The effects of choline supplementation during two time-frames of early development on radial-arm maze performance and the morphology of basal forebrain neurons immunoreactive for the P75 neurotrophin receptor (NTR) in male and female Sprague-Dawley rats were examined. In the first experiment, rats were supplemented with choline chloride from conception until weaning. At 80 days of age, subjects were trained once a day on a 12-arm radial maze for 30 days. Compared to control littermates, supplemented rats made fewer working and reference memory errors; however, the memory enhancing effects of choline supplementation were greater in males than females. A morphometric analysis of NTR-immunoreactive cell bodies at three levels through the medial septum/diagonal band (MS/DBv) of these rats revealed that perinatal choline supplementation caused the somata of cells in the MS/DBv to be larger by 8-15%. In a second experiment, choline supplementation was restricted to embryonic days 12-17. A developmental profile of NTR immunoreactive cell bodies in the MS/DBv of 0-, 8-, 16-, 30- and 90-day old male and female rats again revealed that cell bodies were larger in choline-supplemented rats than controls. As in the behavioral studies, the effect of choline supplementation was greater in male than female rats. These data are consistent with the hypothesis that supplementation with choline chloride during early development leads to an increase in the size of cell bodies of NTR-immunoreactive cells in the basal forebrain and that this change may contribute to long-term improvement in spatial memory.

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) contributes in understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Abeta peptide 42 (Abeta42), calcium ionophore A23187, interleukin (IL)-1beta, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the antiapoptotic proteins of the Bcl-2 family and decreases the expression of proapoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1beta-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-beta (Abeta) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer's disease (AD) cornu ammonis region 1 (CA1) hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A(2) (cPLA(2)), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Abeta42-triggered activation of pro-inflammatory genes and upregulated the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that NPD1 promotes brain and retina cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Abeta42-induced neurotoxicity and other forms of cell injury, which in turn fosters homeostasis during development in aging, as well as during the initiation and progression of neurodegenerative diseases.

Biochemical studies on postmortem brains of patients with Parkinson's disease (PD) have greatly contributed to our understanding of the molecular pathogenesis of this disease. The discovery by 1960 of a dopamine deficiency in the nigro-striatal dopamine region of the PD brain was a landmark in research on PD. At that time we collaborated with Hirotaro Narabayashi and his colleagues in Japan and with Peter Riederer in Germany on the biochemistry of PD by using postmortem brain samples in their brain banks. We found that the activity, mRNA level, and protein content of tyrosine hydroxylase (TH), as well as the levels of the tetrahydrobiopterin (BH4) cofactor of TH and the activity of the BH4-synthesizing enzyme, GTP cyclohydrolase I (GCHI), were markedly decreased in the substantia nigra and striatum in the PD brain. In contrast, the molecular activity (enzyme activity/enzyme protein) of TH was increased, suggesting a compensatory increase in the enzyme activity. The mRNA levels of all four isoforms of human TH (hTH1-hTH4), produced by alternative mRNA splicing, were also markedly decreased. This finding is in contrast to a completely parallel decrease in the activity and protein content of dopamine beta-hydroxylase (DBH) without changes in its molecular activity in cerebrospinal fluid (CSF) in PD. We also found that the activities and/or the levels of the mRNA and protein of aromatic L-amino acid decarboxylase (AADC, DOPA decarboxylase), DBH, phenylethanolamine N-methyltransferase (PNMT), which synthesize dopamine, noradrenaline, and adrenaline, respectively, were also decreased in PD brains, indicating that all catecholamine systems were widely impaired in PD brains. Programmed cell death of the nigro-striatal dopamine neurons in PD has been suggested from the following findings on postmortem brains: (1) increased levels of pro-inflammatory cytokines such as TNF-alpha and IL-6; (2) increased levels of apoptosis-related factors such as TNF-alpha receptor R1 (p 55), soluble Fas and bcl-2, and increased activities of caspases 1 and 3; and (3) decreased levels of neurotrophins such as brain-derived nerve growth factor (BDNF). Immunohistochemical data and the mRNA levels of the above molecules in PD brains supported these biochemical data. We confirmed by double immunofluorescence staining the production of TNF-alpha and IL-6 in activated microglia in the putamen of PD patients. Owing to the recent development of highly sensitive and wide-range analytical methods for quantifying mRNAs and proteins, future assays of the levels of various mRNAs and proteins not only in micro-dissected brain tissues containing neurons and glial cells, but also in single cells from frozen brain slices isolated by laser capture micro-dissection, coupled with toluidine blue, Nissl staining or immunohistochemical staining, should further contribute to the elucidation of the molecular pathogenesis of PD and other neurodegenerative or neuropsychiatric diseases.

Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the central nervous system where it plays several pivotal roles in synaptic plasticity and neuronal survival. As a consequence, BDNF became a key target in the physiopathology of several neurological and psychiatric diseases. Recent studies have reported altered levels of BDNF in the circulation, i.e. serum or plasma, of patients with Alzheimer's disease (AD), and low BDNF levels in the CSF as predictor of future cognitive decline in healthy older subjects. Altered BDNF circulating levels have also been reported in other neurodegenerative and psychiatric disorders, hampering its use as a specific biomarker for AD. Therefore, BDNF seems to be an unspecific biomarker of neuropsychiatric disorders marked by neurodegenerative changes.

The neurotrophin (NT) brain-derived neurotrophic factor (BDNF) plays an essential role in the formation of long-term potentiation (LTP). Here, we address whether this modulation by BDNF requires its continuous presence, or whether a local increase in BDNF is necessary during a specific time period of LTP initiation. Using electrical field stimulation of primary cultures of hippocampal neurons, we demonstrate that short high-frequency bursts of stimuli that induce LTP evoke also an instantaneous secretion of BDNF. In contrast, stimuli at low frequencies, inducing long-term depression, do not enhance BDNF secretion, suggesting that BDNF is specifically present, and thus required, at the time of LTP induction. The field-stimulation-mediated BDNF secretion depends on the formation of action potentials and is induced by IP(3)-mediated Ca(2+) release from intracellular stores. Experiments, aimed at determining the sites of NT secretion that use NT6, showed similar patterns of surface labeling by field stimulation to those shown previously by high potassium.

Among the most critical actions of thyroid hormone in man and other mammals are those exerted on brain development. Severe hypothyroidism during the neonatal period leads to structural alterations, including hypomyelination and defects of cell migration and differentiation, with long-lasting, irreversible effects on behavior and performance. A complex regulatory mechanism operates in brain involving regulation of the concentration of the active hormone, T3, and the control of gene expression. Most brain T3 is formed locally from its precursor, T4, by the action of type II deiodinase which is expressed in glial cells, tanycytes, and astrocytes. Type III deiodinase (DIII) is also involved in the regulation of T3 concentrations, especially during the embryonic and early post-natal periods. DIII is expressed in neurons and degrades T4 and T3 to inactive metabolites. The action of T3 is mediated through nuclear receptors, which are expressed mainly in neurons. The receptors are ligand-modulated transcription factors, and a number of genes have been identified as regulated by thyroid hormone in brain. The regulated genes encode proteins of myelin, mitochondria, neurotrophins and their receptors, cytoskeleton, transcription factors, splicing regulators, cell matrix proteins, adhesion molecules, and proteins involved in intracellular signaling pathways. The role of thyroid hormone is to accelerate changes of gene expression that take place during development. Surprisingly, null-mutant mice for the T3 receptors show almost no signs of central nervous system involvement, in contrast with the severe effects of hypothyroidism. The resolution of this paradox is essential to understand the role of thyroid hormone and its receptors in brain development and function.

