Kathleen M. Guthrie - US grants

Neurotrophic interactions are critical to many developmental processes in the nervous system and are thought to contribute to regenerative events as well. Glial cells are intimately involved in nerve development and may provide appropriate cell substrates and trophic support for axon outgrowth and guidance. The mammalian olfactory nerve contains a unique type of non-myelinating glial cell, termed the ensheathing cell, with phenotypic traits of both Schwann cells and astrocytes. This nerve is unusual in that following injury, it can regenerate its central projections to the olfactory bulb via replacement of sensory neurons in the olfactory epithelium by basal cells which differentiate into sensory neurons. This regenerative capacity may in part be due to specific environmental cues provided to the growing afferents by the ensheathing cells. Little is currently known about potential trophic actions of ensheathing cells on olfactory axon growth, but studies by the applicant have shown that ensheathing glia in the adult rat olfactory bulb express high levels of the neurotrophic factors basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). Both of these factors have been implicated in regeneration of peripheral nerve and central axonal sprouting. These data suggest that expression of neurotrophic factors by ensheathing glia may contribute to axon growth in the olfactory system. The goals of the proposed research are to test aspects Of this hypothesis. The first goal is to characterize expression of CNTF, bFGF and acidic FGF in the developing olfactory system to determine if ensheathing cell expression of trophic factors is correlated with outgrowth of developing sensory axons. The second goal will be to determine if olfactory sensory neurons express appropriate receptors for these trophic factors during this time. The third aim is to determine if ensheathing glia and sensory neurons undergo changes in expression of trophic factors and receptors during periods of olfactory nerve degeneration and regeneration in lesioned adult rats. The final aim is to establish cultures of ensheathing glia from the neonatal rat and to characterize expression of trophic factors by these cells in vitro. Results of these studies could provide valuable insights into potential glial-dependent trophic mechanisms which promote nerve growth.

DESCRIPTION: (Adapted From The Applicant's Abstract) Neurotrophins are known to contribute to critical developmental processes in the nervous system. Recent studies by the applicant have shown that neurotrophins are expressed in the developing mammalian olfactory epithelium during the time that synaptic connections are being made with the developing forebrain. At this time, cells in the rostral telencephalon express the appropriate tyrosine kinase trk receptors for these neurotrophins and the olfactory bulbs begin to form. The spatial and temporal patterns of expression suggest the hypothesis that developing sensory neurons in the olfactory epithelium anterogradely transport specific neurotrophin factors to responsive forebrain neurons and thereby contribute to development of the olfactory bulb. The goals of the proposed research are to test aspects of this hypothesis. The first specific aim is to identify the specific cell types expressing different neurotrophins and their receptors in the epithelium and bulb. In particular we wish to determine if neurotrophin mRNAs are expressed by olfactory sensory neurons and if neurotrophin proteins are localized within olfactory nerve projections. Cell types will be identified by colocalization of neurotrophin/receptor mRNA and protein with phenotypic markers specific for subpopulations of olfactory cells The second aim is to determine if the olfactory nerve anterogradely transports neurotrophins from the epithelium to the bulb, and if this has functional consequences. Studies will determine if colchicine treatment increases neurotrophin immunoreactivity in sensory neurons while decreasing immunoreactivity in olfactory axons and terminals. We will also evaluate the redistribution of radiolabeled neurotrophins applied to the olfactory epithelium, and measure levels of bulb trk phosphorylation following such treatment. The third aim is to determine if early neurotrophic factor deprivation leads to morphological or phenotypic abnormalities in the bulb. This will be accomplished by evaluating olfactory system development in mice carrying targeted mutations in neurotrophin genes. The levels, distribution and cellular localization of neurotrophin and trk expression, and of phenotypic cell markers, will be examined in the normal and neurotrophin-deprived olfactory system. Differences in patterns of cell death in forebrain and bulb neuron morphology will also be examined. The final aim is to determine if early neurotrophin deprivation has functional consequences in this system by evaluating bulb trk phosphorylation and odor-stimulated c-fos expression in knockout mice and control littermates. Results of these studies will contribute to our understanding of the molecular signals that regulate mammalian forebrain development.

