Investigation of neuromuscular abnormalities in neurotrophin-3-deficient mice.

Neurotrophin-3 (NT-3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT-3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency-induced reduction in NT-3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1-43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve-mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1-43 were impaired in mature NT-3-deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype-dependent variation in the number of synaptic vesicles near the active zone. NT-3(+/-) mice had normal soleus muscle fiber numbers but their fibers had smaller cross-sectional areas and were more densely-packed than wild-type littermates. Moreover, the muscles of adult NT-3-deficient animals were weaker than those of wild-type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT-3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.

Neurotrophin-3 deficient Schwann cells impair nerve regeneration.

Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell (SC) survival and differentiation in the absence of axons. Prior studies have failed to define the explicit role of SC versus axon in NT-3 deficiency in relation to nerve regeneration and associated remyelination. In the paradigm we studied, using NT-3 heterozygous (NT3(+/-)) knockout mice capable of survival into adult-life, the experimental design provided a model uniquely capable of differentiating SC/axon influences. In these studies we first identified a defect in nerve regeneration characterized by fewer SCs in the regenerating nerve fibers of crushed sciatic nerves of NT3(+/-) mice. Subsequent experiments differentiated SC versus axonal influences as the culprit in defective nerve regeneration using sciatic nerve transplant paradigms. Results show an impairment in nerve regeneration in NT3(+/-) mice with a retardation of the myelination process, and this defect is associated with decreased SC survival and an increase in the neurofilament packing density of regenerating axons. These observations indicate that NT3(+/-) status of the SCs, but not of the axons, is responsible for impaired nerve regeneration and that NT-3 is essential for SC survival in early stages of regeneration-associated myelination in the adult peripheral nerve.

Mitochondrial malfunction and Ca2+ dyshomeostasis drive neuronal pathology in diabetes.

The World Health Organization (WHO) predicts there will be 300 million people world-wide with diabetes mellitus by 2025. Currently it is estimated that there are 20 and 60 million people suffering from diabetes mellitus in North America and Europe, respectively. Within this huge population of diabetic persons approximately 50% will develop some form of sensory polyneuropathy, which involves the dying back of distal axons and a failure of axons to regenerate. This leads to incapacitating pain, sensory loss and poor wound healing. The end result is lower extremity amputation with approximately 90,000 diabetes-related amputations occurring each year in North America and the expectation of a 5-fold increase over the next 10 years due to increased incidence of type 2 diabetes. Abnormal neuronal Ca(2+) homeostasis and impaired mitochondrial function have been implicated in numerous CNS and PNS diseases including diabetic sensory neuropathy. The endoplasmic reticulum (ER), in part, regulates cellular Ca(2+) homeostasis and this process is linked to regulation of mitochondrial function and activity of anti-apoptotic signal transduction pathways. Here we review the current state of research regarding role of Ca(2+) dyshomeostasis and mitochondrial physiology in neuronal dysfunction in diabetes. The central impact of diabetes-induced alteration of Ca(2+) handling on sensory neurone function is discussed and related to abnormal ER performance. New results are presented showing suboptimal Ca(2+) concentration in the ER lumen in association with reduced SERCA2 expression in sensory neurones from type 1 diabetic rats. We hypothesize that deficits in neurotrophic factor support, specifically linked to diabetes-induced lowered expression of insulin and neurotrophin-3, triggers alterations of sensory neurone phenotype that are critical for the development of abnormal Ca(2+) homeostasis and associated mitochondrial dysfunction. The role of hyperglycaemia in diabetes is also discussed and we propose that high glucose concentration may impact at other sites to contribute to the heterogeneous aetiology of nerve damage in diabetes.

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3.

Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF(-/-), NT3(-/-) and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) mRNA levels were normal in NGF(-/-) embryos, but significantly reduced in NT3(-/-) embryos. In contrast, the beta2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3(-/-) embryos, but significantly reduced in NGF(-/-) embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75(NTR). These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors.

Depleting endogenous neurotrophin-3 enhances myelin formation in the Trembler-J mouse, a model of a peripheral neuropathy.