Hearing is conveyed from the auditory receptors, the hair cells in the organ of Corti, to the brain via the spiral ganglion neurons. Damage or loss of either spiral ganglion neurons or hair cells causes hearing impairment. Such hearing disorders are often permanent and can be caused by therapeutic agents, such as aminoglycoside antibiotics and cisplatin, or by aging, loud sounds, infections and mechanical injury (1). Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrohin family of neurotrophic factors that also include nerve growth factor (NGF) and neurotrophin-4/5 (NT-4), are important in development of the neuronal components of the inner ear. We report here that the loss of target innervation and the degeneration of approximately 90% of the adult spiral ganglion neurons caused by aminoglycoside toxicity can be prevented by infusion of the neurotrophic factor, neurotrophin-3 (NT-3) in the membranous labyrinth in guinea pigs. The potency of NT-3 in protecting spiral ganglion neurons from degenerating suggests that neurotrophins may be useful for the treatment of hearing disorders.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

In this communication, we demonstrate the sequential expression of endogenous molecules, including immediate early genes (IEGs), cytokines, neurotrophins, and neurotrophin receptors in the injured spinal cord. In the acute phase, expression of IEGs and cytokines mRNAs were rapidly upregulated within 1 h in nonneuronal cells in the lesioned sites and the surrounding spinal white and gray matter. Maximal expression was observed at 1 h for c-fos and TNF-alpha mRNAs, at 3 h for c-jun and IL-6 mRNAs, and at 6 h for IL-1 beta mRNA, and these signals were virtually nondetectable after 6-12 h from the onset of the injury. Some of these genes products may promote the degeneration of damaged cells and tissues, while others may be involved in the subsequent repair processes. In the subacute phase, expression of NGF, BDNF, NT-3, p75LNGFR and Trk B mRNAs began to increase in the nonneuronal cells and neuronal cells from 6 h, and peaked at 24-72 h in the area where expression of mRNAs for IEGs and cytokines overlapped. Signals for IL-6 mRNA were also observed in motoneurons at 24-72 h after the injury, with the suggestion that these molecules may be involved in promoting axonal sprouting in the injured spinal cord. Of further interest was the finding that this upregulation of IL-1 beta, BDNF, and NT-3 mRNAs in injured spinal cord was attenuated by treatment with high dose glucocorticoids, with the suggestion that the downregulation of BDNF and NT-3 might be disadvantageous to survival and axonal sprouting of spinal neurons.

Wallerian degeneration is a cascade of stereotypical events in reaction to injury of nerve fibres. These events consist of cellular and molecular alterations, including macrophage invasion, activation of Schwann cells, as well as neurotrophin and cytokine upregulation. This review focuses on cellular and molecular changes distal to various types of peripheral nerve injury which simultaneously contribute to axonal regeneration and neuropathic pain induction. In addition to the stereotypical events of Wallerian degeneration, various types of nerve damage provide different conditions for both axonal regeneration and neuropathic pain induction. Wallerian degeneration of injured peripheral nerve is associated with an inflammatory response including rapid upregulation of the immune signal molecules like cytokines, chemokines and transcription factors with both beneficial and detrimental effects on nerve regeneration or neuropathic pain induction. A better understanding of the molecular interactions between the immune system and peripheral nerve injury would open the possibility for targeting these inflammatory mediators in therapeutic interventions. Understanding the pleiotropic effects of cytokines/chemokines, however, requires investigating their highly specific pathways and precise points of action.

OBJECTIVE

To investigate the potential therapeutic benefit of intravitreally implanted dental pulp stem cells (DPSCs) on axotomized adult rat retinal ganglion cells (RGCs) using in vitro and in vivo neural injury models.

METHODS

Conditioned media collected from cultured rat DPSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were assayed for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) secretion using ELISA. DPSCs or BMSCs were cocultured with retinal cells, with or without Fc-TrK inhibitors, in a Transwell system, and the number of surviving βIII-tubulin⁺ retinal cells and length/number of βIII-tubulin⁺ neurites were quantified. For the in vivo study, DPSCs or BMSCs were transplanted into the vitreous body of the eye after a surgically induced optic nerve crush injury. At 7, 14, and 21 days postlesion (dpl), optical coherence tomography (OCT) was used to measure the retinal nerve fiber layer thickness as a measure of axonal atrophy. At 21 dpl, numbers of Brn-3a⁺ RGCs in parasagittal retinal sections and growth-associated protein-43⁺ axons in longitudinal optic nerve sections were quantified as measures of RGC survival and axon regeneration, respectively.