DESCRIPTION (provided by applicant): New neurons are added to both the adult olfactory bulb and the olfactory sensory neuron (OSN) population throughout life. This continuous neurogenesis allows for adaptive structural responses to sensory experience, olfactory learning, and even injury that continuously shape functional circuitry to fit the behavioral needs of the animal. Production of new bulb interneurons occurs in excess of the numbers that will ultimately integrate, and those that fail are eliminated by apoptosis, a situation reminiscent of developmental neuron death. Activity plays a role in selecting new cells for survival, and impacts the sensory neuron population as well, making it likely that these effects are coordinated to structurally modify responsive olfactory circuits. The underlying cellular mechanisms are not known, but neurotrophic factors are attractive candidate molecules. They exhibit activity-dependent expression, and during development, play essential roles in sculpting neuronal circuitry by regulating neuronal apoptosis and stabilizing connections. In other adult systems that add neurons, notably the song nuclei of male birds, members of the NGF family of neurotrophins regulate seasonal neuronal survival and are obtained from local sources, and by anterograde transport from innervating populations. We hypothesize that similar mechanisms work in the primary olfactory pathway to modulate survival of new neurons in adults. Evidence for this comes from transgenic mice expressing the LacZ reporter under control of neurotrophin promoters;the transgene products are expressed in subpopulations of OSNs, and in their axon projections to the olfactory bulb. Results from studies localizing the native peptides or mRNAs have been inconclusive. To test the hypothesis that OSNs express neurotrophins, and transport the peptides to the adult olfactory bulb, we will 1) use laser capture micro-dissection and quantitative PCR to measure levels of neurotrophin mRNAs in OSNs, and 2) infect OSNs in vivo with adenovirus vectors we have designed to introduce genes encoding neurotrophin- green fluorescent protein (GFP) fusion proteins. Expression of the GFP-tagged peptides will make it possible to monitor neurotrophin trafficking in these cells. These studies will provide insights into mechanisms underlying the plasticity of adult neural circuits that undergo neuronal replacement. PUBLIC HEALTH RELEVANCE: Adult neurogenesis provides the olfactory system with a continuous supply of new neurons throughout life, a process that is regulated by sensory experience. Communication between odor-stimulated sensory neurons and their target neurons coordinates adaptive changes in these populations. Potential signaling molecules involved in this communication include trophic factors. In this proposal, adenovirus vectors will be used to transfer genes encoding trophic factor-GFP fusion peptides into sensory neurons to determine if these peptides are anterogradely supplied to target neurons in vivo.