The heterozygous Trembler-J (TrJ/+) mouse, containing a point mutation in the peripheral myelin protein 22 (Pmp22) gene, is characterized by severe hypomyelination and is a representative model of Charcot-Marie-Tooth 1A (CMT1A) disease/Dejerine-Sottas syndrome (DSS). Given that the neurotrophin-3 (NT3)-TrkC signaling pathway is inhibitory to myelination during development, we investigated the role of the NT3-TrkC pathway in myelination and manipulated this pathway to improve myelin formation in the CMT1A/DSS mouse model. Injection of NT3 to the TrJ/+ mice decreased the myelin protein P(0) level in the sciatic nerves. Suppressing the NT3-TrkC pathway with TrkC-Fc, an NT3 scavenger, enhanced myelination in vitro and in vivo in the TrJ/+ mouse. Furthermore, we found that full-length TrkC was expressed in adult TrJ/+ mouse sciatic nerves but was not detected in the wild-type adults, suggesting that the full-length TrkC is a potential target of treatment to enhance myelination in the TrJ/+ mouse.

Mice heterozygous for neurotrophin-3 display enhanced vulnerability to excitotoxicity in the striatum through increased expression of N-methyl-D-aspartate receptors.

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/-) or brain-derived neurotrophic factor (BDNF +/-) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/- than in BDNF+/- or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/- mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/- mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/- mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.

Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors.

The p75 neurotrophin receptor (p75NTR) binds all four mammalian neurotrophins, including neurotrophin-3 (NT-3) required for the development of select sensory neurons. This study demonstrated that many gustatory and somatosensory neurons of the tongue depend upon p75NTR. Each of thousands of filiform papillae at the front of the tongue as well as each somatosensory prominence at the back of the tongue has a small cluster of p75NTR-positive epithelial cells that is targeted by somatosensory innervation. This expression of p75NTR by epithelial target cells required NT-3 but not adult innervation. NT-3-secreting cells were adjacent to the p75NTR-positive target cells of each somatosensory organ, as demonstrated in NT-3(lacZneo) transgenic mice. In NT-3 null mutant mice, there were few lingual somatosensory neurons. In p75NTR null mutant mice, the lingual somatosensory axons were likewise absent or had deficient terminal arborizations. Cell culture indicated that substrate p75NTR can influence neuronal outgrowth. Specifically, dissociated trigeminal sensory neurons more than doubled their neurite lengths when grown on a lawn of p75NTR-overexpressing fibroblasts. This enhancement of neurite outgrowth by fibroblast p75NTR raises the possibility that epithelial target cell p75NTR may help to promote axonal arborization in vivo. The co-occurrence in p75NTR null mice of a 35% reduction in geniculate ganglion taste neurons and a shortfall of taste buds is consistent with the established role of gustatory innervation in prompting mammalian taste receptor cell differentiation.

NT3 expressed in skin causes enhancement of SA1 sensory neurons that leads to postnatal enhancement of Merkel cells.

To determine the role of NT3 in the postnatal maturation of Merkel cell (MC) sensory neurite complexes (touch domes), we examined the development of their neural and end-organ components in wild-type and transgenic mice that overexpress NT3 (NT3-OE). Touch domes are sensory complexes of the skin that contain specialized MCs innervated by slowly adapting type 1 (SA1) neurons. Touch domes are dependent on NT3 and, though formed in newborn mice that lack NT3, are severely depleted during postnatal maturation. Mice that overexpress NT3 in the skin have larger touch domes characterized by enhanced neural innervation and MC number. In this study, we asked how this NT3-mediated enhancement occurs, whether through stimulatory effects of NT3 on the SA1 neuron, or the MC, or both. The innervation density and number of MCs associated with each touch dome were measured in wild-type and transgenic animals at postnatal times. In newborn NT3-OE mice, touch dome innervation was enhanced. Surprisingly, however, the number of MCs was lower in newborn NT3-OE animals than in wild-type littermates, and equivalent numbers were not reached until postnatal day 8 (PN8). Not until the PN12 and PN16 time points did MCs increase in NT3-OE mice. To examine the neural dependence of MCs in NT3-OE mice, touch domes were chronically denervated by resecting dorsal cutaneous nerves. Both wild-type and NT3-OE animals showed similar depletion in the number of MCs associated with touch domes. These data indicate that NT3 is not a survival factor for MCs and that the NT3-mediated enhancement of MC number is indirect and neurally dependent.

