativen Erkrankungen wie z.B. dem idiopathischen Parkinson-Syndrom sowie einer aktuellen Studie
zu mitochondrialer Pathologie in den mit SPG11 eng verwandten Formen SPG15 und
SPG48 (Denton et al. 2018) nachgewiesen werden. Eine systematische Untersuchung
zu einer möglichen mitochondrialen Pathologie in SPG11 fehlte bisher. Das Ziel dieser
Studie war eine systematische Untersuchung mitochondrialer Morphologie sowie des
axonalen mitochondrialen Transportes in SPG11 in einem für die Krankheit relevanten
humanen Modellsystem, insbesondere mit Modellierung des distalen axonalen Kompartiments. Für diese Studie wurden humane Alpha-Motoneurone verwendet, die aus
induzierten pluripotenten Stammzellen (iPSZ bzw. in Menschen hiPSZ) generiert wurden. Ergänzend wurde eine humane embryonale Stammzell-Linie mit SPG11-Knockout und ihre isogene Kontrolle eingesetzt
Spezifität von Anti-Alpha-Synuclein-Antikörpern - Erkenntnisse aus der durchflusszytometrischen Analyse
Get PDFHintergrund und Ziele
Die Akkumulation von Alpha-Synuclein (aSyn) ist das Charakteristikum einer Gruppe von neurodegenerativen Erkrankungen, die als Synucleinopathien bezeichnet werden. Die physiologischen Funktionen von aSyn, sowohl innerhalb als auch außerhalb des zentralen Nervensystems (ZNS), sind jedoch noch nicht vollständig geklärt. Eine verlässliche und reproduzierbare Bewertung der aSyn-Proteinexpression in verschiedenen Zelltypen, insbesondere in Zellen mit niedriger Expression, wird durch die Vielzahl schlecht charakterisierter Anti-aSyn-Antikörper und das Fehlen einer routinemäßig verwendeten sensitiven und spezifischen Detektionsmethode erschwert. In dieser Studie habe ich einen robusten durchflusszytometrischen Arbeitsablauf für den aSyn-Nachweis und die Antikörper-Validierung entwickelt.
Methoden
Ich habe drei handelsübliche Antikörper (MJFR1, LB509 und 2A7) in einem großen Spektrum humaner Zelltypen, einschließlich induzierter pluripotenter Stammzellen, T-Lymphozyten und Fibroblasten, getestet. Mithilfe von Peptid-Blocking-Experimenten wurde die Sensitivität für die aSyn-Detektion sowie mögliche Kreuzreaktionen gegen weitere Subtypen der Synuclein-Familie sowie Tubulin untersucht. Zusätzlich habe ich eine zell- und antikörperspezifische Kartierung der aSyn-Expression erstellt. Darüber hinaus habe ich den Antikörper 2A7 auf seine Fähigkeit hin untersucht, die physiologische Heterogenität in der aSyn-Expression zwischen neuronalen und nicht-neuronalen Zellen aus kortikalen Organoiden zu detektieren. Abschließend wurde das Potenzial dieses Antikörpers zur Differenzierung des Proteingehalts zwischen gesunden Kontrollen und Parkinson-Patienten mit aSyn-Genlocus-Duplikation in neuronalen Vorläuferzellen und dopaminergen Neuronen des Mesencephalons analysiert.
Ergebnisse und Beobachtungen
In der systematischen Untersuchung konnte ich eine bisher nicht beobachtete Unspezifität des LB509-Antikörpers gegen Tubulin und verschiedene Synucleinformen nachweisen, während der MJFR1-Klon eine spezifische aSyn-Bindung zeigte, allerdings eine geringe Sensitivität aufwies. Im Gegensatz dazu zeigte der Antikörperklon 2A7 eine hohe Spezifität mit einer optimalen Empfindlichkeit für die Detektion von aSyn in einer Reihe von Zelltypen, einschließlich solcher mit geringer aSyn-Expression. Darüber hinaus war dieser imstande, die höheren Konzentrationen von aSyn in neuronalen Zellen zu erfassen und zeigte eine deutlich ausgeprägtere Expression dieses Proteins in der Duplikationslinie des aSyn-Gens.
