Inter- and intracellular resistance and vulnerability in motor neuron diseases

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are fatal diseases presenting with degeneration and loss of lower motor neurons in the brainstem and spinal cord. However, oculomotor and trochlear motor neurons that control eye movement, are resistant until late stages of disease. Moreover, individual motor neurons degenerate in a distinct pattern. Firstly, the distal synapse between motor neuron and muscle, the neuromuscular junction (NMJ), becomes denervated. Subsequently, the neuron degenerates in a retrograde manner, until the cell body in the spinal cord is lost later in disease. This highlights the distal axon and NMJ as the most vulnerable subcellular compartment of motor neurons. We set out to investigate the mechanisms that underlie the resistance of oculomotor neurons, as well as the differential vulnerability within motor neurons themselves. We first validated the resistance of oculomotor neurons in two mouse models of ALS and SMA in Paper I. Next, we performed a large transcriptomic screen of differentially vulnerable motor pools in SMA mice throughout disease progression in Paper II. We used laser capture microdissection (LCM) to dissect motor neurons from different pools in the brainstem and spinal cord. Here we revealed pathways and transcripts that were enriched only in the resistant oculomotor neurons that serve to prevent them from going into apoptosis, as well as promote their regeneration. We showed that applying one of the oculomotor-enriched transcripts, Gdf15, to vulnerable spinal motor neurons derived from stem cells improved their survival. Next, in Paper III we utilised in vitro approaches and generated a model of oculomotor resistance in ALS by deriving both spinal and oculomotor neurons from mouse embryonic stem cells. Here we found that in vitro oculomotor neurons are more resistant to excitotoxic stress and have increased levels of prosurvival Akt-signaling, which also held up in LCM-dissected post-mortem human spinal and oculomotor neurons. To investigate the vulnerability of the distal axon, we generated a technique called Axon-Seq in Paper IV, that allows transcriptomic profiling of isolated motor axons by culturing motor neurons in microfluidic chambers. We subsequently used this technique in Paper V to show that human stem cell-derived motor neurons carrying ALS-causative mutations in FUS and TDP-43 showed unique transcriptome dysregulation in somas and axons. This implies that different pathways of degeneration are at play between subcellular compartments, and provides novel targets for therapeutic intervention directed at different compartments of the motor neuron. LIST OF SCIENTIFIC PAPERS I. Comley LH, Nijssen J, Frost-Nylen J and Hedlund E. Cross-Disease Comparison of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy Reveals Conservation of Selective Vulnerability but Differential Neuromuscular Junction Pathology. J Comp Neurol 524(7):1424-1442 II. Nichterwitz S, Nijssen J*, Storvall H*, Schweingruber C, Comley LH, Allodi I, van der Lee M, Deng Q, Sandberg R and Hedlund E. LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy. In revision III. Allodi I, Nijssen J, Aguila Benitez J, Schweingruber C, Fuchs A, Bonvicini G, Cao M, Kiehn O and Hedlund E. Modeling Motor Neuron Resilience in ALS Using Stem Cells. Stem Cell Rep 12(6):1329-1341 IV. Nijssen J*, Aguila J*, Hoogstraaten R, Kee N and Hedlund E. Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS. Stem Cell Rep 11(6):1565-1578 V. Nijssen J, Aguila J, Schweingruber C, Reber S, Ruepp MD and Hedlund E. Mutations in the ALS-causative genes FUS and TDP-43 cause distinct dysregulation of somatic and axonal transcriptomes. Manuscript * These authors contributed equally to this work SCIENTIFIC PAPERS NOT INCLUDED IN THE THESIS I. Nijssen J, Comley LH and Hedlund E. Motor neuron vulnerability and resistance in amyotrophic lateral sclerosis. Acta Neuropathol 133(6):863-885 II. Lund H, Pieber M, Parsa R, Grommisch D, Ewing E, Kular L, Han J, Zhu K, Nijssen J, Hedlund E, Needhamsen M, Ruhrmann S, Guerreiro-Cacais AO, Berglund R, Forteza MJ, Ketelhuth DFJ, Butovsky O, Jagodic M, Zhang XM and Harris RA. Fatal Demyelinating Disease Is Induced by Monocyte-Derived Macrophages in the Absence of TGF-b Signaling. Nat Immunol 19(5):1-7 III. Nizzardo M, Taiana M, Rizzo F, Benitez JA, Nijssen J, Allodi I, Melzi V, Bresolin N, Comi GP, Hedlund E and Corti S. Synaptotagmin 13 Is Neuroprotective Across Motor Neuron Diseases. Acta Neuropathol 139(5):837-853 CONTENTS