Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury.

The discovery that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) has focused much attention on the function of SOD1 as related to motor neuron survival. Here we describe the creation and characterization of mice completely deficient for this enzyme. These animals develop normally and show no overt motor deficits by 6 months in age. Histological examination of the spinal cord reveals no signs of pathology in animals 4 months in age. However Cu/Zn SOD-deficient mice exhibit marked vulnerability to motor neuron loss after axonal injury. These results indicate that Cu/Zn SOD is not necessary for normal motor neuron development and function but is required under physiologically stressful conditions following injury.

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Hyperinnervation of neuromuscular junctions caused by GDNF overexpression in muscle.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) by muscle greatly increased the number of motor axons innervating neuromuscular junctions in neonatal mice. The extent of hyperinnervation correlated with the amount of GDNF expressed in four transgenic lines. Overexpression of GDNF by glia and overexpression of neurotrophin-3 and neurotrophin-4 in muscle did not cause hyperinnervation. Thus, increased amounts of GDNF in postsynaptic target cells can regulate the number of innervating axons.

Introduction of a neurotrophin-3 transgene into muscle selectively rescues proprioceptive neurons in mice lacking endogenous neurotrophin-3.

To clarify the role of muscle-derived neurotrophin-3 (NT-3) in the development of sensory neurons, we generated transgenic mice selectively overexpressing NT-3 in skeletal muscles under the control of a myogenin promoter (myo-NT-3 mice). The myo-NT-3 transgene was then bred into an NT-3 null mutant (-/-) line to generate myo-NT-3, NT-3(-/-) mice in which NT-3 was expressed in muscles, but not elsewhere. Transient overexpression of NT-3 in developing muscles increased the number of proprioceptive neurons as well as the density of both their central and peripheral projections, resulting in more Ia afferents in spinal cord and more spindles (end organs of Ia afferents) in muscles. NT-3 expression restricted to muscles was sufficient to secure the development of proprioceptive neurons and their central and peripheral projections in myo-NT-3, NT-3(-/-) mice. The loss of nonproprioceptive neurons observed in NT-3(-/-) mice was not reversed by the transgene, suggesting that these neurons are regulated by NT-3 from sources other than muscle. We conclude that target-derived rather than intraganglionic NT-3 is preeminent in supporting the development of proprioceptive neurons. The level of NT-3 in developing muscles may be the principal factor determining the number of proprioceptive neurons in dorsal root ganglions and spindles in skeletal muscles of adults.

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.

Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects.

The potential functional significance of nerve growth factor (NGF) receptors in spinal motoneurons was studied in newborn rats. 125I-NGF was specifically retrogradely transported by motoneurons from their peripheral nerve terminals. This transport was blocked by an excess of unlabeled NGF but not by cytochrome c. 125I-cytochrome c was not transported. The monoclonal anti-rat NGF receptor antibody, but not a control antibody, was also transported. Despite this ability of motoneurons to transport NGF, treatment of newborn rats with this factor did not increase motoneuron size or synthesis of neurotransmitter enzymes and did not prevent cell death after axotomy. We conclude that NGF receptors of spinal motoneurons can bind, internalize, and retrogradely transport NGF. However, these receptors do not mediate the classic trophic effects of NGF.

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development.

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.

Dorsal root ganglion neurons expressing trk are selectively sensitive to NGF deprivation in utero.

In utero immune deprivation of the neurotrophic molecule nerve growth factor (NGF) results in the death of most, but not all, mammalian dorsal root ganglion (DRG) neurons. The recent identification of trk, trkB, and trkC as the putative high affinity receptors for NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively, has allowed an examination of whether their expression by DRG neurons correlates with differential sensitivity to immune deprivation of NGF. In situ hybridization demonstrates that virtually all neurons expressing trk are lost during in utero NGF deprivation. Most, if not all, neurons expressing trkB and trkC survive this treatment. In contrast, the low affinity NGF receptor, p75NGFR, is expressed in both NGF deprivation-resistant and -sensitive neurons. These experiments show that DRG neurons expressing trk require NGF for survival. Furthermore, at least some of the DRG neurons that do not require NGF express the high affinity receptor for another neurotrophin. Finally, these experiments provide evidence that trk, and not p75NGFR, is the primary effector of NGF action in vivo.

GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys.

Glial cell line-derived neurotrophic factor (GDNF) signals through a receptor complex composed of the Ret tyrosine kinase and a glycosylphosphatidylinositol- (GPI-) anchored cell surface coreceptor, either GDNF family receptor alpha1 (GFR alpha1) or GFR alpha2. To investigate the usage of these coreceptors for GDNF signaling in vivo, gene targeting was used to produce mice lacking the GFR alpha1 coreceptor. GFR alpha1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both GDNF- and Ret-deficient mice. Midbrain dopaminergic and motor neurons in GFR alpha1 null mice were normal. Minimal or no neuronal losses were observed in a number of peripheral ganglia examined, including the superior cervical and nodose, which are severely affected in both Ret- and GDNF-deficient mice. These results suggest that while stringent physiologic pairing exists between GFR alpha1 and GDNF in renal and enteric nervous system development, significant cross-talk between GDNF and other GFR alpha coreceptors must occur in other neuronal populations.

