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

Modeling Motor Neuron Resilience in ALS Using Stem Cells

Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Toward this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of PHOX2A in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry, and RNA sequencing, that in vitro-generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro-generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro that display a resilience similar to that seen in vivo.</p

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS

Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500–5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease. In this article, Nijssen, Aguila, and colleagues report an improved method for RNA sequencing of axons, Axon-seq, that incorporates microfluidics and stringent bioinformatic quality control. The authors show that the axonal transcriptome is smaller than and distinct from that of somas and contains genes required for oxidative energy production and ribogenesis as well as a unique set of transcription factors. Axon-seq reveals that the ALS-causing SOD1G93A mutation dysregulates transcripts with axonal functions

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS

Summary: Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500–5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease. : In this article, Nijssen, Aguila, and colleagues report an improved method for RNA sequencing of axons, Axon-seq, that incorporates microfluidics and stringent bioinformatic quality control. The authors show that the axonal transcriptome is smaller than and distinct from that of somas and contains genes required for oxidative energy production and ribogenesis as well as a unique set of transcription factors. Axon-seq reveals that the ALS-causing SOD1G93A mutation dysregulates transcripts with axonal functions. Keywords: RNA sequencing, microfluidic devices, amyotrophic lateral sclerosis, motor neurons, stem cells, transcription factor

LCM-seq reveals unique transcriptional adaptation mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy

Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.</p

Disrupted function of lactate transporter MCT1 , but not MCT4 , in Schwann cells affects the maintenance of motor end‐plate innervation

Recent studies in neuron-glial metabolic coupling have shown that, in the CNS, astrocytes and oligodendrocytes support neurons with energy-rich lactate/pyruvate via monocarboxylate transporters (MCTs). The presence of such transporters in the PNS, in both Schwann cells and neurons, has prompted us to question if a similar interaction may be present. Here we describe the generation and characterization of conditional knockout mouse models where MCT1 or MCT4 is specifically deleted in Schwann cells (named MCT1 and MCT4 cKO). We show that MCT1 cKO and MCT4 cKO mice develop normally and that myelin in the PNS is preserved. However, MCT1 expressed by Schwann cells is necessary for long-term maintenance of motor end-plate integrity as revealed by disrupted neuromuscular innervation in mutant mice, while MCT4 appears largely dispensable for the support of motor neurons