Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Talpha1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors. In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Talpha1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and Talpha1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and Talpha1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.

Neurotrophins are essential for development and maintenance of the vertebrate nervous system. Paradoxically, although mature neurotrophins promote neuronal survival by binding to tropomyosin receptor kinases and p75 neurotrophin receptor (p75(NTR)), pro-neurotrophins induce apoptosis in cultured neurons by engaging sortilin and p75(NTR) in a death-signaling receptor complex. Substantial amounts of neurotrophins are secreted in pro-form in vivo, yet their physiological significance remains unclear. We generated a sortilin-deficient mouse to examine the contribution of the p75(NTR)/sortilin receptor complex to neuronal viability. In the developing retina, Sortilin 1 (Sort1)(-/-) mice showed reduced neuronal apoptosis that was indistinguishable from that observed in p75(NTR)-deficient (Ngfr(-/-)) mice. To our surprise, although sortilin deficiency did not affect developmentally regulated apoptosis of sympathetic neurons, it did prevent their age-dependent degeneration. Furthermore, in an injury protocol, lesioned corticospinal neurons in Sort1(-/-) mice were protected from death. Thus, the sortilin pathway has distinct roles in pro-neurotrophin-induced apoptotic signaling in pathological conditions, but also in specific stages of neuronal development and aging.

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3␣1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, bettercharacterized TRP channels. We selected two splice variants, TRPM3␣1 and TRPM3␣2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3␣1 as well as TRPM3␣2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg 2؉ . However, these two variants are highly different in their ionic selectivity. Whereas TRPM3␣1-encoded channels are poorly permeable for divalent cations, TRPM3␣2-encoded channels are well permeated by Ca 2؉ and Mg 2؉ . Additionally, we found that currents through TRPM3␣2 are blocked by extracellular monovalent cations, whereas currents through TRPM3␣1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ionconducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channelencoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles. The transient receptor potential (TRP)1 gene family comprises at least 28 mammalian genes divided into seven subfamilies (1, 2). Most of the encoded proteins exhibit common structural features such as six predicted transmembrane (TM) domains with a putative pore loop between TM5 and TM6 and the so-called TRP box after TM6 (1, 2). Although all members of this group have been reported to form cationic channels, their mechanisms of activation, their regulation, and their biological functions are remarkably diverse. They also display a large variety of different cation selectivities (1, 2). For example, TRPM4 and TRPM5 have been described as impermeable for divalent cations (3-5), whereas TRPV5 and TRPV6 appear to be exclusively permeable for Ca 2ϩ (6, 7). The diversity of TRP channels is further increased by the fact that most members of the TRP gene family can give rise to several different transcripts due to alternative splicing (8).In a few cases, the functional consequences of these alternative splice events are now beginning to emerge. For example, missplicing of TRPM6 transcripts is associated with a hereditary disorder called hypomagnesemia with secondary hypocalcemia (9, 10), and an amino-terminal-truncated variant of TRPM4 appears to modulate Ca 2ϩ oscillations after receptor stimulation in T lymphocytes (11).However, up to now, the largest number of different splice variants ...

The unprocessed precursor of the neurotrophin nerve growth factor (NGF), proNGF, has been suggested to be a death-inducing ligand for the neurotrophin receptor p75. Whether proNGF is a true pathophysiological ligand that is secreted, binds p75, and activates cell death in vivo, however, has remained unknown. Here, we report that after brain injury, proNGF was induced and secreted in an active form capable of triggering apoptosis in culture. We further demonstrate that proNGF binds p75 in vivo and that disruption of this binding results in complete rescue of injured adult corticospinal neurons. These data together suggest that proNGF binding to p75 is responsible for the death of adult corticospinal neurons after lesion, and they help to establish proNGF as the pathophysiological ligand that activates the celldeath program by means of p75 after brain injury. Interference in the binding of proNGF to p75 may provide a therapeutic approach for the treatment of disorders involving neuronal loss.