BAX is required for neuronal death after trophic factor deprivation and during development.

Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurities; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation-induced death of sympathetic and motor neurons depends on Bax.

IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life.

We have tested the role of glial cell line-derived neurotrophic factor (GDNF) in regulating a group of putatively nociceptive dorsal root ganglion (DRG) neurons that do not express calcitonin gene-related peptide (CGRP) and that downregulate the nerve growth factor (NGF) receptor tyrosine kinase, TrkA, after birth. We show that mRNA and protein for the GDNF receptor tyrosine kinase, Ret, are expressed in the DRG in patterns that differ markedly from those of any of the neurotrophin receptors. Most strikingly, a population of small neurons initiates expression of Ret between embryonic day 15.5 and postnatal day 7.5 and maintains Ret expression into adulthood. These Ret-expressing small neurons are selectively labeled by the lectin IB4 and project to lamina IIi of the dorsal horn. Ret-expressing neurons also express the glycosyl-phosphatidyl inositol-linked (GPI-linked) GDNF binding component GDNFR-alpha and retrogradely transport 125I-GDNF, indicating the presence of a biologically active GDNF receptor complex. In vitro, GDNF supports the survival of small neurons that express Ret and bind IB4 while failing to support the survival of neurons expressing TrkA and CGRP. Together, our findings suggest that IB4-binding neurons switch from dependence on NGF in embryonic life to dependence on GDNF in postnatal life and are likely regulated by GDNF in maturity.

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

Molecular genetics of neuronal survival.

The past year has witnessed remarkable progress towards understanding the molecular genetics of neuronal survival. Gene-targeting experiments in mice have confirmed the long-standing idea that the nerve growth factor model of neuronal survival--that is, neuronal dependence on target-derived molecules during a critical period in development--is broadly applicable. Furthermore, a variety of biochemical and genetic techniques applied to both mammals and invertebrates have identified new genes involved in regulating cell survival during development.

Different signaling pathways mediate regenerative versus developmental sensory axon growth.

Recent advances in defining neurotrophin signaling mediators have provided insights into the signal transduction mechanisms that underlie axon growth. Evidence is accumulating that major Trk effectors regulate the morphological development of embryonic peripheral neurons. Less is known about signaling related to the robust axon extension that follows peripheral axotomy of adult neurons. Regenerative axon growth can be mimicked in vitro by a "conditioning" lesion performed 2 weeks before culture (Smith and Skene, 1997). Previous work has implicated both neurotrophins and cytokines in this response. Because signal transduction mediators of both of these families of growth factors are well characterized, we have compared the role of neurotrophin and cytokine signaling in developmental versus regenerative sensory axon growth. Chemical inhibitors were administrated to embryonic and axotomized sensory neurons in vitro to block the activation of Erk kinase (MEK)-extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI3-K), and janus kinase (JAK) signaling. As expected, both MEK and PI3-K inhibition blocked axon growth from both naive and NGF-stimulated embryonic day 13 sensory neurons, whereas inhibition of JAK phosphorylation had no effect. In contrast, neither MEK nor PI3-K inhibitors blocked elongation of adult sensory neurons after a conditioning lesion. However, the addition of a JAK2 inhibitor prevented the regenerative axon response. Consistent with these pharmacological results, the percentage of neurons showing intense nuclear signal transducers and activators of transcription 3 phosphorylation after a conditioning lesion was markedly increased compared with controls. These observations demonstrate that the signaling mediators that underlie regenerative axon growth are distinct from those used during development and suggest that cytokine signaling may be critical to peripheral nervous system regeneration.

Glial cell line-derived neurotrophic factor and developing mammalian motoneurons: regulation of programmed cell death among motoneuron subtypes.

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF after in utero treatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability. In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRalpha1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRalpha2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRalpha1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRalpha1/GFRalpha2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRalpha2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration.

Evidence garnered from both human autopsy studies and genetic animal models has suggested a potential role for astrocytes in the pathogenesis of amyotrophic lateral sclerosis (ALS). Currently, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) represent the only known cause of motoneuron loss in the disease, producing 21q linked familial ALS (FALS). To determine whether astrocytic dysfunction has a primary role in familial ALS, we have generated multiple lines of transgenic mice expressing G86R mutant SOD1 restricted to astrocytes. In GFAP-m SOD1 mice, astrocytes exhibit significant hypertrophy and increased GFAP reactivity as the animals mature. However, GFAP-mutant SOD1 transgenic mice develop normally and do not experience spontaneous motor deficits with increasing age. Histological examination of spinal cord in aged GFAP-mSOD1 mice reveals normal motoneuron and microglial morphology. These results indicate that 21q linked FALS is not a primary disorder of astrocytes, and that expression of mutant SOD1 restricted to astrocytes is not sufficient to cause motoneuron degeneration in vivo. Expression of mutant SOD1 in other cell types, most likely neurons, is critical for the initiation of disease.