Schlussfolgerungen und Diskussion
Auch wenn zwei der getesteten kommerziell erhältlichen aSyn-Antikörper ihre Anforderungen an die sensitive und spezifische Proteindetektion nicht erfüllten, habe ich den Antikörper 2A7 als geeignetes Werkzeug für die Erforschung der aSyn-Expression in verschiedenen Zelltypen identifiziert. Diese Ergebnisse liefern einen Grundsatzbeweis für den Einsatz der Durchflusszytometrie zur Analyse von aSyn und unterstreichen die Notwendigkeit einer strengen aSyn-Antikörper-Validierung, um die Erforschung der Physiologie und Pathologie dieses Proteins zu ermöglichen
Understanding disease modulators of SPG11‐linked hereditary spastic paraplegia.
Get PDFHereditary spastic paraplegia (HSP) is a diverse group of motor neuron disorders that is characterized by lower-limb weakness and spasticity due to a length-dependent axonopathy of the corticospinal tract. HSP type 11 (SPG11-HSP) is the most frequent form of autosomal recessive HSP and is caused by pathogenic variants in the SPG11 gene, encoding the protein spatacsin. In this complicated form, motor neuron symptoms are accompanied by a thin corpus callosum, white matter abnormalities, and progressive cognitive decline. Currently, there are no effective treatments or reliable biomarkers available. Although inflammation is pivotal in several neurological diseases, SPG11-HSP research has mainly focussed on the neurodegenerative hallmarks. For this reason, we hypothesized that neuroinflammation is a significant disease mechanism in SPG11-HSP patients which can contribute to neurodegeneration. Both aspects of the disease can therefore provide potential biomarkers for monitoring disease progression and validating treatment strategies.
We performed a detailed immunological characterization of SPG11-HSP patients by analyzing human postmortem brains, peripheral blood samples, and induced pluripotent stem cell-derived microglia-like cells (iMGL). Postmortem examination of three SPG11-HSP patient brains revealed profound microgliosis, signatures of disease-associated microglia, and lipid accumulations in myeloid cells. In a larger patient cohort, the ratio of proinflammatory monocytes in the blood was increased along with elevated serum levels of IL-6 that correlated with disease severity. Moreover, we delineated a novel interplay between SPG11 and IFNγ. In control iMGL, IFNγ triggered the upregulation of SPG11, whereas, in patient-specific SPG11 iMGL, IFNγ caused hyperactivation, which was characterized by increased secretion of inflammatory factors, such as CXCL10. Enhanced STAT1 phosphorylation was identified as a mechanism connecting IFNγ-mediated hyperactivation and SPG11 loss of function. Blocking STAT1 signaling by ruxolitinib prevented the upregulation of CXCL10 and rescued the neurotoxic effects of SPG11 iMGL. Examination of human postmortem brain tissue and an Spg11-/- mouse model confirmed increased numbers of STAT1+ cells. These findings highlight the impact of innate immunity in SPG11-HSP and indicate the potential of immunomodulators to slow down disease progression.
To evaluate such therapeutic approaches, biomarkers are essential to monitor disease progression. For other neurodegenerative diseases, peripheral neurofilaments have emerged as promising indicators of neuronal damage. Therefore, we aimed to assess the potential of neurofilament light (NfL) and heavy (NfH) chains as biomarkers of neurodegeneration in SPG11-HSP. Both NfL and NfH were significantly increased in the plasma of SPG11-HSP patients with excellent discriminatory performance from controls and other HSP types. In a follow-up study, the NfL level in SPG11-HSP patients increased further within one year. These data emphasize the potential of neurofilaments as a fluidic biomarker for monitoring disease progression in this complicated subtype of HSP.
Overall, this study demonstrates the role of innate immunity on SPG11-related neurodegeneration and highlights inflammatory factors and neurofilaments as potential biomarkers for SPG11-HSP
Analysis of α-synuclein expression in lymphocytes of healthy donors and patients with sporadic Parkinson's disease
Get PDFBackground and Aims
Parkinson’s disease (PD) is one of the most common neurodegenerative movement diseases primarily affecting the elderly population. PD`s main symptoms - rigor, tremor, and brady- or akinesia - go back to a loss of dopaminergic neurons (DN) in the substantia nigra (SN) pars compacta of the midbrain. DN loss is closely connected with an occurrence of intracellular α-Synuclein (aSyn)-containing protein aggregates – known as Lewy bodies (LB) or Lewy neurites (LN) – and their spreading over the human central nervous system (CNS). The disease was first described in 1817, but still poses various challenges in diagnosis as well as in therapy, even though modern medicine has developed several approaches to pharmacological as well as non-pharmacological symptomatic treatment by now. A causative treatment, however, has not been found up until today. The immune system, consisting of different types of leucocytes as well as of non-cellular defence mechanisms, plays a crucial role in both, acute and chronic diseases and is involved in PD pathology. Preceding studies already showed that there are certain observable reciprocal effects between age, health condition, immune system activation and aSyn expression. Rooting in the main hypothesis that aSyn is expressed in human lymphocytes, my research focused on the questions whether and how aSyn expression rates differ in different lymphocyte subsets, and what influence donor age, health condition and the activation status of the respective cell type might have on aSyn expression. I aimed for a better understanding of the development of PD on the cellular level, because this may offer valuable approaches to the diagnosis and treatment of the disease in the future.
Methods
Lymphocyte subsets were separated under the use of magnetic, cluster of differentiation (CD)-specific beads. Subsequently, activation of the various T cell subsets was conducted by applying specific T cell activation beads (DynaBeads). For observation of aSyn expression on the transcript level, multiple real-time polymerase chain reactions (rtPCR) were conducted, while for observation of the protein level, protein resolubilisation methods were evaluated as well as a combination of intracellular aSyn staining with CD-specific surface staining for fluorescence-activated cell sorting (FACS) analysis. Expression rates on the transcript and protein levels were compared among different lymphocyte subsets of various age groups and an active and at a resting state. Eventually, aSyn expression of PD patients` cells was compared with that of healthy individuals.
Results
While aSyn protein expression analysis in human lymphocytes only offered a perspective of what should be evaluated further in the future, rtPCR analysis allowed an interesting insight in PD pathology on the aSyn transcript level. We were able to show that in activated CD4+ T cells, expression of aSyn transcript seemed to decrease in comparison to that of resting cells – an observation, which was observable in all donor groups, young and healthy, older and healthy and PD patients. Furthermore, aSyn transcript expression showed a correlation with the respective donor age. aSyn transcript expression was on average higher in CD4+ T cells of older and healthy individuals than in those of young and healthy individuals. On the other side, PD patients’ CD4+ T cells appeared to express less aSyn transcript levels than those of healthy individuals.
Discussion
It was shown that aSyn expression in human lymphocytes depends on various influential factors. Age, health condition and the activation state of the respective lymphocytes are important influences on aSyn expression. Those observations give us an idea on how PD might be influenced by activation mechanisms in the human immune system. Our present results appear to contradict the hypothesis of PD as a systemic autoimmune reaction and rather support the theory of a focal inflammatory origin of PD pathomechanism. Further immunological research on cellular mechanisms of aSyn transcript and protein expression as well as regulation might be the future key to an earlier diagnosis of PD and, maybe, also for the treatment or even prevention of the disease
Effekte der Small molecule-mediierten Reduktion von alpha-Synuklein Aggregation auf dopaminerge Neurone in einem Stammzell-basierten Modell des SNCA-assoziierten erblichen Parkinson-Syndroms
No full textBackground: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the accumulation of misfolded α-synuclein (αSyn) aggregates known as Lewy bodies. Developing effective treatments for PD is challenging due to its complex pathophysiology. NPT100-18A, a peptidomimetic compound, has shown promise as an αSyn misfolding inhibitor, potentially targeting a key driver of PD pathology.
Objectives: In this study, we aimed to comprehensively investigate the therapeutic potential of NPT100-18A in a human-induced pluripotent stem cell (iPSC)-derived model of PD. Specifically, we sought to understand its impact on αSyn aggregation, oxidative stress, mitochondrial dysfunction, and neuronal cell survival.
Methods: We employed a panel of five iPSC lines, including two lines derived from PD patients with heterozygous SNCA locus duplication and three age- and sex-matched healthy control lines. We followed a well-established differentiation protocol to generate midbrain dopaminergic neurons (mDANs) from these iPSCs. NPT100-18A was administered from the onset of differentiation, while vehicle-treated (DMSO-treated) cultures served as controls. A series of analyses, including the assessment of αSyn aggregation, quantification of reactive oxygen species (ROS) levels, measurement of adenosine triphosphate (ATP) concentrations, and evaluation of neuronal cell death were performed.
