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Because of its complexity and intricacy, studying the nervous system is often challenging. Fortunately, the small nematode roundworm Caenorhabditis elegans is well established as a model system for basic neurobiological research. The C. elegans model is also the only organism with a supposedly complete connectome, an organism-wide map of synaptic connectivity resolved by electron microscopy, which provides some understanding of how the nervous system works as a whole. However, the number of available data-sets is small and the connectome contains errors and gaps. One example of this concerns electrical synapses. Electrical synapses are formed by gap junctions and difficult to map due to their often ambiguous morphology in electron micrographs, leading to misclassification or omission. On the other hand, chemical synapses are more easily mapped, but many aspects of their mode of operation remain elusive and their role in the C. elegans connectome is oversimplified. A comprehensive understanding of signal transduction of neurons between each other and other cells will be indispensable for a comprehensive understanding of the nervous system. In this thesis, I approach these challenges with a combination of advanced light and electron microscopy techniques.
First, this thesis describes a strategy to increase synaptic specificity in connectomics. Specifically, I classify gap junctions with a high degree of confidence. To achieve this, I utilized array tomography (AT). In this thesis, AT is adapted for high-pressure freezing to optimize for structure preservation and for super-resolution light microscopy; in this manner, I aim to bridge the gap between light and electron microscopy resolutions. I call this adaptation super-resolution array tomography (srAT). The srAT approach made it possible to clearly identify and map gap junctions with high precision and accuracy. The results from this study showcased the feasibility of incorporating electrical synapses into connectomes in a systematic manner, and subsequent studies have used srAT for other models and questions.
As mentioned above, the C. elegans connectomic model suffers from a shortage of datasets. For most larval stages, including the special dauer larval stage, connectome data is completely missing up to now. To obtain the first partial connectome data-set of the C. elegans dauer larva, we used focused ion-beam scanning electron microscopy (FIB-SEM). This technique offers an excellent axial resolution and is useful for acquiring large volumes for connectomics. Together with our collaborators, I acquired several data-sets which enable the analysis of dauer stage-specific “re-wiring” of the nervous system and thus offer valuable insights into connectome plasticity/variability.
While chemical synapses are easy to map relative to electrical synapses, signal transduction via chemical transmitters requires a large number of different proteins and molecular processes acting in conjunction in a highly constricted space. Because of the small spatial scale of the synapse, investigating protein function requires very high resolution, which electron tomography provides. I analyzed electron tomograms of a worm-line with a mutant synaptic protein, the serine/threonine kinase SAD-1, and found remarkable alterations in several architectural features. My results confirm and re-contextualize previous findings and provide new insight into the functions of this protein at the chemical synapse.
Finally, I investigated the effectiveness of our methods on “malfunctioning,” synapses, using an amyotrophic lateral sclerosis (ALS) model. In the putative synaptopathy ALS, the mechanisms of motor neuron death are mostly unknown. However, mutations in the gene FUS (Fused in Sarcoma) are one known cause of the disease. The expression of the mutated human FUS in C. elegans was recently shown to produce an ALS-like phenotype in the worms, rendering C. elegans an attractive disease model for ALS. Together with our collaboration partners, I applied both srAT and electron tomography methods to “ALS worms” and found effects on vesicle docking. These findings help to explain electrophysiological recordings that revealed a decrease in frequency of mini excitatory synaptic currents, but not amplitudes, in ALS worms compared to controls. In addition, synaptic endosomes appeared larger and contained electron-dense filaments in our tomograms. These results substantiate the idea that mutated FUS impairs vesicle docking and also offer new insights into further molecular mechanisms of disease development in FUS-dependent ALS. Furthermore, we demonstrated the broader applicability of our methods by successfully using them on cultured mouse motor neurons.
Overall, using the C. elegans model and a combination of light and electron microscopy methods, this thesis helps to elucidate the structure and function of neuronal synapses, towards the aim of obtaining a comprehensive model of the nervous system.
Motor neuron diseases (MNDs) encompass a variety of clinically and genetically heterogeneous disorders, which lead to the degeneration of motor neurons (MNs) and impaired motor functions. MNs coordinate and control movement by transmitting their signal to a target muscle cell. The synaptic endings of the MN axon and the contact site of the muscle cell thereby form the presynaptic and postsynaptic structures of the neuromuscular junction (NMJ). In MNDs, synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is an early target in the pathophysiological cascade leading to MN death. In this study, we established new experimental strategies to analyze human MNDs by patient derived induced pluripotent stem cells (iPSCs) and investigated pathophysiological mechanisms in two different MNDs.
To study human MNDs, specialized cell culture systems that enable the connection of MNs to their target muscle cells are required to allow the formation of NMJs. In the first part of this study, we established and validated a human neuromuscular co-culture system consisting of iPSC derived MNs and 3D skeletal muscle tissue derived from myoblasts. We generated 3D muscle tissue by culturing primary myoblasts in a defined extracellular matrix in self-microfabricated silicone dishes that support the 3D tissue formation. Subsequently, iPSCs from healthy donors and iPSCs from patients with the progressive MND Amyotrophic Lateral Sclerosis (ALS) were differentiated into MNs and used for 3D neuromuscular co-cultures. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the functionality of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of ALS and found a decrease in neuromuscular coupling, muscle contraction, and axonal outgrowth in co-cultures with MNs harboring ALS-linked superoxide dismutase 1 (SOD1) mutation. In summary, this co-culture system presents a human model for MNDs that can recapitulate aspects of ALS pathophysiology.
In the second part of this study, we identified an impaired unconventional protein secretion (UPS) of Sod1 as pathological mechanisms in Pleckstrin homology domain-containing family G member 5 (Plekhg5)-associated MND. Sod1 is a leaderless cytosolic protein which is secreted in an autophagy-dependent manner. We found that Plekhg5 depletion in primary MNs and NSC34 cells leads to an impaired secretion of wildtype Sod1, indicating that Plekhg5 drives the UPS of Sod1 in vitro. By interfering with different steps during the biogenesis of autophagosomes, we could show that Plekhg5-regulated Sod1 secretion is determined by autophagy. To analyze our findings in a clinically more relevant model we utilized human iPSC MNs from healthy donors and ALS patients with SOD1 mutations. We observed reduced SOD1 secretion in ALS MNs which coincides with reduced protein expression of PLEKHG5 compared to healthy and isogenic control MNs. To confirm this correlation, we depleted PLEKHG5 in control MNs and found reduced extracellular SOD1 levels, implying that SOD1 secretion depends on PLEKHG5. In summary, we found that Plekh5 regulates the UPS of Sod1 in mouse and human MNs and that Sod1 secretion occurs in an autophagy dependent manner. Our data shows an unreported mechanistic link between two MND-associated proteins.