Can Hybrid Synapse be Formed between Rat Spinal Motor Neurons and Major Pelvic Ganglion Neurons in vitro?

The purpose of this study is to determine whether synapses can be formed between spinal motor neurons (SMNs) and major pelvic ganglion (MPG) neurons of a rat in vitro. The green fluorescent protein (GFP)-labelled MPG cells were cultured together with SMNs in a specific medium. The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches. Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro. We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95 (PSD-95). We describe the development process of a defined SMNs-MPG neurons co-culture system. The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.

Anuran Vocal Communication: Effects of Genome Size, Cell Number and Cell Size

Significant variation in genome size occurs among anuran amphibians and can affect cell size and number. In the gray treefrog complex in North America increases in cell size in autotriploids of the diploid (Hyla chrysoscelis) altered the temporal structure of mate-attracting vocalizations and auditory selectivity for these properties. Here we show that the tetraploid species (Hyla versicolor) also has significantly fewer brain neurons than H. chrysoscelis. With regard to cell size in tissues involved in vocal communication, spinal motor neurons were larger in tetraploids than in diploids and comparable to differences in erythrocyte size; smaller increases were found in one of the three auditory centers in the torus semicircularis. Future studies should address questions about how environmental conditions during development affect cell numbers and size and the causal relationships between these cellular changes and the vocal communication system.

The manipulation of spinal motor neuron axonal growth promotes spinal cord repair

The loss of motor function in patients with spinal cord injury (SCI) is primarily due to the severing of the corticospinal tract (CST). Spinal motor neurons are located in the anterior horn of the spinal cord, and as the lower neurons of the CST, they control voluntary movement. Furthermore, its intrinsic axonal growth ability is significantly stronger than that of cerebral cortex pyramid neurons, which are the upper CST neurons. Therefore, we established an axonal regeneration model of spinal motor neurons to investigate the feasibility of repairing SCI by promoting axonal regeneration of spinal motor neurons. We demonstrated that conditionally knocking out pten in mature spinal motor neurons drastically enhanced axonal regeneration in vivo, and the regenerating axons of the spinal motor neurons re-established synapses with other cells in the damaged spinal cord. Thus, this strategy may serve as a novel and effective treatment method for SCI.

A Brainstem reticulotegmental neural ensemble drives acoustic startle reflexes

The reticulotegmental nucleus (RtTg) has long been recognized as a crucial component of brainstem reticular formation (RF). However, the function of RtTg and its related circuits remain elusive. Here, we report a role of the RtTg in startle reflex, a highly conserved innate defensive behaviour. Optogenetic activation of RtTg neurons evokes robust startle responses in mice. The glutamatergic neurons in the RtTg are significantly activated during acoustic startle reflexes (ASR). Chemogenetic inhibition of the RtTg glutamatergic neurons decreases the ASR amplitudes. Viral tracing reveals an ASR neural circuit that the cochlear nucleus carrying auditory information sends direct excitatory innervations to the RtTg glutamatergic neurons, which in turn project to spinal motor neurons. Together, our findings describe a functional role of RtTg and its related neural circuit in startle reflexes, and demonstrate how the RF connects auditory system with motor functions.

Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70's ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43's RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70’s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43’s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

Single-cell transcriptomic analysis unveils spinal motor neuron subtype diversity underpinning the water-to-land transition in vertebrates

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate motor movements in vertebrates. Distinct MN populations and their diversity has long been hypothesized to co-evolve with motor circuit to provide the neural basis from undulatory to ambulatory locomotion during aquatic-to-terrestrial transition of vertebrates. However, how these subtypes are evolved remains largely enigmatic. Using single-cell transcriptomics, we investigate heterogeneity in mouse MNs and discover novel segment-specific subtypes. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. We further uncovered that axial MNs are subdivided by three conserved and molecularly distinct subpopulations, defined by Satb2, Nr2f2 or Bcl11b expression. Although axial MNs are conserved from cephalochordates to humans, subtype diversity becomes prominent in land animals and appears to continue evolving in humans. Overall, our study provides a unified classification system for spinal MNs and paves the way towards deciphering how neuronal subtypes are evolved.

A Novel Supplement Attenuates Oxidative Stress-Induced TDP-43-Related Pathogenesis in TDP-43-Expressed Cells

Amyotrophic lateral sclerosis (ALS) is caused by selective the loss of spinal motor neurons by multifactorial pathological mechanisms and results in muscle atrophy. Incidence rates of ALS are increasing over time, but there are no effective treatments at present due to limitations on approved therapies (riluzole and edaravone). Therefore, this study investigated whether combined treatment with Bojungikgi-tang and riluzole could act synergistically in transactive response DNA-binding protein 43 (TDP-43) stress granule cells. To examine the effect of combined treatment on oxidative stress-induced cell death, the CCK8 assay was performed for the detection of cell viability. The expression of oxidative stress-induced proteins was determined by Western blot. Quantification of sodium arsenite-induced reactive oxygen species (ROS) was measured in TDP-43 stress granular cells using 2,7-diacetyl dichlorofluorescein diacetate. To investigate the effect of combined treatment on TDP-43 aggregation, immunofluorescence and immunoblotting were performed in TDP-43 stress granular cells. This combined treatment alleviated oxidative stress-induced cell death by increasing the expression levels of antioxidation proteins, such as heme oxygenase-1 and B cell lymphoma-2-associated X protein. Furthermore, it reduced oxidative stress-induced TDP-43 aggregates and lowered the levels of autophagy-related proteins, including p62, light chain 3b, and ATG8, in TDP-43-expressing cells. Our results suggest that this combined treatment could be helpful for autophagy regulation in other neurodegenerative diseases.

MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

“Axonal Length Determines distinct homeostatic phenotypes in human iPSC derived motor neurons on a bioengineered platform”

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a “threshold” size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.