Postsynaptic scaffolding molecules modulate the localization of neuroligins.

Previous work has shown an important role for neuroligins in promoting the formation of synaptic connections in cultured cells. Although neuroligins enhance both excitatory and inhibitory synapse formation, individual neuroligin isoforms have been shown to preferentially localize to either glutamatergic or GABAergic synapses. Current evidence points to an important role for both the extracellular and intracellular domains of neuroligins in their synaptic localization. Although postsynaptic density protein 95 (PSD-95) has been shown to be involved in the recruitment of neuroligin 1 to excitatory synapses, the localization of neuroligin 2 (NL2) and neuroligin 3 (NL3) to excitatory and inhibitory synapses is less well defined. We assessed the roles of gephyrin and PSD-95, postsynaptic scaffolding molecules exclusively localized to inhibitory and excitatory synapses, respectively, in localizing NL2 and NL3 in primary neuronal cultures. We demonstrate that knockdown of gephyrin results in a significant shift of NL2 from inhibitory to excitatory synaptic contacts, while knockdown of PSD-95 leads to a partial shift of NL2 and NL3 from excitatory to inhibitory contacts. Furthermore, analysis of specific domain deletions within the C-terminal, intracellular domain of NL2 reveals that the region between amino acids 716 and 782 is required for the normal synaptic clustering of this protein. Together, these data suggest that intracellular mechanisms are involved in the targeting of different neuroligin family members to synapses (216).

Early induction of Talpha1 alpha-tubulin transcription in neurons of the developing nervous system.

In this report, we have examined the relationship between the onset of neuronal gene transcription and neuronal development by characterizing expression of the early panneuronal Talpha1 alpha-tubulin promoter in developing neurons. In the peripheral nervous system, detectable expression of a beta-galactosidase transgene driven by the Talpha1 promoter (Talpha1:nlacZ) was coincident with neuronal birth dates, with the exception of sympathetic neuroblasts, which expressed the transgene prior to terminal mitosis. Similarly, in the central nervous system, the onset of beta-galactosidase expression was coincident with neuronal birth dates in most identifiable populations of central neurons. A small subpopulation of transgene-positive cells localized to ventricular zones, but the vast majority was observed in locations consistent with their identification as migrating and/or differentiating neurons. To determine more precisely the temporal relationship between transgene expression and terminal mitosis, we analyzed cultures of cortical progenitors that become postmitotic neurons in vitro. When initially plated, the vast majority of cells consisted of dividing, nestin-positive progenitors. Neurons differentiated from these progenitors as early as 1 day in vitro, as indicated by immunostaining for betaIII-tubulin, a neuron-specific tubulin isotype that is turned on shortly after terminal mitosis. Double-labeling studies showed that Talpha1:nlacZ expression was detectable in the same cells and at approximately the same time as was betaIII-tubulin, indicating that detectable transcription of the Talpha1 alpha-tubulin promoter commences at the time of terminal mitosis, at least in culture. This promoter, therefore, provides a valuable tool for genetic manipulation of early developing neurons in transgenic mice.

p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors.

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/- or p53-/- mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth.

In this report, we describe a novel local mechanism necessary for optimal axonal growth that involves hepatocyte growth factor (HGF). Sympathetic neurons of the superior cervical ganglion coexpress bioactive HGF and its receptor, the Met tyrosine kinase, both in vivo and in vitro. Exogenous HGF selectively promotes the growth but not survival of cultured sympathetic neurons; the magnitude of this growth effect is similar to that observed with exogenous NGF. Conversely, HGF antibodies that inhibit endogenous HGF decrease sympathetic neuron growth but have no effect on survival. This autocrine HGF is required locally by sympathetic axons for optimal growth, as demonstrated using compartmented cultures. Thus, autocrine HGF provides a local, intrinsic mechanism for promoting neuronal growth without affecting survival, a role that may be essential during developmental axogenesis or after neuronal injury.

Functional evidence that BDNF is an anterograde neuronal trophic factor in the CNS.