Neurotrophin-3 signaling maintains maturational homeostasis between neuronal populations in the olfactory epithelium.

Neurons within the olfactory system undergo functional turnover throughout life. This process of cell death and compensatory neurogenesis requires feedback between neuronal populations of different developmental ages. We examined the role of NT-3 in this process. NT-3 was localized within both the olfactory bulb and olfactory epithelium. Mice null for NT-3 showed increased numbers of immature neurons, without change in the number of mature neurons. This was due to compensatory alterations in apoptosis of mature and immature neuronal populations. Using a primary olfactory neuronal culture, NT-3 was found to directly activate the PI3K/Akt pathway and indirectly activate the MAPK and PLC pathways. Activated PI3K/Akt promoted mature neuronal survival and induced the release of secondary factors, which activated the MAPK and PLC pathways to reduce neuronal precursor proliferation and inhibit neuronal maturation. These effects of NT-3 serve to maintain homeostasis between neuronal populations within the olfactory epithelium.

Reduction of NT-3 or TrkC results in fewer putative vagal mechanoreceptors in the mouse esophagus.

Intraganglionic laminar endings (IGLEs) represent major vagal afferent structures throughout the gastrointestinal tract. Both morphological and functional data suggested a mechanosensory role. Elucidation of their functional significance in a particular organ would be facilitated by the availability of animal models with significantly altered numbers of IGLEs. The present study was aimed at searching for mouse strains fulfilling this criterion in the esophagus. Anterograde wheat germ agglutinin-horseradish peroxidase tracing (WGA-HRP) from nodose ganglion was used in order to label esophageal IGLEs in mice deficient for neurotrophin-3 (NT-3) or tyrosine kinase C-receptor (TrkC) and in control littermates. This approach was feasible only in heterozygous mutants which are viable. IGLEs were counted in tetramethylbenzidine (TMB) processed wholemounts using a standardised protocol. Quantification of myenteric neurons was done in cuprolinic blue-stained specimens. Nodose neuron counts were performed in cryostat sections stained with cresyl violet. Numbers of IGLEs in the esophagus were significantly reduced in both heterozygous NT-3 (NT-3+/-) and heterozygous TrkC (TrkC+/-) mutants (65% and 40% reduction, respectively). Numbers of nodose neurons were also significantly reduced in NT-3+/- mice (48% reduction), while their reduction in TrkC+/- mutants was insignificant (11% reduction). There was no reduction of myenteric neurons in the esophagus of either mutant strain. The numeric deficiency of IGLEs was unlikely to be secondary to reduction of myenteric neurons. Although only heterozygous mutants could be studied, these results suggest that esophageal IGLEs share neurotrophin dependence on NT-3/TrkC with spinal proprioceptors and some cutaneous mechanosensors. This concurs with their proposed function as vagal mechanosensors crucial for reflex peristalsis.

Peripheral NT3 signaling is required for ETS protein expression and central patterning of proprioceptive sensory afferents.

To study the role of NT3 in directing axonal projections of proprioceptive dorsal root ganglion (DRG) neurons, NT3(-/-) mice were crossed with mice carrying a targeted deletion of the proapoptotic gene Bax. In Bax(-/-)/NT3(-/-) mice, NT3-dependent neurons survived and expressed the proprioceptive neuronal marker parvalbumin. Initial extension and collateralization of proprioceptive axons into the spinal cord occurred normally, but proprioceptive axons extended only as far as the intermediate spinal cord. This projection defect is similar to the defect in mice lacking the ETS transcription factor ER81. Few if any DRG neurons from Bax(-/-)/NT3(-/-) mice expressed ER81 protein. Expression of a NT3 transgene in muscle restored DRG ER81 expression in NT3(-/-) mice. Finally, addition of NT3 to DRG explant cultures resulted in induction of ER81 protein. Our data indicate that NT3 mediates the formation of proprioceptive afferent-motor neuron connections via regulation of ER81.

