Structure-function analysis of SAP97, a modular scaffolding protein that drives dendrite growth.

Activation of AMPA receptors assembled with the GluA1 subunit can promote dendrite growth in a manner that depends on its direct binding partner, SAP97. SAP97 is a modular scaffolding protein that has at least seven recognizable protein-protein interaction domains. Several complementary approaches were employed to show that the dendrite branching promoting action of full length SAP97 depends on ligand(s) that bind to the PDZ3 domain. Ligand(s) to PDZ1, PDZ2 and I3 domains also contribute to dendrite growth. The ability of PDZ3 ligand(s) to promote dendrite growth depends on localization at the plasma membrane along with GluA1 and SAP97. These results suggest that the assembly of a multi-protein complex at or near synapses is vital for the translation of AMPA-R activity into dendrite growth.

Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse.

Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.

Induction of a neuronal proteoglycan by the NMDA receptor in the developing spinal cord.

Activation of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors is a critical step in the selection of appropriate synaptic connections in the developing visual systems of cat and frog. Activity-dependent development of mammalian motor neurons was shown to be similarly mediated by activation of the NMDA receptor. The expression of the Cat-301 proteoglycan on motor neurons was developmentally regulated and could be specifically inhibited by blockade of the NMDA receptor at the spinal segmental level. In the adult, Cat-301 immunoreactivity on motor neurons was not diminished by NMDA receptor blockade. The NMDA receptor may regulate the expression of a class of neuronal proteins (of which Cat-301 is one example) that underlie the morphological and physiological features of activity-dependent development.

Sensitization to morphine induced by viral-mediated gene transfer.

Repeated administration of morphine sensitizes animals to the stimulant and rewarding properties of the drug. It also selectively increases expression of GluR1 (an AMPA glutamate receptor subunit) in the ventral tegmental area, a midbrain region implicated in morphine action. By viral-mediated gene transfer, a causal relation is shown between these behavioral and biochemical adaptations: Morphine's stimulant and rewarding properties are intensified after microinjections of a viral vector expressing GluR1 into the ventral tegmental area. These results confirm the importance of AMPA receptors in morphine action and demonstrate specific locomotor and motivational adaptations resulting from altered expression of a single localized gene product.

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301.

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animal's postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of integral of 680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.

Experience-dependent development of spinal motor neurons.

Locomotor activity in many species undergoes pronounced alterations in early postnatal life, and environmental cues may be responsible for modifying this process. To determine how these events are reflected in the nervous system, we studied rats reared under two different conditions-the presence or absence of gravity-in which the performance of motor operations differed. We found a significant effect of rearing environment on the size and complexity of dendritic architecture of spinal motor neurons, particularly those that are likely to participate in postural control. These results provide evidence that neurons subserving motor function undergo activity-dependent maturation in early postnatal life in a manner analogous to sensory systems.

Neuropilin-1 extracellular domains mediate semaphorin D/III-induced growth cone collapse.

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). Here, semD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, we show that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1, the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein that in turn transduces a semD signal into axon repulsion.

Semaphorins A and E act as antagonists of neuropilin-1 and agonists of neuropilin-2 receptors.

Neuropilin-1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, we demonstrate that neuropilins are sufficient to determine the functional specificity of semaphorin responsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity at NP-1 receptors, whereas they act as agonists at receptors containing NP-2.

Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2-5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.

Activity-dependent structural changes during neuronal development.

Activity during the early postnatal period can have a pronounced effect on the structure of neurons in the central nervous system. Recent studies in the cat visual system and in the vertebrate and invertebrate neuromuscular system, have provided new insights into the cellular and molecular features of this process.

Immunofluorescence localization of plasma protein synthesis in cultured chick hepatocytes.

