91 research outputs found
Modeling of FUS- and C9ORF72-associated cortical neuropathology using patient-specific induced pluripotent stem cells
Amyotrophe Lateralsklerose (ALS) ist eine neurodegenerative Erkrankung, bei welcher speziell erste (kortikospinal) und zweite (spinal) Motorneurone (MN) von Neurodegeneration betroffen sind. Gegenwärtig bleibt ALS eine unheilbare Erkrankung. Der Tod tritt durchschnittlich 2 bis 5 Jahre nach Auftreten der Symptome ein. Circa 90% der Fälle treten sporadisch auf (sALS), während 10% familiär sind (fALS). Es ist von großem Interesse monogenetische Formen der fALS zu untersuchen um zugrundeliegende Pathologien und Mechanismen zu verstehen. Bislang wurden über 20 Gene mit ALS in Verbindung gebracht, einschließlich Fused in sarcoma (FUS) und Chromsosome 9 open reading frame (C9ORF72). Circa 4% der fALS Fälle sind durch dominante Mutationen in FUS verursacht und repräsentieren damit die dritthäufigste Form der fALS in Deutschland. Die G4C2 hexanucleotide repeat expansion (HRE) in C9ORF72 ist die häufigste Ursache für ALS und Frontotemporale Demenz (FTD). ALS Patienten unterscheiden sich erheblich in der Präsentation ihrer klinischen Symptome wie Ausbruchsort, Progressionsrate und Auftreten kognitiver Störungen. Diese Faktoren sind auch stark abhängig von der zugrundeliegenden Mutation in fALS. Ziel dieser Doktorarbeit ist die Modellierung von FUS- und C9ORF72-assozierter ALS in einem krankheits-relevanten in vitro Model von speziell kortikaler Neuropathologie mit Hilfe von Patienten-spezifischen iPSZs. Die Hypothese der vorliegenden Arbeit ist das in einer Zelltyp-abhängigen Art und Weise zugrundeliegende Erkrankungsmechanismen in kortikalen vs. spinalen Neuronen unterschiedlich betroffen sind. Humane iPSZ, generiert von gesunden Kontrollen und ALS Patienten mit FUS oder C9ORF72 Mutation, wurden für die gerichtete kortikale und spinale Differenzierung genutzt. Zusätzlich wurden zwei neue FUS-WT- und FUS-P525L-EGFP-markierte isogene Linien mittels CRISPR/Cas9n Technik generiert. Methoden basierend auf Immunfluoreszenz Färbungen und Lebendzell-Mikroskopie wurden angewendet um Krankheits-relevante Proteine, DNA Schäden und axonale Organell-Mobilität zu analysieren. In diesem Projekt konnte ein deutlicher Zelltyp-abhängiger Effekt auf analysierte Phänotypen beobachtet werden, während ALS-assoziierte Mutationen scheinbar nur geringfügige Effekte zeigten. Dementsprechend wurde ein Zelltyp-abhängiger Anstieg des basalen DNA Schadens in kortikalen Astrozyten vs. Neuronen und spinalen vs. kortikalen Neuronen detektiert. Jedoch konnte in FUS oder C9ORF72 mutierten kortikalen Zellen kein erhöhter DNA Schaden nachgewiesen werden, wie es zuvor in spinalen MN beobachtet wurde. Des Weiteren beeinflussen FUS Mutationen die Rekrutierung von FUS zu DNA-geschädigten Stellen, die Organell-Mobilität und die zytoplasmatische Fehllokalisation des Proteins in Abhängigkeit vom Zelltyp. In kortikalen Neuronen wurde in Bezug auf die Rekrutierung von mutiertem FUS und Organell-Mobilität nur leichte Mutations-abhängige und wesentlich schwächer ausgeprägte Effekte beobachtet als in spinalen MN. Zusammenfassend kann gesagt werden, dass Patienten-spezifische Zellmodelle ein wichtiges Instrument in der ALS Forschung sind und das vor allem Unterschiede zwischen kortikalen und spinalen MN weiter untersucht werden müssen, um zugrundeliegende Krankheits-relevante Mechanismen zu entschlüsseln und wie diese zum Fortschreiten der Erkrankung beitragenAmyotrophic lateral sclerosis (ALS) is a of neurodegenerative diseases, in which neurodegeneration specifically affects upper (corticospinal) and lower (spinal) motor neurons (MNs). At present, ALS remains an incurable disease. Death occurs on average 2 to 5 years after symptom onset. About 90% are sporadic cases (sALS) and 10% are familial cases (fALS). It is of great