RESULTS

Both DPSCs and BMSCs secreted NGF, BDNF, and NT-3, with DPSCs secreting significantly higher titers of NGF and BDNF than BMSCs. DPSCs, and to a lesser extent BMSCs, promoted statistically significant survival and neuritogenesis/axogenesis of βIII-tubulin⁺ retinal cells in vitro and in vivo where the effects were abolished after TrK receptor blockade.

CONCLUSIONS

Intravitreal transplants of DPSCs promoted significant neurotrophin-mediated RGC survival and axon regeneration after optic nerve injury.

Spiral ganglion neurons (SGNs) are postsynaptic to hair cells and project to the brainstem. The inner hair cell (IHC) to SGN synapse is susceptible to glutamate excitotoxicity and to acoustic trauma, with potentially adverse consequences to long-term SGN survival. We used a cochlear explant culture from P6 rat pups consisting of a portion of organ of Corti maintained intact with the corresponding portion of spiral ganglion to investigate excitotoxic damage to IHC-SGN synapses in vitro. The normal innervation pattern is preserved in vitro. Brief treatment with NMDA and kainate results in loss of IHC-SGN synapses and degeneration of the distal type 1 SGN peripheral axons, mimicking damage to SGN peripheral axons caused by excitotoxicity or noise in vivo. The number of IHC presynaptic ribbons is not significantly altered. Reinnervation of IHCs occurs and regenerating axons remain restricted to the IHC row. However, the number of postsynaptic densities (PSDs) does not fully recover and not all axons regrow to the IHCs. Addition of either neurotrophin-3 (NT-3) or BDNF increases axon growth and synaptogenesis. Selective blockade of endogenous NT-3 signaling with TrkC-IgG reduced regeneration of axons and PSDs, but TrkB-IgG, which blocks BDNF, has no such effect, indicating that endogenous NT-3 is necessary for SGN axon growth and synaptogenesis. Remarkably, TrkC-IgG reduced axon growth and synaptogenesis even in the presence of BDNF, indicating that endogenous NT-3 has a distinctive role, not mimicked by BDNF, in promoting SGN axon growth in the organ of Corti and synaptogenesis on IHCs.

Adherent cultures of E10.5 rat neuroepithelial cells (NEP cells) from the caudal neural tube require FGF (fibroblast growth factor) and CEE (chick embryo extract) to proliferate and maintain an undifferentiated phenotype in culture. Epidermal growth factor (EGF) does not support E10.5 NEP cells in adherent culture and NEP cells do not form EGF-dependent neurospheres. NEP cells, however, can be grown as FGF-dependent neurospheres. NEP cells express nestin and lack all lineage-specific markers for neuronal and glial sublineages, retain their pleuripotent character over multiple passages, and can differentiate into neurons, astrocytes, and oligodendrocytes when plated on laminin in the absence of CEE. In clonal culture, NEP cells undergo self-renewal and generate colonies that vary in size from single cells to several thousand cells. With the exception of a few single-cell clones, all other NEP-derived clones contain more than one identified phenotype, with over 40% of the colonies containing A2B5, beta-111 tubulin, and GFAP-immunoreactive cells. Thus, NEP cells are multipotent and capable of generating multiple neural derivatives. NEP cells also differentiate into motoneurons immunoreactive for choline acetyl transferase (ChAT) and the low-affinity neurotrophin receptor (p75) in both mass and clonal culture. Double labeling of clones for ChAT and glial, neuronal, or oligodendrocytic lineage markers shows that motoneurons always arose in mixed cultures with other differentiated cells. Thus, NEP cells represent a common progenitor for motoneurons and other spinal cord cells. The relationship of NEP cells with other neural stem cells is discussed.

Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits.

