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Chronic pain conditions are a major reason for the utilization of the health care system. Inflammatory pain states can persist facilitated by peripheral sensitization of nociceptors. The voltage-gated sodium channel 1.9 (NaV1.9) is an important regulator of neuronal excitability and is involved in inflammation-induced pain hypersensitivity. Recently, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (OxPAPC) was identified as a mediator of acute inflammatory pain and persistent hyperalgesia, suggesting an involvement in proalgesic cascades and peripheral sensitization. Peripheral sensitization implies an increase in neuronal excitability. This thesis aims to characterize spontaneous calcium activity in neuronal compartments as a proxy to investigate neuronal excitability, making use of the computational tool Neural Activity Cubic (NA3). NA3 allows automated calcium activity event detection of signal-close-to-noise calcium activity and evaluation of neuronal activity states. Additionally, the influence of OxPAPC and NaV1.9 on the excitability of murine dorsal root ganglion (DRG) neurons and the effect of OxPAPC on the response of DRG neurons towards other inflammatory mediators (prostaglandin E2, histamine, and bradykinin) is investigated. Using calcium imaging, the presence of spontaneous calcium activity in murine DRG neurons was established. NA3 was used to quantify this spontaneous calcium activity, which revealed decreased activity counts in axons and somata of NaV1.9 knockout (KO) neurons compared to wildtype (WT). Incubation of WT DRG neurons with OxPAPC before calcium imaging did not show altered activity counts compared to controls. OxPAPC incubation also did not modify the response of DRG neurons treated with inflammatory mediators. However, the variance ratio computed by NA3 conclusively allowed to determine neuronal activity states. In conclusion, my findings indicate an important function of NaV1.9 in determining the neuronal excitability of DRG neurons in resting states. OxPAPC exposition does not influence neuronal excitability nor sensitizes neurons for other inflammatory mediators. This evidence reduces the primary mechanism of OxPAPC-induced hyperalgesia to acute effects. Importantly, it was possible to establish an approach for unbiased excitability quantification of DRG neurons by calcium activity event detection and calcium trace variance analysis by NA3. It was possible to show that signal-close-to-noise calcium activity reflects neuronal excitability states.
Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
Spinal muscular atrophy and amyotrophic lateral sclerosis are the two most common devastating motoneuron diseases. The mechanisms leading to motoneuron degeneration are not resolved so far, although different hypotheses have been built on existing data. One possible mechanism is disturbed axonal transport of RNAs in the affected motoneurons. The underlying question of this study was therefore to characterize changes in transcript levels of distinct RNAs in cell culture models of spinal muscular atrophy and amyotrophic lateral sclerosis, especially in the axonal compartment of primary motoneurons.
To investigate this in detail we first established compartmentalized cultures of Primary mouse motoneurons. Subsequently, total RNA of both compartments was extracted
separately and either linearly amplified and subjected to microarray profiling or whole transcriptome amplification followed by RNA-Sequencing was performed. To make
the whole transcriptome amplification method suitable for compartmentalized cultures, we adapted a double-random priming strategy. First, we applied this method
for initial optimization onto serial dilutions of spinal cord RNA and later on to the compartmentalized motoneurons.
Analysis of the data obtained from wildtype cultures already revealed interesting results. First, the RNA composition of axons turned out to be highly similar to the somatodendritic compartment. Second, axons seem to be particularly enriched for transcripts related to protein synthesis and energy production. In a next step we
repeated the experiments by using knockdown cultures. The proteins depleted hereby are Smn, Tdp-43 and hnRNP R. Another experiment was performed by knocking down the non-coding RNA 7SK, the main interacting RNA of hnRNP R.
Depletion of Smn led to a vast number of deregulated transcripts in the axonal and somatodendritic compartment. Transcripts downregulated in the axons upon Smn depletion were especially enriched for GOterms related to RNA processing and encode proteins located in neuron projections including axons and growth cones.
Strinkingly, among the upregulated transcripts in the somatodendritic compartment we mainly found MHC class I transcripts suggesting a potential neuroprotective role.
In contrast, although knockdown of Tdp-43 also revealed a large number of downregulated transcripts in the axonal compartment, these transcripts were mainly
associated with functions in transcriptional regulation and RNA splicing. For the hnRNP R knockdown our results were again different. Here, we observed
downregulated transcripts in the axonal compartment mainly associated with regulation of synaptic transmission and nerve impulses. Interestingly, a comparison between deregulated transcripts in the axonal compartment of both hnRNP R and 7SK knockdown presented a significant overlap of several transcripts suggesting
some common mechanism for both knockdowns.
Thus, our data indicate that a loss of disease-associated proteins involved in axonal RNA transport causes distinct transcriptome alterations in motor axons.
Motoneurons played an essential role in establishing the concept of target-mediated support of innervating neurons. However, it took several decades until molecules were identined which trophically support motoneurons in vitro and in vivo. The most potent molecule identined so far is ciliary neurotrophic factor (CNTF). It is expressed as a cytosolic molecule in myelinating Schwann cells rather than in skeletal muscle in the postnatal period and therefore does not qualify as a target-derived neurotrophic factor regulating motoneuron survival during embryonic development. However, the inactivation of CNTF by gene targeting experiments results in progressive atrophy and degeneration of motoneurons, demonstrating that CNTF plays an essential role as a maintenance factor for motoneurons postnatally. Secretory molecules which are expressed in skeletal muscle during embryonic development and which support motoneurons in culture and partially also in vivo include members of the NGF gene family (BDNF, NT-3, NT-4/S) , FGF-S, IGF-I, and UF. The evaluation of the physiological importance of these molecules is under investigation.
The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.
In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today.
In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP?
Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process.
In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription.
The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia
(2019)
Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α‐helix 5, presumably disrupting the integrity of this α‐helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild‐type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non‐symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild‐type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α‐helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium‐binding site of the transporter.