Stabilization of V1 interneuron-motor neuron connectivity ameliorates motor phenotype in a mouse model of ALS.

Loss of connectivity between spinal V1 inhibitory interneurons and motor neurons is found early in disease in the SOD1G93A mice. Such changes in premotor inputs can contribute to homeostatic imbalance of motor neurons. Here, we show that the Extended Synaptotagmin 1 (Esyt1) presynaptic organizer is downregulated in V1 interneurons. V1 restricted overexpression of Esyt1 rescues inhibitory synapses, increases motor neuron survival, and ameliorates motor phenotypes. Two gene therapy approaches overexpressing ESYT1 were investigated; one for local intraspinal delivery, and the other for systemic administration using an AAV-PHP.eB vector delivered intravenously. Improvement of motor functions is observed in both approaches, however systemic administration appears to significantly reduce onset of motor impairment in the SOD1G93A mice in absence of side effects. Altogether, we show that stabilization of V1 synapses by ESYT1 overexpression has the potential to improve motor functions in ALS, demonstrating that interneurons can be a target to attenuate ALS symptoms.

Spinal inhibitory neurons degenerate before motor neurons and excitatory neurons in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of somatic motor neurons. A major focus has been directed to motor neuron intrinsic properties as a cause for degeneration, while less attention has been given to the contribution of spinal interneurons. In the present work, we applied multiplexing detection of transcripts and machine learning-based image analysis to investigate the fate of multiple spinal interneuron populations during ALS progression in the SOD1G93A mouse model. The analysis showed that spinal inhibitory interneurons are affected early in the disease, before motor neuron death, and are characterized by a slow progressive degeneration, while excitatory interneurons are affected later with a steep progression. Moreover, we report differential vulnerability within inhibitory and excitatory subpopulations. Our study reveals a strong interneuron involvement in ALS development with interneuron specific degeneration. These observations point to differential involvement of diverse spinal neuronal circuits that eventually may be determining motor neuron degeneration.

Neural circuit and synaptic dysfunctions in ALS-FTD pathology.

Action selection is a capital feature of cognition that guides behavior in processes that range from motor patterns to executive functions. Here, the ongoing actions need to be monitored and adjusted in response to sensory stimuli to increase the chances of reaching the goal. As higher hierarchical processes, these functions rely on complex neural circuits, and connective loops found within the brain and the spinal cord. Successful execution of motor behaviors depends, first, on proper selection of actions, and second, on implementation of motor commands. Thus, pathological conditions crucially affecting the integrity and preservation of these circuits and their connectivity will heavily impact goal-oriented motor behaviors. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders known to share disease etiology and pathophysiology. New evidence in the field of ALS-FTD has shown degeneration of specific neural circuits and alterations in synaptic connectivity, contributing to neuronal degeneration, which leads to the impairment of motor commands and executive functions. This evidence is based on studies performed on animal models of disease, post-mortem tissue, and patient derived stem cells. In the present work, we review the existing evidence supporting pathological loss of connectivity and selective impairment of neural circuits in ALS and FTD, two diseases which share strong genetic causes and impairment in motor and executive functions.

Locomotor deficits in a mouse model of ALS are paralleled by loss of V1-interneuron connections onto fast motor neurons.

ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.

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.

Spinal motoneurones are intrinsically more responsive in the adult G93A SOD1 mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Although in vitro recordings using neonatal preparations from mouse models of amyotrophic lateral sclerosis (ALS) suggest increased motoneurone excitability, in vivo recordings in adult ALS mouse models have been conflicting. In adult G93A SOD1 models, spinal motoneurones have previously been shown to have deficits in repetitive firing, in contrast to the G127X SOD1 mouse model. Our in vivo intracellular recordings in barbiturate-anaesthetized adult male G93A SOD1 mice reveal that the incidence of failure to fire with current injection was equally low in control and ALS mice (∼2%). We show that failure to fire repetitively can be a consequence of experimental protocol and should not be used alone to classify otherwise normal motoneurones as hypo-excitable. Motoneurones in the G93A SOD1 mice showed an increased response to inputs, with lower rheobase, higher input-output gains and increased activation of persistent inward currents. ABSTRACT: In vitro studies from transgenic amyotrophic lateral sclerosis models have suggested an increased excitability of spinal motoneurones. However, in vivo intracellular recordings from adult amyotrophic lateral sclerosis mice models have produced conflicting findings. Previous investigations using barbiturate anaesthetized G93A SOD1 mice have suggested that some motoneurones are hypo-excitable, defined by deficits in repetitive firing. Our own previous recordings in G127X SOD1 mice using different anaesthesia, however, showed no repetitive firing deficits and increased persistent inward currents at symptom onset. These discrepancies may be a result of differences between models, symptomatic stage, anaesthesia or technical differences. To investigate this, we repeated our original experiments, but in adult male G93A SOD1 mice, at both presymptomatic and symptomatic stages, under barbiturate anaesthesia. In vivo intracellular recordings from antidromically identified spinal motoneurones revealed that the incidence of failure to fire with current injection was equally low in control and G93A SOD1 mice (∼2%). Motoneurones in G93A SOD1 mice fired significantly more spontaneous action potentials. Rheobase was significantly lower and the input resistance and input-output gain were significantly higher in both presymptomatic and symptomatic G93A SOD1 mice. This was despite a significant increase in the duration of the post-spike after-hyperpolarization in both presymptomatic and symptomatic G93A SOD1 mice. Finally, evidence of increased activation of persistent inward currents was seen in both presymptomatic and symptomatic G93A SOD1 mice. Our results do not confirm previous reports of hypo-excitability of spinal motoneurones in the G93A SOD1 mouse and demonstrate that the motoneurones show an increased response to inputs.

