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Megakaryocytes (MKs) are the largest cells of the hematopoietic system and the precursor cells of platelets. During proplatelet formation (PPF) bone marrow (BM) MKs extent large cytoplasmic protrusions into the lumen of sinusoidal blood vessels. Under homeostatic conditions PPF occurs exclusively in the direction of the sinusoid, while platelet generation into the marrow cavity is prevented. So far, the mechanisms regulating this process in vivo are still not completely understood, especially when PPF is deregulated during disease. This thesis investigated the mechanisms of PPF in native BM and after myeloablation by total body irradiation (TBI).
First, we have identified a specialized type of BM stromal cells, so called CXCL12-abundant reticular (CAR) cells, as novel possible regulators of PPF. By using complementary high-resolution microscopy techniques, we have studied the morphogenetic events at the MK/vessel wall interface in new detail, demonstrating that PPF formation preferentially occurs at CAR cell-free sites at the endothelium.
In the second part of this thesis, we analyzed the processes leading to BM remodeling in response to myeloablation by TBI. We used confocal laser scanning microscopy (CLSM) to study the kinetic of radiation-triggered vasodilation and mapped extracellular matrix (ECM) proteins after TBI. We could demonstrate that collagen type IV and laminin α5 are specifically degraded at BM sinusoids. At the radiation-injured vessel wall we observed ectopic release of platelet-like particles into the marrow cavity concomitantly to aberrant CAR cell morphology, suggesting that the balance of factors regulating PPF is disturbed after TBI. ECM proteolysis is predominantly mediated by the matrix metalloproteinase MMP9, as revealed by gelatin-zymography and by a newly established BM in situ zymography technique. In transgenic mice lacking MMP9 vascular recovery was delayed, hinting towards a role of MMP9 in vessel reconstitution after myeloablation.
In a third series of experiments, we studied the irradiated BM in the context of hematopoietic stem cell transplantation (HSCT). By using mice as BM donors that ubiquitously express the fluorescent reporter protein dsRed we tracked engraftment of donor cells and especially MKs in the recipient BM. We found a distinct engraftment pattern and cluster formation for MKs, which is different from other blood cell lineages.
Finally, we assessed platelet function after TBI and HSCT and were the first to demonstrate that platelets become massively hyporeactive, particularly upon stimulation of the collagen receptor GPVI.
In summary, our findings shed light on the processes of PPF during health and disease which will help to develop treatments for aberrant thrombopoiesis.
While the healthy brain works through balanced synaptic communication between
glutamatergic and GABAergic neurons to coordinate excitation (E) and inhibition (I), disruption
of E/I balance interferes with synaptic communication, information processing, and ultimately
cognition. Multiple line of evidence indicates that E/I imbalance represents the
pathophysiological basis of a wide spectrum of mental disorders. Genetic screening
approaches have identified Cadherin-13 (CDH13). as a risk gene across neurodevelopmental
and mental disorders. CDH13 regulates several cellular and synaptic processes in brain
development and neuronal plasticity in adulthood. In addition to other functions, it is specifically
localized at inhibitory synapses of parvalbumin- and somatostatin-expressing GABAergic
neurons. In support of CDH13’s function in moderating E/I balance, electrophysiological
recordings of hippocampal slices in a CDH13-deficient mouse model revealed an increase in
basal inhibitory but not excitatory synaptic transmission. Moreover, the search for genetic
variants impacting functional expression of the CDH13 gene identified SNP (single nucleotide
polymorphism)) rs2199430 in intron 1 to be associated with differential mRNA concentrations
in human post-mortem brain across the three genotypes CDH13G/G, CDH13A/G and CDH13A/A
.
This work therefore aimed to further validate these findings in a complementary human model
by using induced pluripotent stem cells (iPSCs). The application of human iPSCs in research
has replaced the use of embryonic cells, resolving the ethical conflict of destructive usage of
human embryos. Investigating CDH13’s mode of action in inhibitory synapses was predicted
to facilitate mechanistic insight into the effects of CDH13 gene variants on E/I network activity,
which can then be targeted to reinstate balance.
