Thomas W. Gould

Complete Dissociation of Motor Neuron Death from Motor Dysfunction by Bax Deletion in a Mouse Model of ALS

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Cytokines Promote Motoneuron Survival through the Janus Kinase-Dependent Activation of the Phosphatidylinositol 3-Kinase Pathway ☆

To determine which intracellular pathways mediate the survival effects of ciliary neurotrophic factor and cardiotrophin-1 cytokines on motoneurons, we studied the activation of the Jak/STAT, the PI 3-kinase/Akt, and the ERK pathways. At shorter time points, cytokines induced the activation of STAT3 and ERK, but not PI 3-kinase. Jak3 inhibitor suppressed cytokine- and muscle extract-induced survival. In contrast, PD 98059, a MEK inhibitor, was not able to prevent cytokine-induced survival, demonstrating that ERK is not involved. Surprisingly, the PI 3-kinase inhibitor LY 294002 prevented the survival-promoting effects of cytokines. When assays of PI 3-kinase activity were performed at later stages following cytokine treatment a significant increase was observed compared to control cultures. This delayed increase of activity could be completely prevented by treatment with protein synthesis or Jak3 inhibitors. Collectively, these results demonstrate that cytokines induce motoneuron survival through a PI 3-kinase activation requiring de novo protein synthesis dependent on Jak pathway.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

ABSTRACT The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

The Neurotrophic Effects of Glial Cell Line-Derived Neurotrophic Factor on Spinal Motoneurons Are Restricted to Fusimotor Subtypes

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor α1 (GFRα1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRα1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRα1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRα1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

Neuromuscular Development in the Absence of Programmed Cell Death: Phenotypic Alteration of Motoneurons and Muscle

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from α- to γ-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Phosphorylation of c-Jun in Avian and Mammalian Motoneurons In Vivo during Programmed Cell Death: An Early Reversible Event in the Apoptotic Cascade

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo.We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.

The Function of Neurotrophic Factor Receptors Expressed by the Developing Adductor Motor Pool In Vivo

We examined the spatio-temporal relationship between neurotrophic factor receptor (NTF-R) expression and motoneuron (MN) survival in the developing avian spinal cord and observed heterogeneity in the expression of NTF-Rs between, but not within, pools of MNs projecting to individual muscles. We then focused on the role of NTFs in regulating the survival of one motor pool of MNs, all of which innervate a pair of adductor muscles in the thigh and hence compete for survival during the period of programmed cell death (PCD). The complete NTF-R complement of these MNs was analyzed and found to include many, but not all, NTF-Rs. Treatment with exogenous individual NTFs rescued some, but not all, adductor MNs expressing appropriate NTF-Rs. In contrast, administration of multiple NTFs completely rescued adductor MNs from PCD. Additionally, adductor MNs were partially rescued from PCD by NTFs for which they failed to express receptors. NTF-Rs expressed by the nerve but not in the muscle target were capable of mediating survival signals to MNs in trans. Finally, the expression of some NTF-Rs by adductor MNs was not required for MN survival. These studies demonstrate the complexity in NTF regulation of a defined subset of competing MNs and suggest that properties other than NTF-R expression itself can play a role in mediating trophic responses to NTFs.

Nestin negatively regulates postsynaptic differentiation of the neuromuscular synapse

Positive and negative regulation of neurotransmitter receptor aggregation on the postsynaptic membrane is a critical event during synapse formation. Acetylcholine (ACh) and agrin are two opposing signals that regulate ACh receptor (AChR) clustering during neuromuscular junction (NMJ) development. ACh induces dispersion of AChR clusters that are not stabilized by agrin via a cyclin-dependent kinase 5 Cdk5)-mediated mechanism, but regulation of Cdk5 activation is poorly understood. We found that the intermediate filament protein nestin physically interacts with Cdk5 and is required for ACh-induced association of p35, the co-activator of Cdk5, with the muscle membrane. Blockade of nestin-dependent signaling inhibited ACh-induced Cdk5 activation and the dispersion of AChR clusters in cultured myotubes. Similar to the effects of Cdk5 gene inactivation, knockdown of nestin in agrin-deficient mouse embryos ubstantially restored AChR clusters. These results suggest that nestin is required for ACh-induced, Cdk5-dependent dispersion of AChR clusters during NMJ development.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Neurotrophic Modulation of Motor Neuron Development

Neurotrophic factors (NTFs) are a pleiotropic group of secreted growth factors that regulate multiple aspects of neuronal development, including the regressive event of cell death. Skeletal muscleinnervating lower motoneurons (MNs) of the brain stem and spinal cord comprise one population of central neurons in which programmed cell death (PCD) during embryogenesis has been actively investigated, as much for reasons of technical facility as clinical relevance. The precise identity of NTF-dependent MNs has remained unclear, with most studies simply reporting losses or gains across the entire spinal cord or individual brain-stem nuclei. However, MNs are grouped into highly heterogenous populations based on transcriptional identity, target innervation, and physiological function. Therefore, recent work has focused on the effects of NTF overexpression or deletion on the survival of these MN subpopulations. Together with the recent progress attained in the generation of conditional mutant mice, in which the function of an NTF or its receptor can be eliminated specifically in MNs, these recent studies have begun to define the differential trophic requirements for MN subpopulations during PCD. The intent of this review is to summarize these recent findings and to discuss their significance with respect to neurotrophic theory.