Regional expression of p75NTR contributes to neurotrophin regulation of cerebellar patterning.

Neurotrophins were initially identified as critical regulators of neuronal survival. However, these factors have many additional functions. In the developing cerebellum the roles of the neurotrophins BDNF and NT3 include a surprising effect on patterning, as revealed by changes in foliation in neurotrophin-deficient mice. Here we examine the potential role of p75NTR in cerebellar development and patterning. We show that p75NTR is expressed at highest levels in the region of the cerebellum where foliation is altered in BDNF and NT3 mutants. Although the cerebellar phenotype of p75NTR mutant animals is indistinguishable from wild type, mutation of p75NTR in BDNF heterozygotes results in defects in foliation and in Purkinje cell morphologic development. Taken together, these data suggest that p75NTR activity is critical for cerebellar development under pathologic circumstances where neurotrophin levels are reduced.

Neurotrophin-3 is required for appropriate establishment of thalamocortical connections.

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Formation of the thalamocortical tract occurs during pre- and postnatal development, with distinct thalamic nuclei projecting to specific cortical regions. The molecular forces that underlie the invasion by axons into specific cortical layers followed by activity-dependent maturation of synapses are poorly understood. We show that genetic ablation of neurotrophin-3 (NT-3) in the mouse neocortex results in reduction of a set of anatomically distinct axonal bundles projecting from thalamus through cortical white matter. These bundles include thalamocortical axons that normally establish connections with retrosplenial and visual cortex, sites of early postnatal NT-3 expression. These results implicate neurotrophins in the critical stage of precise thalamocortical connections.

Neurotrophin-3 modulates noradrenergic neuron function and opiate withdrawal.

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

Increased synaptic inhibition in dentate gyrus of mice with reduced levels of endogenous brain-derived neurotrophic factor.

The aim of this study was to explore the role of endogenous neurotrophins for inhibitory synaptic transmission in the dentate gyrus of adult mice. Heterozygous knockout (+/-) mice or neurotrophin scavenging proteins were used to reduce the levels of endogenous brain-derived neurotrophic factor and neurotrophin-3. Patch-clamp recordings from dentate granule cells in brain slices showed that the frequency, but not the kinetics or amplitude, of miniature inhibitory postsynaptic currents was modulated in brain-derived neurotrophic factor +/- compared to wild-type (+/+) mice. Furthermore, paired-pulse depression of evoked inhibitory synaptic responses was increased in brain-derived neurotrophic factor +/- mice. Similar results were obtained in brain slices from brain-derived neurotrophic factor +/+ mice incubated with tyrosine receptor kinase B-immunoglobulin G, which scavenges endogenous brain-derived neurotrophic factor. The increased inhibitory synaptic activity in brain-derived neurotrophic factor +/- mice was accompanied by decreased excitability of the granule cells. No differences in the frequency, amplitude or kinetics of miniature inhibitory postsynaptic currents were seen between neurotrophin-3 +/- and +/+ mice. From these results we suggest that endogenous brain-derived neurotrophic factor, but not neurotrophin-3, has acute modulatory effects on synaptic inhibition onto dentate granule cells. The site of action seems to be located presynaptically, i.e. brain-derived neurotrophic factor regulates the properties of inhibitory interneurons, leading to increased excitability of dentate granule cells. We propose that through this mechanism, brain-derived neurotrophic factor can change the gating/filtering properties of the dentate gyrus for incoming information from the entorhinal cortex to hippocampus. This will have consequences for the recruitment of hippocampal neural circuitries both under physiological and pathological conditions, such as epileptogenesis.

Inactivation of one copy of the mouse neurotrophin-3 gene induces cardiac sympathetic deficits.