Genome-wide association studies have identified rare copy number variants (CNVs) resulting
in a deletion (or duplication) of CDH13. To reduce genetic background variance, a set of
isogenic iPSC lines with a gene dose-dependent deficiency of CDH13 (CDH13-/- and CDH13+/-
) was generated by using the Clustered Regulatory Interspaced Short Palindromic
Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. These CRISPRed iPSCs
carrying a single or two allele(s) with CDH13 inactivation facilitate investigation of CDH13
function in cellular processes, at inhibitory synapses and in neuronal network activity. In
addition, iPSCs carrying allelic SNP rs2199430 variants were used to study the effects of
common genetic variation of CDH13. These cell lines were differentiated into pure
glutamatergic and GABAergic neurons and co-cultured to generate neuronal networks allowing
its activity to be measured and correlated with electrophysiological signatures of differential
CDH13 genotypes. The work towards assessment of neuronal network activity of the iPSC
lines was subdivided into three major steps: first, generating rtTA/Ngn2 and rtTA/Ascl1-positive
iPSCs via a lentivirus-mediated approach; second, differentiating pure glutamatergic and
GABAergic neurons from the genetically transduced iPSCs and co-culturing of pure
glutamatergic and GABAergic neurons in a pre-established ratio (65:35) by direct
differentiation upon supplementation with doxycycline and forskolin on a microelectrode array
(MEA) chip; and, finally, recording of neuronal network activity of iPSC lines after 49 days in
vitro, followed by extraction and analyses of multiple MEA parameters.
x
Based on the MEA parameters, it was confirmed that complete CDH13 knockout as well as
heterozygous deficiency influence E/I balance by increasing inhibition. It was further revealed
that common SNP variation alters the signature of neuronal network activity. Specifically,
CDH13 deficiency resulted in a significant reduction in network burst duration (NBD), reduced
number of detected spikes within a network burst and reduction in network burst rate (NBR)
compared to the control (CDH13G/G). CDH13A/G and CDH13A/A showed similarities with the
CRISPRed CDH13-deficient networks by showing a significant reduction in the NBD and a
reduced number of detected spikes within a network compared to CDH13G/G. Strikingly. there
was a significant increase in the NBR of the CDH13A/G and CDH13A/A compared to CDH13G/G
networks. CDH13A/G networks exhibited significant differences in both parameters. At the
cellular level, this indicates that signalling pathways which determine the length and frequency
of network bursts differ among allelic variants of SNP rs2199430, thus confirming functional
relevance of this intronic SNP.
In summary, CDH13-deficient isogenic iPSC lines were generated using CRISPR/Cas9, iPSCs
were genetically transduced via a lentivirus approach, direct differentiation of
glutamatergic/GABAergic neurons derived from transduced iPSCs were used to establish a
scalable co-culture system, and network activity was recorded by MEA using pre-established
parameters to extract and analyze activity information. The results indicate that iPSC-derived
neuronal networks following CRISPR/Cas9-facilitated CDH13 inactivation, as well as networks
with allelic SNP variants of CDH13, moderate E/I balance, thus advancing understanding of
CDH13 function at inhibitory synapses and elucidating the effects of rare and common CDH13
gene variation.
Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.
RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease “Neurodevelopmental Disorder with severe Motor Impairment and Absent Language” (NEDMIAL;OMIM#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3’ → 5’ RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome’s mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5’TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.
Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets.
Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin.
In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.
Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research.
Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition.
Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 %, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 %. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments.
Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation.
As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.
Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin (‘ubiquitylation’), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.
This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.
Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.
∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially
contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the
expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been
extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to
maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer
resistance toward therapies inducing DNA damage.
SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been
treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited,
and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities,
the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a
potential alternative to improve the therapeutic response and clinical outcomes of SCC patients.
However, ∆Np63 is considered “undruggable.” As is commonly observed in transcription factors, ∆Np63 does
not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of
different pathways and cellular processes, making it difficult to counteract its function by targeting
downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin–proteasome system (UPS), the
development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target
∆Np63 in SCC treatment.
This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability,
and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to
maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time,
∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of
USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in
preclinical mouse models.
Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic
alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented
within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance,
thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28
inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such
as Cisplatin.