Kharen L. Doyle

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus

Adult dentate neurogenesis is important for certain types of hippocampal‐dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety‐related behaviour and learning and memory. We report that adult NPY receptor knock‐out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin‐positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1‐receptor mediated and involves extracellular signal‐regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY‐releasing interneurons may modulate dentate neurogenesis.

Critical role for Y1 receptors in mesenchymal progenitor cell differentiation and osteoblast activity.

The neuropeptide Y (NPY) system has been implicated in the regulation of bone homeostasis and osteoblast activity, but the mechanism behind this is unclear. Here we show that Y1 receptor signaling is directly involved in the differentiation of mesenchymal progenitor cells isolated from bone tissue, as well as the activity of mature osteoblasts. Importantly, the mRNA levels of two key osteogenic transcription factors, runx2 and osterix, as well as the adipogenic transcription factor PPAR‐γ, were increased in long bones of Y1−/− mice compared with wild‐type mice. In vitro, bone marrow stromal cells (BMSCs) isolated from Y1−/− mice formed a greater number of mineralized nodules under osteogenic conditions and a greater number of adipocytes under adipogenic conditions than controls. In addition, both the number and size of fibroblast colony‐forming units formed in vitro by purified osteoprogenitor cells were increased in the absence of the Y1 receptors, suggestive of enhanced proliferation and osteogenesis. Furthermore, the ability of two specific populations of mesenchymal progenitor cells isolated from bone tissue, an immature mesenchymal stem cell population and a more committed osteoprogenitor cell population, to differentiate into osteoblasts and adipocytes in vitro was enhanced in the absence of Y1 receptor signaling. Finally, Y1 receptor deletion also enhanced the mineral‐producing ability of mature osteoblasts, as shown by increased in vitro mineralization by BMSCs isolated from osteoblast‐specific Y1−/− mice. Together these data demonstrate that the NPY system, via the Y1 receptor, directly inhibits the differentiation of mesenchymal progenitor cells as well as the activity of mature osteoblasts, constituting a likely mechanism for the high‐bone‐mass phenotype evident in Y1−/− mice.

Osteoblast specific Y1 receptor deletion enhances bone mass

Neuropeptide Y, Y1 receptors are found in neuronal as well as bone tissue and Y1 signalling has been implicated in the regulation of bone mass. However, the contribution of Y1 receptors located in these different tissues, particularly that of the bone-specific Y1 receptors, to the regulation of bone homeostasis is unclear. Here we demonstrate that osteoblast-specific Y1 receptor deletion resulted in a marked increase in femoral cancellous bone volume, trabecular thickness and trabecular number. This is the result of elevated osteoblast activity as shown by increased mineral apposition rate and bone formation rate, and is associated with an upregulation in the mRNA expression levels of alkaline phosphatase, osteocalcin and dentin matrix protein-1. Furthermore, osteoblastic Y1 receptor deletion also led to increased mineral apposition rate on both the endocortical and the periosteal surfaces resulting in increased femoral diameter. Together these data demonstrate a direct role for the Y1 receptor on osteoblasts in the regulation of osteoblast activity and bone formation in vivo and suggest that targeting Y1 receptor signalling directly in the bone may have potential therapeutic implications for stimulating bone accrual in diseases such as osteoporosis.

Differentiation of adult mouse olfactory precursor cells into hair cells in vitro.

Many forms of deafness result from degeneration of the sensory cells for hearing, the hair cells in the cochlea. Stem cells offer a potential cell‐based therapy for the treatment of deafness. Here, we investigate whether adult olfactory precursor cells can differentiate into hair cells in culture. Precursor cells were isolated from mouse olfactory neuroepithelium, were sphere‐forming, showed proliferative capacity, and contained cells expressing neuronal and non‐neuronal proteins. To induce differentiation, precursor cells were cocultured with cochlear cells and/or cochlear supernatant. Differentiated precursor cells were immunopositive for specific hair cell markers, including myosin VIIa, FM1‐43, calretinin, phalloidin, and espin, and resembled hair cells anatomically and immunocytochemically in culture. The results demonstrate for the first time that adult olfactory precursor cells can differentiate into hair cell‐like cells, thus providing a potential autotransplantation therapy for hearing loss.