Results: iPSC-derived mDANs from PD patients exhibited several hallmark features of the disease, including increased αSyn aggregation, elevated ROS levels, mitochondrial dysfunction, and heightened rates of neuronal cell death. Remarkably, treatment with NPT100-18A produced significant therapeutic effects. It effectively reduced the levels of insoluble αSyn aggregates, demonstrating its potential to mitigate αSyn aggregation, a central pathological event in PD. Additionally, NPT100-18A displayed a specific antioxidative effect within mitochondria, substantially lowering mitochondrial ROS levels. While not significantly altering cytoplasmic ROS levels, NPT100-18A showed a promising trend toward increasing ATP concentrations, hinting at potential benefits for mitochondrial function and overall cellular energy metabolism. Importantly, the compound rescued neurons from the increased cell death observed in patient-derived mDANs.
Conclusion: Our findings highlight the therapeutic potential of NPT100-18A in mitigating αSyn aggregation and rescuing neurons in a human iPSC-derived model of PD. By specifically targeting αSyn misfolding and reducing mitochondrial oxidative stress, NPT100-18A demonstrates promise as a novel treatment strategy for PD. These results offer valuable insights into the neuroprotective mechanisms of NPT100-18A, suggesting that it may act through multiple pathways, including αSyn aggregation and mitochondria-specific antioxidant effects. Further research is warranted to comprehensively characterize its effects on mitochondrial function and to explore its potential for further clinical translation. NPT100-18A represents a promising candidate for future PD therapeutics, addressing critical aspects of the disease's pathogenesis and offering new avenues for intervention.Hintergrund: Die Parkinson-Krankheit (PD) ist eine neurodegenerative Erkrankung, die durch den fortschreitenden Verlust dopaminerger Neuronen in der Substantia nigra und die Anhäufung fehlgefalteter α-Synuclein (αSyn)-Aggregate, so genannter Lewy-Körper, gekennzeichnet ist. Die Entwicklung wirksamer Behandlungen für Parkinson ist aufgrund der komplexen Pathophysiologie eine Herausforderung. NPT100-18A, ein Peptidomimetikum, hat sich als vielversprechender Hemmstoff für die Fehlfaltung von α-Synuclein erwiesen, der möglicherweise einen der Hauptfaktoren der Parkinson-Pathologie angreift.
Ziele: Ziel dieser Studie war es das therapeutische Potenzial von NPT100-18A in einem aus menschlichen pluripotenten Stammzellen (iPSC) abgeleiteten Parkinson-Modell umfassend zu untersuchen. Insbesondere sollten Auswirkungen auf die αSyn-Aggregation, oxidativen Stress, mitochondriale Dysfunktion und das neuronale Zellüberleben verstanden werden.
Methoden: Hierzu wurden insgesamt fünf iPSC-Linien, darunter zwei Linien, die von PD-Patienten mit heterozygoter SNCA-Lokus-Duplikation stammen, und drei alters- und geschlechtsgleiche Kontrolllinien gesunder Probanden herangezogen. Ein etabliertes Differenzierungsprotokoll diente als Vorlage, um aus diesen iPSCs dopaminerge Mittelhirnneurone (mDANs) zu generieren. NPT100-18A wurde von Beginn der Differenzierung an verabreicht, während mit Vehikel (DMSO) behandelte Kulturen als Kontrolle dienten. Nach Abschluss der neuronalen Differenzierung und Maturierung wurde eine Reihe von Analysen durchgeführt, darunter die Quantifizierung und Charakterisierung von αSyn-Aggregaten, die Quantifizierung reaktiver Sauerstoffspezies (ROS), die Messung der Adenosintriphosphat (ATP)-Konzentration und Quantifizierung neuronalen Zelltods.
Ergebnisse: iPSC-derivierte mDANs von Parkinson-Patienten wiesen mehrere charakteristische Merkmale der Krankheit auf, darunter eine erhöhte αSyn-Aggregation, erhöhte ROS-Werte, mitochondriale Dysfunktion und erhöhte Raten neuronalen Zelltods. Die Behandlung mit NPT100-18A resultierte in signifikanten therapeutischen Effekten. NPT100-18A verringerte wirksam die Menge an unlöslichen αSyn-Aggregaten und zeigte damit sein Potenzial, die αSyn-Aggregation, ein zentrales pathologisches Ereignis in der Pathogenese des Morbus Parkinson, zu limitieren. Darüber hinaus zeigte NPT100-18A eine spezifische antioxidative Wirkung in Mitochondrien und senkte mitochondriale ROS-Konzentration signifikant. Obwohl NPT100-18A die zytoplasmatischen ROS-Konzentrationen nicht signifikant veränderte, zeigte sich ein deutlicher Trend hin zur Normalisierung verringerter ATP-Konzentrationen, was auf mögliche Vorteile für die mitochondriale Funktion und den gesamten zellulären Stoffwechsel hindeutet. Darüber hinaus, konnte gezeigt werden, dass der Wirkstoff erhöhte Raten neuronaler Apoptose auf das Niveau von Kontroll-Zelllinien senken und somit einen signifikanten und relevanten, positiven Effekt auf den wichtigsten Outcome-Parameter in dieser Studie erzielen konnte.
Schlussfolgerung: Unsere Ergebnisse demonstrieren das therapeutische Potenzial von NPT100-18A bei der Abschwächung von αSyn-Aggregation und der Reduktion neuronalen Zelltods in einem humanen iPSC-basierten Model des Morbus Parkinson. Durch die gezielte Beeinflussung der Fehlfaltung von αSyn und die Verringerung des damit assoziierten mitochondrialen oxidativen Stress erweist sich NPT100-18A als vielversprechende neue Behandlungsstrategie für Parkinson. Diese Ergebnisse bieten wertvolle Einblicke in die neuroprotektiven Mechanismen von NPT100-18A und deuten darauf hin, dass mehrere zelluläre Signal- und Stoffwechselwege positiv beeinflusst werden, einschließlich der αSyn-Aggregation und mitochondrienspezifischer, antioxidativer Effekte. Weitere Forschungsarbeiten sind erforderlich, um die Auswirkungen auf andere Parameter mitochondrialer Funktionen umfassend zu charakterisieren und das Potenzial für die weitere klinische Translation zu erforschen. NPT100-18A ist ein vielversprechender Kandidat für künftige Parkinson-Therapeutika, der kritische Aspekte der Pathogenese der Krankheit anspricht und neue Perspektiven auf krankheitsmodifizierende Interventionen eröffnet
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The roles of Voltage-Gated Sodium Channels and β subunits in mechanosensitivity and neuronal electrical excitability
Get PDFBackground and aims:
The voltage-gated sodium channels (Nav) are responsible for action potential generation and propagation in excitable cells like cardiac myocytes and neurons. The modulation of Navs affects the properties of action potentials and cellular excitability. The main Nav in cardiac myocytes is Nav1.5 and is modulated by mechanical stimuli. Previous studies showed that also Nav 1.4, Nav 1.6 and Nav1.7 are mechanosensitive, and a recent study showed that the Nav β1 subunit modulates the mechanosensitivity of Nav1.7.
This work aimed to investigate whether the mechanosensitivity of Nav1.5 is regulated by β subunits. For this purpose, I characterized the gating and kinetic properties of Nav1.5 in the presence of β1 and β3 subunits in the HEK293 cell line.
While cell lines offer an accessible and effective model for studying Nav properties and cellular excitability, they do not fully recapitulate the in vivo properties of human excitable cells like cardiomyocytes and neurons. Therefore, to better understand excitability in models of neurological diseases, I also conducted electrophysiological studies on human induced pluripotent stem cells (hiPSCs)-derived nociceptors and midbrain dopaminergic neurons.
Methods and results:
To investigate the modulation of Nav mechanosensitivity by β subunits, Nav1.5 was heterologously expressed in HEK293 cells, alone or combined with either β1 or β3 subunits. Patch-clamp recordings were performed in the Nav1.5, Nav1.5 β1, and Nav1.5 β3 expression groups, with or without mechanical stimulation.
This study demonstrates that β1 and β3 subunits elicit specific differences in gating and kinetic properties of Na+ currents under mechanical stimulation. The β1 subunit enhanced the effect of mechanical stimulation on fast inactivation. In contrast, the β3 subunit showed a stronger effect on the kinetics of current decay under mechanical stimulation.