DESCRIPTION (provided by applicant): Finding ways to preserve or replace brain neurons that are vulnerable to disease or injury remains an important therapeutic goal. The mammalian olfactory bulb is the only brain structure identified to date that is known to regularly replace neurons in adulthood. Proliferating neural stem cells in the forebrain subventricular zone (SVZ) give rise to thousands of neuroblasts daily that migrate in the rostral migratory stream to the olfactory bulb and differentiate as inhibitory interneurons. The vast majority of new neurons develop as granule cells, and these modulate the activity of excitatory output neurons, the mitral cells, through synapses that form on their dendritic processes. Pre-existing granule cells gradually die, and their replacement by adult-born granule cells maintains the overall integrity of olfactory bulb circuitry, and the functional capabilities of this sensory system. About half of new granule cells die over the first few weeks of their development in the bulb, with only a fraction o the population surviving long- term. How this cell turnover is controlled within the bulb, what determines which neurons, and how many, will live or die, and how new neurons form synapses within pre-established circuits is not well understood. Much more is known is about how these processes are controlled during early development, and there may be similarities in the mechanisms involved. Trophic factor signaling is one such mechanism that operates to sculpt the sizes of neuronal populations and their connections during brain development, and continues to maintain neuron morphology and synaptic plasticity in adults. Using transgenic mice, the work described in this proposal will first determine if increasing endogenous trophic factor signaling in the bulb promotes greater survival of new granule cells under normal conditions. Secondly, granule cell survival is reduced by suppressing neural activity and by pathology associated with Huntington's disease, and trophic factor over-expression will be tested for its ability to rescue new neurons under these adverse conditions. Finally, the morphological development of new cells will be monitored to determine whether increased trophic signaling can foster their functional integration by enhancing dendrite growth and synapse formation. Identifying factors in the adult CNS that promote the survival and development of SVZ-derived progenitors under normal, as well as pathological conditions, will have important implications for adapting adult neural stem cells for therapeutic purposes. PUBLIC HEALTH RELEVANCE: In adult brain, only the olfactory system has the capacity to regularly replace older neurons with new neurons generated from SVZ stem cells. Understanding the mechanisms that control this process and favor the survival and integration of new neurons has practical implications for the design of cell-based therapeutic strategies aimed at treating neurological disorders. PUBLIC HEALTH RELEVANCE: The olfactory forebrain contains the only population of neurons that are regularly replaced by new neurons born in the adult brain. The mutation that causes Huntington's disease (HD), and conditions that reduce neural activity, inhibit the survival of new neurons in this system. This research project will test if neurotrophic factor signaling regulates the normal neuron replacement process, and if increasing trophic factor availability rescues adult-born neurons from death caused by HD pathology or suppressed network activity.

Project Abstract Spine defects and synaptic dysfunction are common to neurodevelopmental disorders characterized by cognitive impairments in language, learning and sensory processing. Angelman Syndrome (AS) is a disorder caused by mutation of the maternal Ube3a allele, resulting in neuronal loss of the encoded ubiquitin ligase via developmental paternal imprinting. Delayed milestones are evident at 6-12 months of age, with progression to seizures and autistic features that include impaired speech, intellectual disability, altered social behaviors, and aberrant responses to sensory stimuli, including aversions to certain odors, flavors and textures. AS pathogenesis is not well understood; Ube3a participates in multiple cellular processes, including turnover of synaptic proteins. Spine defects, impaired synaptic plasticity, and deficits in cortical inhibitory drive are found in adult and juvenile AS mouse models, however it is not known how Ube3a loss impacts early stages of neuronal maturation and circuit integration. Gene reinstatement has established an early requirement for Ube3a in which the critical window for full phenotypic rescue occurs during prenatal/neonatal development, and we hypothesize that pathogenesis begins at this time, while newly-imprinted neurons are still maturing and engaged in circuit assembly. Intriguingly, selective loss of Ube3a in GABAergic interneurons reproduces features of circuit dysfunction caused by pan-neuronal loss, highlighting GABAergic neurons potential therapeutic targets. Adult-born interneurons that integrate in the adult olfactory bulb (OB) are subject to the same mutation effects as those born in embryo. In our work, we found that while Ube3a is absent in most brain regions in AS mice, the adult olfactory system shows a pattern of Ube3a expression that allows us to monitor paternal imprinting as new GABAergic granule cells (GCs) are generated, migrate, and mature in the OB. Moreover, we find that new, 35 day-old GCs show spine abnormalities. In this exploratory work, we propose to test the hypothesis that imprinting-mediated loss of Ube3a disrupts subsequent GC maturation, and that as a consequence, spine development is impaired. Study aims will test this by defining a precise temporal profile of paternal imprinting in birth- dated GCs, and cell reconstruction/quantitative morphological analyses will identify emerging structural abnormalities relative to the timing of Ube3 loss. How the mutation affects olfaction is unknown, and in the final aim we will use a modified social interaction test to evaluate innate responses to conspecific odors. The overall goal of this project is to identify developmental defects that emerge from Ube3a loss in GCs to gain an understanding of both its normal role in GC maturation, and interneuron pathobiology in AS.