Fatal demyelinating disease is induced by monocyte-derived macrophages in the absence of TGF-ß signaling.

The cytokine transforming growth factor-ß (TGF-ß) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-ß is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature1,2. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS)3-5. Whether monocytes require TGF-ß for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-ß signaling in CX3CR1+ monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocytosis. TGF-ß is thus crucial for the functional integration of monocytes into the CNS microenvironment.

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.

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. Concomitant to detected structural alterations, lumbar motor neurons from MCT1 cKO mice show transcriptional changes affecting cytoskeletal components, transcriptional regulators, and mitochondria related transcripts, among others. Together, our data indicate that MCT1 plays a role in Schwann cell-mediated maintenance of motor end-plate innervation thus providing further insight into the emerging picture of the biology of the axon-glia metabolic crosstalk.

Motor neuron vulnerability and resistance in amyotrophic lateral sclerosis.

In the fatal disease-amyotrophic lateral sclerosis (ALS)-upper (corticospinal) motor neurons (MNs) and lower somatic MNs, which innervate voluntary muscles, degenerate. Importantly, certain lower MN subgroups are relatively resistant to degeneration, even though pathogenic proteins are typically ubiquitously expressed. Ocular MNs (OMNs), including the oculomotor, trochlear and abducens nuclei (CNIII, IV and VI), which regulate eye movement, persist throughout the disease. Consequently, eye-tracking devices are used to enable paralysed ALS patients (who can no longer speak) to communicate. Additionally, there is a gradient of vulnerability among spinal MNs. Those innervating fast-twitch muscle are most severely affected and degenerate first. MNs innervating slow-twitch muscle can compensate temporarily for the loss of their neighbours by re-innervating denervated muscle until later in disease these too degenerate. The resistant OMNs and the associated extraocular muscles (EOMs) are anatomically and functionally very different from other motor units. The EOMs have a unique set of myosin heavy chains, placing them outside the classical characterization spectrum of all skeletal muscle. Moreover, EOMs have multiple neuromuscular innervation sites per single myofibre. Spinal fast and slow motor units show differences in their dendritic arborisations and the number of myofibres they innervate. These motor units also differ in their functionality and excitability. Identifying the molecular basis of cell-intrinsic pathways that are differentially activated between resistant and vulnerable MNs could reveal mechanisms of selective neuronal resistance, degeneration and regeneration and lead to therapies preventing progressive MN loss in ALS. Illustrating this, overexpression of OMN-enriched genes in spinal MNs, as well as suppression of fast spinal MN-enriched genes can increase the lifespan of ALS mice. Here, we discuss the pattern of lower MN degeneration in ALS and review the current literature on OMN resistance in ALS and differential spinal MN vulnerability. We also reflect upon the non-cell autonomous components that are involved in lower MN degeneration in ALS.

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Single-Cell mRNA-Seq of In Vitro-Derived Human Neurons Using Smart-Seq2.

Single-cell mRNA sequencing can dissect heterogeneous cell populations as it can identify cell types and cellular states based on their unique transcriptional signatures. We use fluorescence-activated cell sorting (FACS) to isolate individual cultured neurons derived from human-induced pluripotent stem cells (hiPSCs) followed by polyA-based Smart-Seq2 RNA sequencing to analyze the single-cell transcriptional profiles. We provide protocols and guidelines on dissociation, cell selection, and library preparation that can be readily adapted to other cell types or tissue samples.

Muscle-secreted neurturin couples myofiber oxidative metabolism and slow motor neuron identity.

Endurance exercise promotes skeletal muscle vascularization, oxidative metabolism, fiber-type switching, and neuromuscular junction integrity. Importantly, the metabolic and contractile properties of the muscle fiber must be coupled to the identity of the innervating motor neuron (MN). Here, we show that muscle-derived neurturin (NRTN) acts on muscle fibers and MNs to couple their characteristics. Using a muscle-specific NRTN transgenic mouse (HSA-NRTN) and RNA sequencing of MN somas, we observed that retrograde NRTN signaling promotes a shift toward a slow MN identity. In muscle, NRTN increased capillary density and oxidative capacity and induced a transcriptional reprograming favoring fatty acid metabolism over glycolysis. This combination of effects on muscle and MNs makes HSA-NRTN mice lean with remarkable exercise performance and motor coordination. Interestingly, HSA-NRTN mice largely recapitulate the phenotype of mice with muscle-specific expression of its upstream regulator PGC-1ɑ1. This work identifies NRTN as a myokine that couples muscle oxidative capacity to slow MN identity.

Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes.

Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single microfluidic devices. We also detail how to generate motor neurons from mouse and human pluripotent stem cells for sequencing, but Axon-seq is applicable to any neuronal cell. In Axon-seq, the axons are recruited through a growth factor gradient, lysed and directly processed to cDNA without RNA isolation. A careful bioinformatic step ensures that any soma-contaminated samples are easily identified and removed.

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.

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.

Cross-disease comparison of amyotrophic lateral sclerosis and spinal muscular atrophy reveals conservation of selective vulnerability but differential neuromuscular junction pathology.

Neuromuscular junctions are primary pathological targets in the lethal motor neuron diseases spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Synaptic pathology and denervation of target muscle fibers has been reported prior to the appearance of clinical symptoms in mouse models of both diseases, suggesting that neuromuscular junctions are highly vulnerable from the very early stages, and are a key target for therapeutic intervention. Here we examined neuromuscular pathology longitudinally in three clinically relevant muscle groups in mouse models of ALS and SMA in order to assess their relative vulnerabilities. We show for the first time that neuromuscular junctions of the extraocular muscles (responsible for the control of eye movement) were resistant to degeneration in endstage SMA mice, as well as in late symptomatic ALS mice. Tongue muscle neuromuscular junctions were also spared in both animal models. Conversely, neuromuscular junctions of the lumbrical muscles of the hind-paw were vulnerable in both SMA and ALS, with a loss of neuronal innervation and shrinkage of motor endplates in both diseases. Thus, the pattern of selective vulnerability was conserved across these two models of motor neuron disease. However, the first evidence of neuromuscular pathology occurred at different timepoints of disease progression, with much earlier evidence of presynaptic involvement in ALS, progressing to changes on the postsynaptic side. Conversely, in SMA changes appeared concomitantly at the neuromuscular junction, suggesting that mechanisms of neuromuscular disruption are distinct in these diseases. J. Comp. Neurol. 524:1424-1442, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.