Fibrinogen, albumin and the major apoprotein of high density lipoprotein (apoprotein A) were localized in a primary embryonic chick liver cell culture by indirect immunofluorescence staining. Changes in the pattern of plasma protein synthesis under a variety of conditions, as measured by the accumulation of secreted plasma proteins in the culture medium, could be studied at the cellular level because relative fluorescence intensities were shown to reflect synthetic rates. In all cases studied, the immunofluorescence of the hepatic parenchymal cells was of a similar intensity throughout the monolayers, indicating that the cells in culture constitute a homogeneous population with respect to the synthesis of these plasma proteins.

Synchronized overproduction of AMPA, kainate, and NMDA glutamate receptors during human spinal cord development.

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [3H]glutamate, kainic acid (KA) receptors were labeled with [3H]KA, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [3H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [3H]KA and [3H]AMPA binding is not developmentally regulated. In contrast, the affinity of [3H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury.

Molecular evidence for nitric oxide-mediated motor neuron development.

The complex morphological, electrophysiological and molecular properties of the adult vertebrate nervous system emerge over an extended period in prenatal and early postnatal life. Numerous studies have shown that synaptic activity plays a key role in the postnatal acquisition of mature neuronal phenotype. The cellular and molecular mechanisms subserving activity-dependent development are largely unknown. Several lines of evidence suggest that a rise in intracellular Ca2+ as a consequence of synaptic activity may regulate neuronal differentiation through its interactions with calcium-activated signal transduction molecules such as calcium/calmodulin kinase type II, protein kinase C or nitric oxide synthase (NOS). The aim of the present study is to identify potential signal transduction events subserving postnatal motor neuron development. Here we show that NOS antagonists block the molecular maturation of motor neurons and this effect is likely to be mediated by a subpopulation of ventral horn cells that express NOS transiently during early postnatal life. These results suggest that the local production of nitric oxide within the ventral horn may contribute to a late phase in motor neuron differentiation.

Large diameter primary afferent input is required for expression of the Cat-301 proteoglycan on the surface of motor neurons.

The expression of a cell surface proteoglycan, recognized by monoclonal antibody Cat-301, is regulated by neuronal activity in early life. Here we report that the expression of the Cat-301 proteoglycan on motor neurons depends on primary afferent input in the early postnatal period. Previously we showed that in two different systems, Y-cells in the cat lateral geniculate nucleus and motor neurons in the hamster spinal cord, the expression of the Cat-301 antigen requires neuronal activity during a circumscribed period in development. Disrupting the activity of Y-cells (by dark rearing or by monocular lid suture) or of motor neurons (by sciatic nerve crush or by spinal cord lesion) during the early postnatal period prevents Cat-301 expression. Disrupting neuronal activity in adults has no effect on Cat-301 expression. The onset of Cat-301 expression corresponds to the end of the period of activity-dependent development. In order to further dissect the components of the segmental reflex are required for the expression of Cat-301 on motor neurons, here we evaluated the effect of deafferentation by dorsal rhizotomy. In adult animals two weeks after deafferentation all sciatic motor neurons continue to express Cat-301. In contrast, in neonates two weeks after deafferentation the normal developmental expression of Cat-301 is reduced and less than 50% of sciatic motor neurons express Cat-301. We next selectively lesioned the small diameter afferents using the neurotoxin capsaicin. In contrast to rhizotomy, neonatal deletion of small diameter afferents has no effect on the development of Cat-301 expression on motor neurons. These results imply that input relayed by large diameter primary afferents (probably those conveying muscle and/or joint information) is required for normal maturation of motor neuronal properties during early life. They also provide further evidence for activity-dependent maturation of motor neurons.

Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord.

Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

In situ hybridization analysis of AMPA receptor subunit gene expression in the developing rat spinal cord.