interest to investigate monogenetic forms causing fALS to understand its underlying disease pathologies and mechanisms. Over 20 genes have been linked to ALS until now, including Fused in sarcoma (FUS) and Chromosome 9 open reading frame 72 (C9ORF72). About 4% of fALS cases are caused by dominant mutations within FUS, representing the third most common fALS form in Germany. The G4C2 hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause for ALS and Frontotemporal dementia (FTD). ALS patients differ significantly in their presentation of clinical symptoms, including site of onset, rate of progression, and presence of cognitive dysfunction. Those factors were also shown to highly depend on the underlying mutation in fALS cases. Aim of this thesis work is the modeling of FUS- and C9ORF72-associated ALS in a disease-related in vitro model of particularly cortical neuropathology using patient-derived iPSCs. The hypothesis of the current work is that underlying disease mechanisms do differentially affect cortical vs. spinal neurons and act in a cell type-dependent manner. Human iPSCs derived from healthy controls and ALS patients carrying mutations within FUS or C9ORF72 were used for directed cortical and spinal differentiation. Additionally, two new FUS-WT- and FUS-P525L-EGFP-tagged isogenic iPSC lines were generated by CRISPR/Cas9n gene editing. Immunofluorescence staining and live cell imaging approaches were implemented to analyze disease-associated proteins, DNA damage, and axonal trafficking. Within this project, a clear cell type-dependent effect on analyzed phenotypes was observed, while ALS-associated mutations seemed to have only minor effects. Accordingly, cell type-dependent increased basal DNA damage levels in cortical astrocytes vs. neurons and spinal vs. cortical neurons were detected. However, FUS or C9ORF72 mutant cortical cells do not recapitulate increased DNA damage levels as they have been observed in spinal MNs. Furthermore, FUS mutation affected recruitment to DNA damage sites, axonal trafficking, and cytoplasmic mislocalization differentially, depending on the analyzed cell type. In cortical neurons, recruitment and trafficking of mutant FUS showed only slight mutation-dependent effects and also less pronounced phenotypes than observed in spinal MNs. In conclusion, patient-specific cellular models are an important tool in ALS research and particularly differences between cortical and spinal MNs need to be further investigated to decipher underlying disease mechanisms, the interplay of cell types affected by the disease, and how they participate in disease progression
Intestinal Acid Sphingomyelinase Protects From Severe Pathogen-Driven Colitis
Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory Th1 and Th17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium
NMR metabolomics highlights sphingosine kinase-1 as a new molecular switch in the orchestration of aberrant metabolic phenotype in cancer cells
Sphingosine 1-phosphate modulates antigen capture by murine langerhans cells via the S1P2 receptor subtype
Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions
Poly-glycine-alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine-arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine-alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB
Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice
Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance
C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.
Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics
Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation
Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS
Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS-associated amyotrophic lateral sclerosis
Signaling of Sphingosine-1-Phosphate and its influence on endocytotic capacity of dendritic cells.