In this report, we provide evidence that NGF and BDNF have functionally antagonistic actions on sympathetic neuron growth and target innervation, with NGF acting via TrkA to promote growth and BDNF via p75NTR to inhibit growth. Specifically, in cultured sympathetic neurons that themselves synthesize BDNF, exogenous BDNF inhibits and function-blocking BDNF antibodies enhance process outgrowth. Both exogenous and autocrine BDNF mediate this effect via p75NTR because (1) BDNF does not inhibit growth of neurons lacking p75NTR, (2) function-blocking p75NTR antibodies enhance NGF-mediated growth, and (3) p75NTR-/- sympathetic neurons grow more robustly in response to NGF than do their wild-type counterparts. To determine the physiological relevance of this functional antagonism, we examined the pineal gland, a well defined sympathetic target organ. BDNF is present in the pineal gland during target innervation, and incoming sympathetic axons are p75NTR positive. Moreover, the pineal glands of BDNF+/- and BDNF-/- mice are hyperinnervated with sympathetic fibers, and tyrosine hydroxylase (TH) levels are elevated. Increased tyrosine hydroxylase is also observed in the BDNF+/- carotid artery, another sympathetic neuron target. Thus, BDNF, made by sympathetic neurons and/or their target organs, acts via p75NTR to antagonize NGF-mediated growth and target innervation, suggesting that sympathetic target innervation is determined by the balance of positively and negatively acting neurotrophins present in developing and potentially mature targets.

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)-IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP-dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses.

Lymphocytes respond to myelin proteins after spinal cord injury (SCI) and may contribute to post-traumatic secondary degeneration. However, there is increasing evidence that autoreactive T-lymphocytes may also convey neuroprotection and promote functional recovery after CNS injury. To clarify the role of myelin autoreactive lymphocytes after SCI, we performed contusion injuries in the thoracic spinal cord of transgenic (Tg) mice in which >95% of all CD4+ T-lymphocytes are reactive with myelin basic protein (MBP). We observed significantly impaired recovery of locomotor and reflex function in Tg mice compared with non-Tg (nTg) littermates. Measures of functional impairment in Tg mice correlated with significantly less white matter at the injury site, and morphometric comparisons of injured Tg and nTg spinal cords revealed increased rostrocaudal lesion expansion (i.e., secondary degeneration) in Tg mice. Rostrocaudal to the impact site in SCI-nTg mice, demyelination was restricted to the dorsal funiculus, i.e., axons undergoing Wallerian degeneration. The remaining white matter appeared normal. In contrast, lymphocytes were colocalized with regions of demyelination and axon loss throughout the white matter of SCI-Tg mice. Impaired neurological function and exacerbated neuropathology in SCI-Tg mice were associated with increased intraspinal production of proinflammatory cytokine mRNA; neurotrophin mRNA was not elevated. These data suggest that endogenous MBP-reactive lymphocytes, activated by traumatic SCI, can contribute to tissue injury and impair functional recovery. Any neuroprotection afforded by myelin-reactive T-cells is likely to be an indirect effect mediated by other non-CNS-reactive lymphocytes. Similar to the Tg mice in this study, a subset of humans that are genetically predisposed to autoimmune diseases of the CNS may be adversely affected by vaccine therapies designed to boost autoreactive lymphocyte responses after CNS trauma. Consequently, the safe implementation of such therapies requires that future studies define the mechanisms that control T-cell function within the injured CNS.

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits long-term Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.

Neurotrophins are known to regulate dendritic development, but the mechanisms that mediate neurotrophin-dependent dendrite formation are largely unknown. Here we show that brain-derived neurotrophic factor (BDNF) induces the formation of primary dendrites in cortical neurons by a protein synthesis-independent mechanism. BDNF leads to the rapid activation of PI3-kinase, MAP kinase, and PLC-gamma in cortical neurons, and pharmacological inhibition of PI3-kinase and MAP kinase in dissociated cell cultures and cortical slice cultures suppresses the ability of BDNF to induce dendrite formation. A constitutively active form of PI3-kinase, but not MEK, is sufficient to induce primary dendrite formation in cortical neurons. These observations indicate that BDNF induces primary dendrite formation via activation of the PI3-kinase and MAP kinase pathways and provide insight into the mechanisms that mediate the morphological effects of neurotrophin signaling.

Although the effects of neurotrophins on survival and differentiation of various neuronal populations have been well studied, little is known about their role in synaptic development and function. We have investigated the long-term effects of neurotrophins in the maturation of neuromuscular synapses in Xenopus nerve-muscle cocultures. BDNF and NT-3, but not NGF, elicited significant changes in several properties of spontaneous synaptic currents (SSCs), indicative of more mature synapses. Most synapses treated by the neurotrophins exhibited a bell-shaped distribution of SSC amplitudes, which reflects mature quantal secretion. The neurotrophins also potentiated the efficacy and reliability of stimulus-induced synaptic transmission. Moreover, BDNF and NT-3 increased the levels of the synaptic vesicle proteins, synaptophysin, and synapsin 1 in the spinal neurons. The number of varicosities per neuron also showed a significant increase after neurotrophin treatment. The effects of the neurotrophins appear to be mediated by the Trk family of receptor tyrosine kinases, primarily through a presynaptic mechanism. These results suggest that BDNF and NT-3 promote functional maturation of synapses.

OBJECTIVE

To determine if exogenous neurotrophins can prevent spiral ganglion neuron degeneration in the rat cochlea.

BACKGROUND

The loss of hair cells resulting in sensorineural hearing loss also leads to the secondary degeneration of spiral ganglion neurons. The effectiveness of cochlear implantation in patients with profound sensorineural hearing loss relies in part on the survival of spiral ganglion neurons; therefore, any therapy that can prevent or halt the loss of these neurons would be of potential clinical benefit. Previous research has shown that intracochlear infusion with neurotrophins can provide trophic support to SGNs in deafened guinea pigs. Whether this effect is seen in other species remains to be determined.

METHODS

After documenting the rate of spiral ganglion neuron degeneration after ototoxic deafening, we investigated the trophic effects of exogenous brain-derived neurotrophic factor (BDNF) on rat spiral ganglion neurons. The left cochleae of profoundly deafened rats were implanted with a drug delivery system connected to a mini-osmotic pump. BDNF or artificial perilymph was infused for 28 days; then the cochleae were prepared for histological study.

RESULTS

Treatment with BDNF led to a statistically significant increase in spiral ganglion neuron density and a highly significant increase in spiral ganglion neuron soma area compared with artificial perilymph-treated and untreated deafened cochleae.

CONCLUSIONS

The study has demonstrated the trophic advantage of exogenous BDNF in the mature rat cochlea and provides confidence that spiral ganglion neuron rescue after sensorineural hearing loss with exogenous BDNF may have clinical application.

Neurotrophins are a family of proteins which act as survival and differentiative factors in the developing and mature nervous system. Extensive evidence has been provided for their retrograde action following incorporation into nerve terminals and transport to the cell body. In contrast, we now demonstrate that one neurotrophin, brain-derived neurotrophic factor, is transported anterogradely via both peripheral and central processes of spinal sensory neurons. Using newly generated antisera, we have examined the distribution of brain-derived neurotrophic factor immunoreactivity and found it to be present within a subpopulation of sensory somata, primarily those with a small-to-medium diameter. The immunoreactivity was accumulated on both the distal and proximal sides of a ligature on the sciatic nerve. The accumulation on the distal side, but not on the proximal side, was substantially reduced by pretreatment with brain-derived neurotrophic factor antibodies in vivo. In contrast to the periphery, the immunoreactivity only accumulated on the proximal side of a lesion of the dorsal root. In the spinal cord, most nerve terminals immunoreactive for brain-derived neurotrophic factor were identified in lamina II. Lesion of the dorsal root led to a reduction of these nerve terminals. These studies indicate that the factor is transported not only retrogradely to, but also anterogradely from, the spinal ganglia to terminals in the periphery and spinal cord. The findings add a new dimension to the role of neuronal growth factors, since anterograde transport has not been observed previously for any endogenous survival factor.