Glial cell line-derived neurotrophic factor administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) in embryonic muscle fibers causes dramatic hyperinnervation of neuromuscular junctions. However, it is not known whether GDNF induces the extra innervation by regulation of axonal branching and/or synaptic maintenance. To address this issue, high levels of circulating GDNF were established by administering subcutaneous injections starting either at birth or later and continuing for up to 40 d. Treatment with exogenous GDNF beginning in the first week, but not later, increased the number of axons converging at neuromuscular junctions. The effect of GDNF on the branching pattern of individual motor axons was determined by reconstructing labeled axonal arbors from transgenic mice expressing yellow fluorescent protein in subsets of motor neurons. Whereas, at postnatal day 8 (P8) individual axons in control animals branched to sporadically innervate junctions within circumscribed regions of the muscle, motor units from GDNF injected animals had significantly more axonal branches and exhibited a high degree of localized arborization such that adjacent muscle fibers were often innervated by the same axon. Administration beginning at P0 and continuing through P40 prolonged multiple innervation of most fibers throughout the period of injection. Between P30 and P40 there was no net change in multiple innervation, although there was evidence of retraction bulbs, suggesting that axon extension and retraction were in equilibrium. We conclude that GDNF has a developmentally regulated effect on presynaptic branching and that sustained administration of GDNF induces a state of continuous synaptic remodeling.

The transmembrane protein semaphorin 6A repels embryonic sympathetic axons.

Semaphorin 6A (Sema6A) (previously named Semaphorin VIa) is the originally described member of the vertebrate semaphorin class 6, a group of transmembrane semaphorins homologous to the insect semaphorin class 1. Although Sema-1a (previously named semaphorin I) has been implicated in axon guidance in insects, the function of Sema6A is currently unknown. We have expressed the extracellular domain of Sema6A in mammalian cells as either a monomeric or a dimeric fusion protein and tested for potential axon guidance effects on two populations of embryonic neurons in growth cone collapse and collagen matrix chemorepulsion assays. Sema6A was observed to induce growth cone collapse of sympathetic neurons with an EC50 of approximately 200 pM, although a 10-fold higher (EC50 of approximately 2 nM) concentration was necessary to induce growth cone collapse of dorsal root ganglion neurons. The activity of Sema6A is likely to depend on protein dimerization or oligomerization. Although Sema6A mRNA is expressed in complex patterns during embryonic development, it is strikingly absent from sympathetic ganglia. Sema6A is, however, expressed in areas avoided by sympathetic axons and in areas innervated by sympathetics, but before their arrival. Our results demonstrate that transmembrane semaphorins, like the secreted ones, can act as repulsive axon guidance cues. Our findings are consistent with a role for Sema6A in channeling sympathetic axons into the sympathetic chains and controlling the temporal sequence of sympathetic target innervation.

Rostrocaudal differences in dendritic growth and synaptogenesis in rat sympathetic chain ganglia.

Vertebrate embryos show a rostral to caudal gradient of morphogenesis. I have investigated the effect of this developmental gradient on neuronal maturation and synapse formation by comparing the development of rostral and caudal sympathetic chain ganglia in the rat. In young adults the superior cervical and lumbar ganglia are almost identical in terms of neuronal morphology and quantitative aspects of innervation. In neonates, however, substantial rostrocaudal differences are apparent in dendritic complexity, number of axons innervating ganglion cells, and synaptic density. There is an associated delay in ganglion cell-target interactions in the lumbar region. My results show that rostrocaudal position is significantly correlated with the time course of dendritic growth and synaptogenesis and suggest that ganglion cell-target interactions may be important in these developmental processes. This difference in developmental rate, however, does not lead to differences in neuronal morphology or synaptic density between rostral and caudal ganglia in maturity.

Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons.

Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is required for the survival of all small and many intermediate-sized murine dorsal root ganglion (DRG) neurons during development, accounting for 80% of the total DRG population. Surprisingly, NGF/TrkA-dependent neurons include a large population that does not express TrkA in adult mice (Silos-Santiago et al., 1995). This finding suggests two hypotheses: Neurons lacking TrkA in the adult may express TrkA during development, or they may be maintained through a paracrine mechanism by TrkA-expressing neurons. To determine whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood, we examined the distribution of TrkA protein during development. We show here that TrkA expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at postnatal day (P) 21. Because the period of TrkA down-regulation corresponds with a critical period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell [1993] Trends Neurosci. 16:353-359), we examined whether NGF modulates the down-regulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the extent of TrkA down-regulation. Our results demonstrate a novel form of regulation of neurotrophin receptor expression that occurs late in development. All DRG neurons that require NGF for survival express TrkA during embryogenesis, and many continue to express TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The subsequent down-regulation of TrkA is likely to be importantly related to functional distinctions among nociceptive neurons in maturity.