Whether two copies of the neurotrophin-3 (NT3) gene are necessary for proper development of cardiac sympathetic innervation was investigated in mice carrying a targeted inactivation of the NT3 gene. Heterozygous (+/-) and null (-/-) mutant mice had fewer stellate ganglion neurons than did wild-type (+/+) mice at postnatal day 0 (P0 or birth), and this deficit was maintained between adult (P60) +/- and +/+ mice. The sympathetic innervation of the heart matured postnatally in +/+ and +/- mice. Tyrosine hydroxylase (TH)-positive axons were restricted largely to the epicardium at P0, were concentrated around large blood vessels in the myocardium at P21, and were present among cardiac myocytes at P60. Cardiac norepinephrine (NE) concentrations paralleled the growth of the sympathetic axons into the heart. NE concentrations were equivalent among +/+, +/-, and -/- mice at birth, but differences between +/- and +/+ mice increased with age. Adult +/- mice also exhibited lower resting heart rates and sympathetic tonus than +/+ mice. Thus deletion of one copy of the NT3 gene translates into anatomical, biochemical, and functional deficits in cardiac sympathetic innervation of postnatal mice, thereby indicating a gene-dosage effect for the NT3 gene.

Afferent-specific modulation of short-term synaptic plasticity by neurotrophins in dentate gyrus.

Neurotrophins modulate synaptic transmission and plasticity in the adult brain. We here show a novel feature of this synaptic modulation, i.e. that two populations of excitatory synaptic connections to granule cells in the dentate gyrus, lateral perforant path (LPP) and medial perforant path (MPP), are differentially influenced by the neurotrophins BDNF and NT-3. Using field recordings and whole-cell patch-clamp recordings in hippocampal slices, we found that paired-pulse (PP) depression at MPP-granule cell synapses was impaired in BDNF knock-out (+/-) mice, but PP facilitation at LPP synapses to the same cells was not impaired. In accordance, scavenging of endogenous BDNF with TrkB-IgG fusion protein also impaired PP depression at MPP-granule cell synapses, but not PP facilitation at LPP-granule cell synapses. Conversely, in NT-3+/- mice, PP facilitation was impaired at LPP-granule cell synapses whilst PP depression at MPP-granule cell synapses was unaffected. These deficits could be reversed by application of exogenous neurotrophins in an afferent-specific manner. Our data suggest that BDNF and NT-3 differentially regulate the synaptic impact of different afferent inputs onto single target neurons in the CNS.

Alterations in neurotrophin and neurotrophin receptor gene expression patterns in the rat central nervous system following perinatal Borna disease virus infection.

Infection of newborn rats with Borna disease virus (BDV) leads to persistence in the absence of overt signs of inflammation. BDV persistence, however, causes cerebellar hypoplasia and hippocampal dentate gyrus neuronal cell loss, which are accompanied by diverse neurobehavioral abnormalities. Neurotrophins and their receptors play important roles in the differentiation and survival of hippocampal and cerebellar neurons. We have examined whether BDV can cause alterations in the neurotrophin network, thus promoting neuronal damage. We have used RNase protection assay to measure mRNA levels of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their trkC and trkB receptors, as well as the growth factors insulin-like growth factor I (IGF-1) and basic fibroblast growth factor (bFGF), in the cerebellum and hippocampus of BDV-infected and control rats at different time points p.i. Reduced mRNA expression levels of NT-3, BDNF and NGF were found after day 14 p.i. in the hippocampus, but not in the cerebellum, of newborn infected rats. Three weeks after infection, trkC mRNA expression levels were reduced in both hippocampus and cerebellum of infected rats, whereas decreased trkB mRNA levels were only observed in the cerebellum. Reduced trkC mRNA expression was confined to the dentate gyrus of the hippocampus, as assessed by in situ hybridization. TUNEL assay revealed massive apoptotic cell death in the dentate gyrus of infected rats at days 27 and 33 p.i. Increased numbers of apoptotic cells were also detected in the cerebellar granular layer of infected rats after 8 days p.i. Moreover, a dramatic loss of cerebellar Purkinje cells was seen after day 27 p.i. Our results support the hypothesis, that BDV-induced alterations in neurotrophin systems might contribute to selective neuronal cell death.