In early postnatal life the acquisition of mature morphological and molecular features of motor neurons is influenced by synaptic activity within the spinal cord. Glutamatergic synaptic neurotransmission is believed to play a central role in this process. We hypothesize that the repertoire of glutamate receptors expressed by neurons in the young spinal cord differ from those expressed in adults and such receptors support activity-dependent developmental plasticity. To explore this idea, we used in situ hybridization histochemistry to determine the distribution, temporal expression, and potential subunit composition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the developing rat spinal cord and compared these findings with those in adult rats. We find qualitative and quantitative changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit gene expression over the first month of postnatal life. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit genes GluR1, 2 and 4 are expressed at greater levels throughout the spinal cord of the neonate versus the adult animals. The developmental down-regulation is most pronounced for GluR1 transcripts, less for GluR2 and GluR4 transcripts, and minimal for GluR3 transcripts. Analysis of flip and flop splice variants of each subunit show that receptors expressed by adult motor neurons are potentially composed of the subunits GluR1 flop, GluR2 flip, GluR3 flip and flop, and GluR4 flip. In neonatal motor neuron all subunits are potentially expressed (except GluR2 flop) with quantitatively the dominent subunits being the flip splice variants of GluR1, 2 and 4. Receptors in the substantia gelatinosa undergo equally dramatic, developmentally independent changes. Changes in the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit composition are likely to have an important effect on the electrophysiological properties of motor neurons and may form part of the molecular identity of neurons capable of undergoing activity-dependent developmental plasticity.

Introduction of the glutamate receptor subunit 1 into motor neurons in vitro and in vivo using a recombinant herpes simplex virus.

We developed and characterized a recombinant herpes simplex virus vector and used it to introduce the complementary DNA encoding glutamate receptor subunit 1 flip into postmitotic motor neurons. Infection of purified motor neurons in vitro with this vector resulted in selective, high-level expression of glutamate receptor subunit 1 immunoreactivity in nearly 100% of the neurons. Patch-clamp experiments demonstrated that the protein product of the glutamate receptor subunit 1 flip transgene assembles into functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels. Herpes simplex virus-glutamate receptor subunit 1 flip was introduced into spinal cord cells by direct injection into the ventral horn and selectively into motor neurons by sciatic nerve injection. High levels of expression were sustained for at least one week and were accompanied by changes in the ionic permeability of AMPA receptors in transgene-expressing neurons. Throughout the first week of infection, there was little evidence for toxicity. Herpes simplex virus provides a versatile tool for manipulating the glutamate receptor phenotype of postmitotic neurons and will permit study of the role of individual glutamate receptor subunits in neuronal physiology and pathophysiology.

Quantitative and qualitative changes in AMPA receptor expression during spinal cord development.

Synaptic activity in early postnatal life is important for the acquisition of mature structural and functional properties of neurons. Previous studies indicate that the mature molecular features of spinal motor neurons emerge during a period of activity-dependent development in early postnatal life. Since glutamatergic synaptic transmission provides the major excitatory drive into motor neurons, glutamate receptors are likely to play a central role in motor neuron activity-dependent development. To gain insight into this process, we have used receptor autoradiography, immunoblotting and immunohistochemistry to determine the distribution, temporal expression and potential subunit composition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors in the developing rat spinal cord. Using two different ligands, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and [3H]-6-cyano-7-nitroquinoxaline-2,3-dione, we find that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites in the adult are largely restricted to the substantia gelatinosa. In marked contrast, during early postnatal life, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites are transiently expressed at high levels in the ventral horn. This parallels previous findings on the developmental regulation of N-methyl-D-aspartate receptor expression. Using alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit-specific antibodies we show by immunoblot analysis and immunohistology that, to varying degrees, the expression patterns of glutamate receptor subunit 1 and glutamate receptor subunits 2/3 are significantly developmentally regulated. The most conspicuous change is the downregulation of glutamate receptor 1 expression within motor neurons over the first three weeks of postnatal life. The qualitative and quantitative changes we observe in glutamate receptor expression in early postnatal life are likely to have a major impact on the electrophysiological properties of young motor neurons and thus may contribute to their activity-dependent development.

Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly.