Inmaculada Silos-Santiago

The role of neurotrophic factors in regulating the development of inner ear innervation

Several neurotrophins and their receptors regulate the survival of vestibular and cochlear neurons and probably also the efferent and autonomic neurons that innervate the inner ear. Mice lacking either brain-derived neurotrophic factor (BDNF) or its associated receptor, TrkB, lose all innervation to the semicircular canals and have reduced innervation of the outer hair cells in the apical and middle turns of the cochlea. Mice lacking neurotrophin-3 (NT-3) or its receptor, TrkC, lose many spiral ganglion cells predominantly in the basal turn of the cochlea. Nerve fibers from spiral ganglion cells in the middle turn extended to inner hair cells of the base. In mice lacking both BDNF and NT-3, or both TrkB and TrkC, there is a complete loss of innervation to the inner ear. Thus, these two neurotrophins and their associated receptors have been shown to be absolutely necessary for the normal development of afferent innervation of the inner ear. Current research efforts are testing the therapeutic potential for neurotrophins to treat hearing loss.

Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain.

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 α-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1–3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific “ball” domain of the Kvβ1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

Severe sensory deficits but normal CNS development in newborn mice lacking TrkB and TrkC tyrosine protein kinase receptors.

Analysis of mice carrying targeted mutations in genes encoding neurotrophins and their signalling Trk receptors has provided critical information regarding the role that these molecules play in the mammalian nervous system. In this study we generated mice defective in both TrkB and TrkC tyrosine kinase receptors to determine the biological effects of these receptors in the absence of compensatory mechanisms. trkB(–/–);trkC(–/–) double‐mutant mice were born at the expected frequency, indicating that TrkB and TrkC signalling are not required for embryonic survival. However, these double‐mutant mice had a significantly shorter lifespan and displayed more severe sensory defects than their single‐mutant trkB(–/–) and trkC(–/–) littermates. The most dramatic sensory deficit observed in trkB(–/–); trKC(–/–) mutant mice was the absence of vestibular and cochlear ganglia. Interestingly, these mice developed inner ear sensory epithelia in spite of the complete absence of sensory innervation. Analysis of the CNS in trkB(–/–); trkC(–/–) mutant mice revealed a well formed hippocampus, cortex and thalamus. Moreover, the pattern of expression of several neuronal markers appeared normal in these animals. These observations suggest that neurotrophin signalling through TrkB and TrkC receptors is essential for the development of sensory ganglia: however, it does not play a major role in the differentiation and survival of CNS neurons during embryonic development.

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.

Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds.

The alleged ability of taste afferents to induce taste buds in developing animals is investigated using a mouse model with a targeted deletion of the tyrosine kinase receptor trkB for the neurotrophin BDNF. This neurotrophin was recently shown to be expressed in developing taste buds and the receptor trkB has been shown to be expressed in the developing ganglion cells that innervate the taste buds. Our data show a reduction of geniculate ganglion cells to about 5% of control animals in neonates. Degeneration of ganglion cells starts when processes reach the central target (solitary tract) but before they reach the peripheral target (taste buds). Degeneration of ganglion cells is almost completed in trkB knockout mice before taste afferents reach in control animals the developing fungiform papillae. Four days later the first taste buds can be identified in fungiform papillae of both control and trkB knockout mice in about equal number and density. Many taste buds undergo a normal maturation compared to control animals. However, the more lateral and caudal fungiform papillae grow less in size and become less conspicuous in older trkB knockout mice. No intragemmal innervation can be found in trkB knockout taste buds but a few extragemmal fibers enter the apex and end between taste bud cells without forming specialized synapses. Taste buds of trkB knockout mice appear less well organized than those of control mice, but some cells show similar vesicle accumulations as control taste bud cells in their base but no synaptic contact to an afferent. These data strongly suggest that the initial development of many fungiform papillae and taste buds is independent of the specific taste innervation. It remains to be shown why others appear to be more dependent on proper innervation.

Corneal innervation and sensitivity to noxious stimuli in trkA knockout mice

Most primary sensory neurones depend on neurotrophins for survival. Mutant mice in which TrkA, the high‐affinity receptor for nerve growth factor (NGF), has been inactivated lack nociceptive neurones in sensory ganglia and do not respond to noxious stimuli. The cornea of the eye is innervated by trigeminal neurones that are activated by noxious mechanical, thermal and chemical stimuli. In the human cornea, these stimuli evoke only sensations of pain. We have analysed the innervation pattern and the response to noxious stimulation of the cornea of trkA (–/–) mutant mice. Corneal nerves were stained with the gold chloride impregnation method. Corneal sensitivity to noxious stimuli was assessed by counting blinking movements evoked by von Frey hairs, topical application of saline at different temperatures and application of acetic acid and capsaicin at different concentrations. In the cornea of trkA (–/–) mutant animals, we observed a drastic reduction in the number of nerve trunks and branches in the corneal stroma. Furthermore, quantitative analysis of the number of thin nerve terminals revealed a marked decrease in the corneal epithelium of trkA (–/–) mice when compared to those present in wild type and trkA (+/–) animals. The blinking response of trkA (–/–) mice to mechanical, thermal and chemical noxious stimuli was also significantly reduced. These results indicate that the population of corneal sensory neurones is markedly depleted in trkA (–/–) mutant mice. However, a small portion of corneal sensory neurones survive in these mice suggesting that they may be NGF independent. On the basis of our results, we propose that these surviving cells are polymodal nociceptive neurones, sensitive to mechanical stimulation, noxious heat and acid.

Localization of pleiotrophin and its mRNA in subpopulations of neurons and their corresponding axonal tracts suggests important roles in neural-glial interactions during development and in maturity

Trophic factors are being increasingly recognized as important contributors to growth, differentiation, and maintenance of viability within the mammalian nervous system during development. Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein that stimulates mitogenesis and angiogenesis and neurite and glial process outgrowth guidance activities in vitro. We localized the sites and time course of expression of the Ptn gene and its protein product in developing and adult mouse nervous system. Expression of the Ptn gene was first observed at embryo day 8.5 (E8.5). At E12.5, transcripts of the Ptn gene were localized in developing neuroepithelium at sites of active cell division in the spinal cord and brain. At E15.5, transcripts were found in the somata of some but not all neurons and glia whereas in the adult its pattern of expression was nearly exclusively restricted to the brain. The PTN protein was found almost entirely in association with the axonal tracts during development and in adults. Furthermore, as opposed to the finding of PTN in both central and peripheral nervous systems during development, PTN was not expressed beyond the exit where axonal tracts become the peripheral nervous system in adults. At all sites and times examined, the somata that contained Ptn transcripts corresponded with the axonal tracts that contained the PTN protein. The results establish that Ptn is expressed in early development at sites of active mitogenesis in developing neuroepithelium and later in both glial cells and neurons at sites of neuronal and glial process formation in developing axonal tracts. The findings establish a correspondence in the localization of PTN within the nervous system at sites of normal developmental processes that correlate with the functional activities of PTN previously described in vitro.

Selective glial cell line-derived neurotrophic factor production in adult dopaminergic carotid body cells in situ and after intrastriatal transplantation

Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinsons disease (PD). The clinical applicability of this therapy is, however, hampered by the need of a durable and stable GDNF source allowing the safe and continuous delivery of the trophic factor into the brain parenchyma. Intrastriatal carotid body (CB) autografting is a neuroprotective therapy potentially useful in PD. It induces long-term recovery of parkinsonian animals through a trophic effect on nigrostriatal neurons and causes amelioration of symptoms in some PD patients. Moreover, the adult rodent CB has been shown to express GDNF. Here we show, using heterozygous GDNF/lacZ knock-out mice, that unexpectedly CB dopaminergic glomus, or type I, cells are the source of CB GDNF. Among the neural or paraneural cells tested, glomus cells are those that synthesize and release the highest amount of GDNF in the adult rodent (as measured by standard and in situ ELISA). Furthermore, GDNF expression by glomus cells is maintained after intrastriatal grafting and in CB of aged and parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. Thus, glomus cells appear to be prototypical abundant sources of GDNF, ideally suited to be used as biological pumps for the endogenous delivery of trophic factors in PD and other neurodegenerative diseases.

The combined effects of trkB and trkC mutations on the innervation of the inner ear.

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer DiI. In the vestibular system, trkC+/− heterozygosity aggravates the trkB−/− mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthals canal of the cochlea. trkC−/− mutation alone or in combination with trkB+/− heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB−/− mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB−/− mutant mouse combined with trkC+/− heterozygosity. In addition, the trkB−/− mutation combined with trkC+/− heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC−/− mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type : trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.

Developmental dependency of Meissner corpuscles on trkB but not trkA or trkC.

The distribution of S100-immunoreactive (ir) corpuscular endings was examined in the palate of wildtype and knockout mice for trkA, trkB or trkC. In wildtype mice, S100-ir corpuscular endings were abundant at the top of palatal rugae. The endings contained 2–4 parallel arrays of S100-ir neurites. The distribution of S100-ir nerve endings in trkA and trkC knockout mice was similar to that in wildtype mice; S100-ir corpuscular endings were abundant in palates of the mutant mice. In trkB knockout mice, the palate was devoid of corpuscular endings, An immunoelectron microscopic method indicated that S100-ir corpuscular endings were identical to Meissner corpuscles. The normal development of Meissner corpuscles is probably dependent on trkB but not trkA or trkC.