Expression of the intermediate filament protein nestin by sustentacular cells in mature olfactory neuroepithelium

The intermediate filament protein nestin has been widely used as a marker for proliferating neural progenitor cells in the nervous system. The mammalian olfactory neuroepithelium is a region of the nervous system that robustly supports ongoing neurogenesis, yet where nestin has not been reported to mark proliferating progenitors. Using immunohistochemistry, we examined nestin expression in the mature olfactory neuroepithelium and found it to be tightly restricted to the basal compartment where the olfactory neuronal progenitor cell population resides. The pattern of nestin immunoreactivity was consistent with expression by the endfeet and inferior processes of sustentacular cells rather than basal cells. Using a bank of defined antibody markers, we confirmed nestins pattern of distribution to be different from that of cytokeratin, vimentin, GBC‐1, GAP43, and carnosine. It was highly similar to the pattern of SUS‐4 immunoreactivity in the basal region of the neuroepithelium. Following surgical bulbectomy, nestin expression was up‐regulated and became evident in the cell bodies of sustentacular cells situated more apically in the neuroepithelium. We have shown nestin to be present in the basal region of the adult olfactory neuroepithelium in the zone that supports ongoing neurogenesis in the adult, but its expression is restricted to the inferior parts of sustentacular cells rather than the neuronal progenitor cells. Nestin may play a potential role in the migration of recently proliferated olfactory neurons on the scaffolding of sustentacular cells in a manner analogous to its proposed role in radial glia during embryonic development of the central nervous system. J. Comp. Neurol. 437:186–195, 2001.

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.

Neuropeptide Y and peptide YY have distinct roles in adult mouse olfactory neurogenesis

Neuropeptide Y (NPY) and peptide YY (PYY) are differentially expressed throughout the olfactory neuroepithelium (ON), with NPY expression present in sustentacular cells, olfactory ensheathing cells, and olfactory receptor neurons and PYY expressed only in sustentacular cells. Examination of the anatomical morphology of the ON in NPY knockout (NPY−/−) and PYY knockout (PYY−/−) mice shows that there are significantly more neurons in PYY−/− mice and significantly fewer neurons in NPY−/− mice. Interestingly, the mature neurons of NPY−/− mice were undergoing apoptosis. The transcription factor Mash1, which is critical in the production of olfactory precursors, is also differentially expressed in NPY−/− and PYY−/− ON. It is upregulated in the neurons of NPY−/− mice and unchanged in PYY−/− mice. Furthermore, significantly fewer olfactory neurospheres are present in cultures prepared from PYY−/− mice in the first 2 weeks compared with NPY−/− and wild‐type mice. Together these results suggest that, during olfactory neurogenesis, NPY acts as a trophic factor for the maturation and survival of olfactory receptor neurons, whereas PYY has an important role in the regulation of olfactory neuron differentiation.

Morphological and behavioural changes occur following the X-ray irradiation of the adult mouse olfactory neuroepithelium.

BackgroundThe olfactory neuroepithelium lines the upper nasal cavity and is in direct contact with the external environment and the olfactory bulbs. The ability to self-renew throughout life and the reproducible recovery after injury, make it a model tissue to study mechanisms underlying neurogenesis. In this study, X-rays were used to disrupt proliferating olfactory stem cell populations and to assess their role in the cellular and morphological changes involved in olfactory neurogenic processes.ResultsWe have analysed the histological and functional effects of a sub-lethal dose of X-rays on the adult mouse olfactory neuroepithelium at 2 hours, 24 hours, 1 week, 2 weeks and 5 weeks. We have shown an immediate cessation of proliferating olfactory stem cells as shown by BrdU, Ki67 and pH3 expression. At 24 hours there was an increase in the neural transcription factors Mash1 and Pax6 expression, and a disruption of the basal lamina and increase in glandular cell marker expression at 1 week post-irradiation. Coincident with these changes was an impairment of the olfactory function in vivo.ConclusionsWe have shown significant changes in basal cell proliferation as well as morphological changes in the olfactory neuroepithelium following X-ray irradiation. There is involvement of the basal lamina as well as a clear role for glandular and sustentacular cells.

Role of neuropeptide Y (NPY) in the differentiation of Trpm-5-positive olfactory microvillar cells

The mouse olfactory neuroepithelium (ON) is comprised of anatomically distinct populations of cells in separate regions; apical (sustentacular and microvillar), neuronal (olfactory sensory neurons) and basal (horizontal and globose basal cells). The existence of microvillar cells (MVCs) is well documented but their nature and function remains unclear. An important transcription factor for the differentiation of MVCs is Skn-1a, with loss of function of Skn-1a in mice resulting in a complete loss of Trpm-5 expressing MVCs, while olfactory sensory neuron differentiation is normal. Our previous research has shown that neuropeptide Y (NPY) is expressed in MVCs and is important in the neuroproliferation of olfactory precursors. This study showed that following X-ray irradiation of the snout of wildtype mice, which decreases the proliferation of basal precursor cells, the numbers of Trpm-5-positive MVCs is increased at 2 and 5 weeks post-irradiation compared to controls. Skn-1a expression in the ON following X-ray irradiation also increases at 2 weeks post-irradiation in a regionally specific manner matching the expression pattern of Trpm-5-positive MVCs. In parallel, NPYCre knock-in mice were used to examine the expression of Skn-1a following activation of NPY unilaterally in the ON (unilateral nasal irrigation of AAV-NPY-FLEX). These experiments demonstrated that Skn-1a is only expressed when NPY is activated in MVCs. Therefore the expression of NPY is necessary for the transcription factor-mediated differentiation of olfactory MVCs.