Intact single muscle fibres from SOD1G93A amyotrophic lateral sclerosis mice display preserved specific force, fatigue resistance and training-like adaptations.

KEY POINTS: How defects in muscle contractile function contribute to weakness in amyotrophic lateral sclerosis (ALS) were systematically investigated. Weakness in whole muscles from late stage SOD1G93A mice was explained by muscle atrophy as seen by reduced mass and maximal force. On the other hand, surviving single muscle fibres in late stage SOD1G93A have preserved intracellular Ca2+ handling, normal force-generating capacity and increased fatigue resistance. These intriguing findings provide a substrate for therapeutic interventions to potentiate muscular capacity and delay the progression of the ALS phenotype. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of motor neurons, leading to severe muscle weakness and paralysis. The SOD1G93A mouse model of ALS displays motor neuron degeneration and a phenotype consistent with human ALS. The purpose of this study was to determine whether muscle weakness in ALS can be attributed to impaired intrinsic force generation in skeletal muscles. In the current study, motor neuron loss and decreased force were evident in whole flexor digitorum brevis (FDB) muscles of mice in the late stage of disease (125-150 days of age). However, in intact single muscle fibres, specific force, tetanic myoplasmic free [Ca2+ ] ([Ca2+ ]i ), and resting [Ca2+ ]i remained unchanged with disease. Fibre-type distribution was maintained in late-stage SOD1G93A FDB muscles, but remaining muscle fibres displayed greater fatigue resistance compared to control and showed increased expression of myoglobin and mitochondrial respiratory chain proteins that are important determinants of fatigue resistance. Expression of genes central to both mitochondrial biogenesis and muscle atrophy where increased, suggesting that atrophic and compensatory adaptive signalling occurs simultaneously within the muscle tissue. These results support the hypothesis that muscle weakness in SOD1G93A is primarily attributed to neuromuscular degeneration and not intrinsic muscle fibre defects. In fact, surviving muscle fibres displayed maintained adaptive capacity with an exercise training-like phenotype, which suggests that compensatory mechanisms are activated that can function to delay disease progression.

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.

Neurturin is a PGC-1α1-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation.

OBJECTIVE: We examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. METHODS: A microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. RESULTS: Skeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. CONCLUSIONS: Our findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons.

Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3ß phosphorylation and ß-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

Schwann cells transduced with a lentiviral vector encoding Fgf-2 promote motor neuron regeneration following sciatic nerve injury.

Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF-2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF-2 (LV-FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF-2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV-FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV-FGF2 into the rat sciatic nerve resulted in increased expression of FGF-2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF-2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV-FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV-FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow-up.

Directed midbrain and spinal cord neurogenesis from pluripotent stem cells to model development and disease in a dish.

Induction of specific neuronal fates is restricted in time and space in the developing CNS through integration of extrinsic morphogen signals and intrinsic determinants. Morphogens impose regional characteristics on neural progenitors and establish distinct progenitor domains. Such domains are defined by unique expression patterns of fate determining transcription factors. These processes of neuronal fate specification can be recapitulated in vitro using pluripotent stem cells. In this review, we focus on the generation of dopamine neurons and motor neurons, which are induced at ventral positions of the neural tube through Sonic hedgehog (Shh) signaling, and defined at anteroposterior positions by fibroblast growth factor (Fgf) 8, Wnt1, and retinoic acid (RA). In vitro utilization of these morphogenic signals typically results in the generation of multiple neuronal cell types, which are defined at the intersection of these signals. If the purpose of in vitro neurogenesis is to generate one cell type only, further lineage restriction can be accomplished by forced expression of specific transcription factors in a permissive environment. Alternatively, cell-sorting strategies allow for selection of neuronal progenitors or mature neurons. However, modeling development, disease and prospective therapies in a dish could benefit from structured heterogeneity, where desired neurons are appropriately synaptically connected and thus better reflect the three-dimensional structure of that region. By modulating the extrinsic environment to direct sequential generation of neural progenitors within a domain, followed by self-organization and synaptic establishment, a reductionist model of that brain region could be created. Here we review recent advances in neuronal fate induction in vitro, with a focus on the interplay between cell intrinsic and extrinsic factors, and discuss the implications for studying development and disease in a dish.