In this report, we have tested the hypothesis that brain-derived neurotrophic factor (BDNF) is an anterograde neurotrophic factor in the CNS and have focused on central noradrenergic neurons that synthesize BDNF. Double-label immunocytochemistry for BDNF and dopamine-beta-hydroxylase (DBH), a marker for noradrenergic neurons, demonstrated that BDNF is partially localized to noradrenergic nerve fibers and terminals in the adult rat brain. To test the functional importance of this anterograde BDNF, we analyzed transgenic mice carrying a DBH-BDNF minigene. Increased synthesis of BDNF in noradrenergic neurons of DBH-BDNF mice caused elevated TrkB tyrosine kinase activation throughout postnatal life in the neocortex, a noradrenergic target region. This afferently regulated increase in TrkB receptor activity led to long-lasting alterations in cortical morphology. To determine whether noradrenergic neuron-expressed BDNF also anterogradely regulated neuronal survival, we examined a second noradrenergic target, neonatal facial motoneurons. One week after axotomy, 72% of facial motoneurons were lost in control animals, whereas only 30-35% were lost in DBH-BDNF transgenic mice. Altogether, these results indicate that BDNF is anterogradely transported to fibers and terminals of noradrenergic neurons, that anterogradely secreted BDNF causes activation of TrkB in target regions, and that this secretion has functional consequences for target neuron survival and differentiation. This presynaptic secretion of BDNF may provide a cellular mechanism for modulating neural circuitry, in either the developing or mature nervous system.

The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death.

To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF-/- and p75NTR-/- mice. In BDNF-/- mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR-/- mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR-/- neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.

Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis.

We have asked whether p75(NTR) may play a role in neuronal apoptosis by producing transgenic mice that express the p75(NTR) intracellular domain within peripheral and central neurons. These animals showed profound reductions in numbers of sympathetic and peripheral sensory neurons as well as cell loss in the neocortex, where there is normally little or no p75(NTR) expression. Developmental loss of facial motor neurons was not observed, but induced expression of the p75(NTR) intracellular domain within adult animals led to increased motor neuron death after axotomy. Biochemical analyses suggest that these effects were not attributable to a p75(NTR)-dependent reduction in trk activation but instead indicate that the p75(NTR) intracellular domain may act as a constitutive activator of signaling cascades that regulate apoptosis in both peripheral and central neurons.

Synaptic innervation density is regulated by neuron-derived BDNF.

In this report, we have examined the role of neuron-derived BDNF at an accessible synapse, that of preganglionic neurons onto their sympathetic neuron targets. Developing and mature sympathetic neurons synthesize BDNF, and preganglionic neurons express the full-length BDNF/TrkB receptor. When sympathetic neuron-derived BDNF is increased 2- to 4-fold in transgenic mice, preganglionic cell bodies and axons hypertrophy, and the synaptic innervation to sympathetic neurons is increased. Conversely, when BDNF synthesis is eliminated in BDNF -/- mice, preganglionic synaptic innervation to sympathetic neurons is decreased. Together these results indicate that variations in neuronal neurotrophin synthesis directly regulate neuronal circuitry by selectively modulating synaptic innervation density.

Comparison of the expression of a T alpha 1:nlacZ transgene and T alpha 1 alpha-tubulin mRNA in the mature central nervous system.

We have previously demonstrated that one member of the alpha-tubulin multigene family, termed T alpha 1 in rats, is a panneuronal gene that is regulated as a function of neuronal growth and regeneration. Moreover, 1.1 kb of the 5' upstream region from this gene is sufficient to direct expression of a marker gene to growing neurons in transgenic mice. In this report, we have characterized the distribution of the T alpha 1:nlacZ transgene in the mature central nervous system in two lines of transgenic mice and have compared its expression to that of the endogenous T alpha 1 alpha-tubulin mRNA. These results demonstrate that the pattern of expression of the T alpha 1:nlacZ transgene is similar to that of T alpha 1 mRNA, with a few notable differences. Furthermore, expression of the transgene and the mRNA within the mature brain is panneuronal and, in many cases, is highest in those populations of neurons that show some capacity for morphological growth. These results, together with our previous studies on mature regenerating neurons (Gloster et al. [1994] J. Neurosci. 14:7319-7330; Wu et al. [1994] Soc. Neurosci. Abstr. 20:542) suggest that the T alpha 1:nlacZ transgene will provide a useful marker of growth-associated gene expression in the mature nervous system.