Sphingosin-1-Phosphat (S1P) stellt einen neuen biologischen Mediator dar, für
den eine multilaterale Modulation vieler immunologischer Mechanismen bereits
nachgewiesen werden konnte. In diesem Zusammenhang führt die topische
Applikation von S1P in einem Tiermodell der experimentellen Atopischen
Dermatitis zu einer abgeschwächten Hautentzündungsreaktion. Im Rahmen dieser
Arbeit kann zum ersten Mal eine Modulation der dendritischen Zellen (DC`s)
durch S1P nachgewiesen werden, die bei einer topischen Behandlung zu einer
anti-inflammatorischen Wirkung beiträgt. Diese beruht auf einer Beeinflussung
der essentiellen Phase der immunologischen DC-Aktivierung, die durch die
Endozytose von Antigenen geprägt ist. Tatsächlich kann eine
konzentrationsabhängige Reduktion der endozytotischen Kapazität in unreifen
DC`s durch S1P ermittelt werden. In der vorliegenden Arbeit war es zudem von
großem Interesse die molekularen Mechanismen aufzuklären, die zu der
entsprechenden Reduktion der Endozytose durch S1P führen. Für die Aufnahme von
Antigenen durch die unreifen DC`s aus ihrer unmittelbaren Umgebung stehen
mehrere Endozytosemechanismen zur Verfügung. Tatsächlich kann für S1P die
Reduktion der unspezifischen Flüssigkeitsaufnahme und der darin gelösten
Antigene bzw. der Makropinozytose nachgewiesen werden. Die Untersuchung des
dafür verantwortlichen Signalwegs zeigt die bereits etablierte Regulation der
Phosphoinositid 3-Kinase-Aktivität als Ursache für das veränderte
Endozytoseverhalten der unreifen DC`s, welche durch die Stimulation des
S1P2-Rezeptorsubtyps bewerkstelligt wird. Darüber hinaus liefert die
Erniedrigung der S1P2-Rezeptorexpression einen Hinweis auf eine endogene
Regulation der Endozytose durch S1P in unreifen DC`s. So kann im Zuge dieser
Arbeit ein autokriner Mechanismus unter Beteiligung des Lipidmediators in den
DC`s postuliert werden. Dabei zeigt die Steigerung der endogenen S1P-Synthese
mittels pharmakologischer Sphingosinkinase1-Aktivierung die erwartete
Reduktion der endozytotischen Kapazität. Die Analyse des für die
transmembranäre Translokation von S1P verantwortlichen Mechanismus kann
ebenfalls näher identifiziert werden. Der Einsatz verschiedener, spezifischer
Hemmstoffe bestätigt die Einbindung des ABCC1-Transporters. Die vorliegenden
Untersuchungen zeigen somit eine essentielle Rolle von S1P bei der Endozytose
der unreifen DC`s. Da die Antigenaufnahme den Initialschritt in der
Immunkaskade darstellt, kann die Aufklärung der damit verbundenen Signalwege
zur Etablierung neuer Therapien entzündlicher Erkrankungen wie der Atopischen
Dermatitis maßgeblich beitragen.The lipid mediator sphingosine 1-phosphate (S1P) has been identified as a new
biological molecule that is involved in the modulation of multilateral
immunological processes. In this context, topically administrated S1P inhibits
the inflammatory reaction in an animal model of atopic dermatitis.
Furthermore, for the first time the present work supplies clear evidence that
a modulation of dendritic cell (DC) function contributes to the observed anti-
inflammatory effect of local S1P application. The presence of S1P influences
the essential step of DC activation, which is characterized by endocytosis of
antigens. Stimulation of immature DC`s with S1P demonstrates a dose dependant
reduction of antigen capture by these antigen presenting cells. In the present
work, it was of great interest to specify the molecular mechanisms that lead
to the S1P-induced reduction of endocytosis in immature skin DC`s. In an
immature stage, DC`s are able to take up antigens via several different
mechanisms. An examination of a mechanism affected by S1P indicated that this
sphingolipid inhibits the screening of large volumes of fluid for antigens
during macropinocytosis in DC`s. The present study shows that macropinocytosis
is mediated by modulation of the PI3K activity, allowing a fine-tuned
regulation of antigen capture. Furthermore, the present work indicates that
S1P is able to reduce PI3K activity via the S1P2 receptor subtype. Most
interestingly, down regulation of S1P2 receptor subtype not only prevents the
inhibitory effect of S1P on antigen uptake but also increases the basal level
of macropinocytotic capacity. These results provide evidence that S1P could be
involved in the endogenous regulation of endocytosis in immature DC`s. This
hypothesis has been substantiated by enhancing the endogenous biosynthesis of
S1P using a direct inducer of SphK1 activity. As expected, observed reduction
of endocytosis by DC`s in the presence of the SphK1 activator was accompanied
by diminished phosphoinositid 3-kinase activity. In agreement with previous
studies the present work provides evidence, that an ABCC1 transporter is
involved in the secretion of the sphingolipid. In summary, the present work
demonstrates an essential role of S1P in antigen capture by immature DC`s.
Endocytosis is the initial step of the adaptive immune response. Thus,
elucidation of related signalling pathways could significantly contribute to
the establishment of new treatments for inflammatory diseases such as atopic
dermatitis
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