C3 exoenzyme lacks effects on peripheral axon regeneration in vivo.

Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP-ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3bot suggesting an inflammatory component in the inhibition of axon growth. C3bot or C3E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3E174Q . Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth-promoting environment.

Neuroprotection and axonal regeneration after lumbar ventral root avulsion by re-implantation and mesenchymal stem cells transplant combined therapy.

Ventral spinal root avulsion causes complete denervation of muscles in the limb and also progressive death of segmental motoneurons (MN) leading to permanent paralysis. The chances for functional recovery after ventral root avulsion are very poor owing to the loss of avulsed neurons and the long distance that surviving neurons have to re-grow axons from the spinal cord to the corresponding targets. Following unilateral avulsion of L4, L5 and L6 spinal roots in adult rats, we performed an intraspinal transplant of mesenchymal stem cells (MSC) and surgical re-implantation of the avulsed roots. Four weeks after avulsion the survival of MN in the MSC-treated animals was significantly higher than in vehicle-injected rats (45% vs. 28%). Re-implantation of the avulsed roots in the injured spinal cord allowed the regeneration of motor axons. By combining root re-implantation and MSC transplant the number of surviving MN at 28 days post-injury was higher (60%) than in re-implantation alone animals (46%). Electromyographic tests showed evidence of functional re-innervation of anterior tibialis and gastrocnemius muscles by the regenerated motor axons only in rats with the combined treatment. These results indicate that MSC are helpful in enhancing neuronal survival and increased the regenerative growth of injured axons. Surgical re-implantation and MSC grafting combined had a synergic neuroprotective effect on MN and on axonal regeneration and muscle re-innervation after spinal root avulsion.

FGF-2 low molecular weight selectively promotes neuritogenesis of motor neurons in vitro.

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.

Specificity of peripheral nerve regeneration: interactions at the axon level.

Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs.

Effects of activity-dependent strategies on regeneration and plasticity after peripheral nerve injuries.

Peripheral nerve injuries result in loss of motor, sensory and autonomic functions of the denervated limb, but are also accompanied by positive symptoms, such as hyperreflexia, hyperalgesia and pain. Strategies to improve functional recovery after neural injuries have to address the enhancement of axonal regeneration and target reinnervation and also the modulation of the abnormal plasticity of neuronal circuits. By enhancing sensory inputs and/or motor outputs, activity-dependent therapies, like electrostimulation or exercise, have been shown to positively influence neuromuscular functional recovery and to modulate the plastic central changes after experimental nerve injuries. However, it is important to take into account that the type of treatment, the intensity and duration of the protocol, and the period during which it is applied after the injury are factors that determine beneficial or detrimental effects on functional recovery. The adequate maintenance of activity of neural circuits and denervated muscles results in increased trophic factor release to act on regenerating axons and on central plastic changes. Among the different neurotrophins, BDNF seems a key player in the beneficial effects of activity-dependent therapies after nerve injuries.

In vitro comparison of motor and sensory neuron outgrowth in a 3D collagen matrix.

In this work we set up an in vitro model, based on organotypic cultures of spinal cord slices and dorsal root ganglia explants from P7 rats, embedded in a collagen matrix and cultured under the same conditions. As specific reinnervation of end-organs is still an unresolved issue in peripheral nerve research, we characterized a model that allows us to compare under the same conditions motor and sensory neuron regeneration. RT97 labeling was used to visualize the regenerating neurites that extended in the collagen gel from both motor neurons in the spinal cord slices and sensory neurons in the DRG explants after a few days in vitro. By adding different neurotrophic factors in the collagen matrix, we evaluated the reliability of DRG and spinal cord preparations. Moreover, we also set up a co-culture with dissociated Schwann cells to further mimic the permissive environment of the peripheral nerve. Thus, these in vitro models can be useful tools to investigate mechanisms for the selective regeneration of sensory and motor neurons, which can be translated into in vivo models.