Using homology modeling and residue interaction network (RIN) analysis, this study identified differences in the number, strength, and distribution of bonds that β1 and β3 subunits establish with DI, DIII, and DIV (Domain I-IV of Navs) of Nav1.5 channel. In the RIN analysis, β3 established a stronger binding to Nav1.5 compared to β1. These binding differences could account for the effects on fast inactivation and kinetics of current decay since it has been shown that the movement of the voltage sensing domain within the DIV of Nav channels is important for the onset of inactivation.
Additionally, the electrophysiological properties of hiPSCs-derived midbrain dopaminergic neurons and nociceptors were analyzed using conventional patch-clamp and multi-electrode-arrays. These results show that both midbrain dopaminergic neurons and nociceptors differentiated from stem cells in dual-SMAD inhibition are electrically active. Finally, slow inactivation and modulation of electrical excitability by Lacosamide were evaluated using stem cell-derived nociceptors from an SFN patient bearing a Nav1.9 variant.
Discussion:
This study demonstrates that the mechanosensitivity of Navs and their subunits is subtype-specific. The study shows that Navs mechanosensitivity is differentially modulated by distinct β subunits and that one β subunit may have specific effects in modulating the mechanosensitivity of different Navs subtypes.
These results provide insights into the role of Nav in the mechanosensitive regulation of cardiac function through mechano-electric feedback. The β1 and β3 subunits are differentially expressed in myofibroblasts and cardiomyocytes and are also expressed in different cellular compartments. This differential expression pattern offers a perspective on how electrical excitability is modulated mechanically by the β3 subunit at the plasma membrane and by β1 at intercalated disks and in myofibroblasts.
The investigation of electrophysiological properties of hiPSCs-derived midbrain dopaminergic neurons and nociceptors showed that the obtained neurons are electrically active. Furthermore, no alteration in slow inactivation potentially contributing to the patient’s pain phenotype was detected in nociceptors derived from the SFN patient’s hiPSCs. Using multi-electrode-array, I show that Lacosamide normalizes the altered electrical activity observed in the patient’s nociceptors
Einfluss verschiedener T-Zell Subtypen auf die α-Synuklein-Aggregation beim idiopathischen Parkinson Syndrom
Get PDFRole of T cell subsets in α-synuclein aggregation in Parkinson’s disease
Background and Aims
Parkinson’s disease is the second most common neurodegenerative disease
after Alzheimer’s disease. Its pathological hallmarks are neurodegeneration in
the substantia nigra and the resulting loss of dopaminergic neurons in addition
to α-synuclein aggregation being neurotoxic. In the last decades,
neuroinflammation gained attention as potential mechanism for progression of
this devastating disease. Thus, the innate and the adaptive immune systems are
claimed to play a crucial role in Parkinson’s disease pathogenesis. However,
which lymphocyte subset is involved in pathomechanisms and whether they
interplay with microglia, remains elusive. In the present thesis, I investigated the
influence of lymphocyte subtypes (T and B cells) on α-synuclein aggregation in
mouse models of Parkinson`s disease.
Methods
Mice overexpressing human α-synuclein under the murine Thy1 promoter were
crossed with those lacking CD8+ T cells, B cells or αβ T cells to investigate,
which lymphocyte subset influences α-synuclein aggregation. To characterize α-
synuclein aggregation, I performed immunohistochemistry (IHC) for human α-
synuclein (15G7), Western blot (WB) analysis, solubility assay and size
exclusion chromatography (SEC). These results were compared within different
mouse groups. Additionally, I used immunofluorescence for microglia and human
α-synuclein to investigate the role of lymphocytes on microglia phagocytosis of
α-synuclein aggregates.
Results
CD8+ T cells, B cells, and αβ T cells were lacking any influence on α-synuclein
aggregation rate and species. Moreover, microglia activation and phagocytosis activity were not altered in mice lacking CD8+ T cells and B cells, while αβ T
cells were not further investigated.
Discussion
CD8+ T cells, B cells, and αβ T cells showed no influence on α-synuclein
aggregation rate and species composition. However, a complete knockout of
mature lymphocytes leads to a reduction of α-synuclein aggregation in the
substantia nigra. This indicates, that since CD8+ T cells, B cells, and αβ T cells
have no influence on α-synuclein aggregation, this reduction may be caused by
γδ T cells or Natural Killer (NK) T cells. A previous study on human-derived stem
cells showed an influence of interleukin 17 (IL-17)-producing T cells on
Parkinson’s disease neurons. Since γδ T cells and NK T cells are secreting IL-
17, this result may indicate that γδ T cells play a role in Parkinson’s disease
pathology and might thus pave the path for new potential therapeutic targets.Einfluss verschiedener T-Zell Subtypen auf die α-Synuklein-Aggregation beim
idiopathischen Parkinson Syndrom
Hintergrund und Ziele
Das idiopathische Parkinson Syndrom ist nach der Alzheimer Demenz die
zweithäufigste neurodegenerative Erkrankung. Seine pathologischen Merkmale
sind die Neurodegeneration in der Substantia Nigra und der daraus resultierende
Verlust von dopaminergen Neuronen, sowie die neurotoxische Aggregation von
α-Synuklein. In den letzten Jahrzehnten hat die Neuroinflammation als
potenzieller Mechanismus für das Fortschreiten dieser verheerenden
Erkrankung an Bedeutung gewonnen. Daher wird angenommen, dass das
angeborene und das adaptive Immunsystem eine entscheidende Rolle bei der
Pathogenese des idiopathischen Parkinson Syndroms spielen. Welcher
Lymphozyten-Subtyp an diesem Pathomechanismus beteiligt ist und welche
Rolle die Mikroglia spielen, bleibt jedoch unklar. In der vorliegenden Arbeit habe
ich den Einfluss von Lymphozyten-Subtypen (T- und B-Zellen) auf die α-
Synuklein-Aggregation in Mausmodellen des Parkinson Syndroms untersucht.
Methoden
Mäuse, die humanes α-Synuklein unter dem murinen Thy1-Promotor
überexprimieren wurden mit solchen, denen CD8+ T-Zellen, B-Zellen und αβ TZellen
fehlten, gekreuzt, um zu untersuchen, welche Lymphozyten-Untergruppe
die α-Synuklein-Aggregation beeinflusst. Um die α-Synuklein-Aggregation zu
charakterisieren, führte ich eine Immunhistochemie (IHC) für humanes α-
Synuklein (15G7), eine Western-Blot (WB)-Analyse, einen Löslichkeitstest und
eine Größenausschlusschromatographie (SEC) durch. Diese Ergebnisse wurden
innerhalb der verschiedenen Maus-Gruppen verglichen. Zusätzlich verwendete
ich Immunfluoreszenz für Mikroglia und humanes α-Synuklein, um den Einfluss der Lymphozyten-Untergruppen auf Mikroglia-Phagozytose der α-Synuklein-
Aggregate zu untersuchen.
Ergebnisse und Beobachtungen
CD8+ T-Zellen, B-Zellen und αβ T-Zellen zeigten keinen Einfluss auf die
Aggregationsrate und die Aggregationsspezien von α-Synuklein. Darüber hinaus
wurden die Mikroglia-Aktivierung und die Phagozytoseaktivität in Abwesenheit
von CD8+ T-Zellen und B-Zellen nicht verändert, während αβT-Zellen nicht auf
unterschiedliche Mikrogliaaktivierungen untersucht wurden.
Schlussfolgerungen
CD8+ T-Zellen, B-Zellen und αβ T-Zellen zeigten keinen Einfluss auf die
Aggregationsrate und die Aggregationsspezien von α-Synuklein. Das vollständige
Fehlen reifer Lymphozyten führte jedoch zu einer Verringerung der α-Synuklein-
Aggregation in der Substantia Nigra. Dies lässt annehmen, dass diese Reduktion
durch γδ T-Zellen oder Natürliche-Killer-T-Zellen (NK T-Zellen) verursacht werden
könnte. Eine frühere Studie an humanen Stammzellen zeigte einen Einfluss von
IL17-produzierenden T-Zellen auf die Neurone von Parkinson-Patienten. Da γδ TZellen
und NK T-Zellen Interleukin 17 (IL-17) sezernieren, könnte dieses Ergebnis
darauf hindeuten, dass γδ T-Zellen oder NK T-Zellen eine Rolle bei der Pathologie
des idiopathischen Parkinson Syndroms spielen und somit den Weg für neue
potenzielle therapeutische Angriffspunkte ebnen
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