Stem cells and their niche in the adult olfactory mucosa.

It is well known that new neurons are produced in the adult brain, in the hippocampus and in the subventricular zone. The neural progenitors formed in the subventricular zone migrate forward and join in neural circuits as interneurons in the olfactory bulb, the target for axons from the olfactory sensory neurons in the nose. These neurons are also continually replaced during adulthood from a stem cell in a neurogenic niche in the olfactory epithelium. The stem cell responsible can regenerate all the cells of the olfactory epithelium if damaged by trauma or toxins. This stem cell, the horizontal basal cell, is in a niche defined by the extra cellular matrix of the basement membrane as well as the many growth factors expressed by surrounding cells and hormones from nearby vasculature. A multipotent cell has been isolated from the olfactory mucosa that can give rise to cells of endodermal and mesodermal origin as well as the expected neural lineage. Whether this is an additional stem cell or the horizontal basal cell is still an open question.

Autologous olfactory ensheathing cell transplantation in human paraplegia: a 3-year clinical trial.

Olfactory ensheathing cells show promise in preclinical animal models as a cell transplantation therapy for repair of the injured spinal cord. This is a report of a clinical trial of autologous transplantation of olfactory ensheathing cells into the spinal cord in six patients with complete, thoracic paraplegia. We previously reported on the methods of surgery and transplantation and the safety aspects of the trial 1 year after transplantation. Here we address the overall design of the trial and the safety of the procedure, assessed during a period of 3 years following the transplantation surgery. All patients were assessed at entry into the trial and regularly during the period of the trial. Clinical assessments included medical, psychosocial, radiological and neurological, as well as specialized tests of neurological and functional deficits (standard American Spinal Injury Association and Functional Independence Measure assessments). Quantitative test included neurophysiological tests of sensory and motor function below the level of injury. The trial was a Phase I/IIa design whose main aim was to test the feasibility and safety of transplantation of autologous olfactory ensheathing cells into the injured spinal cord in human paraplegia. The design included a control group who did not receive surgery, otherwise closely matched to the transplant recipient group. This group acted as a control for the assessors, who were blind to the treatment status of the patients. The control group also provided the opportunity for preliminary assessment of the efficacy of the transplantation. There were no adverse findings 3 years after autologous transplantation of olfactory ensheathing cells into spinal cords injured at least 2 years prior to transplantation. The magnetic resonance images (MRIs) at 3 years showed no change from preoperative MRIs or intervening MRIs at 1 and 2 years, with no evidence of any tumour of introduced cells and no development of post-traumatic syringomyelia or other adverse radiological findings. There were no significant functional changes in any patients and no neuropathic pain. In one transplant recipient, there was an improvement over 3 segments in light touch and pin prick sensitivity bilaterally, anteriorly and posteriorly. We conclude that transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to 3 years of post-implantation, however, this conclusion should be considered preliminary because of the small number of trial patients.

Neurotrophin expression in the adult olfactory epithelium.

Published reports of neurotrophin expression in the olfactory system are incomplete because of missing data and conflicting results. Previous studies used a variety of fixation procedures and antibodies on different species and different ages. The aim of the present study was to examine expression of neurotrophins and their receptors using optimized methodologies: five methods of fixation, multiple antibodies, a variety of immunochemical protocols, and RT-PCR. We show here that (i) transcripts for all neurotrophins and their receptors are found in the adult olfactory epithelium; (ii) all neurotrophins are expressed in the supporting cells and the neuronal layers of the undisturbed adult olfactory epithelium while NT4 is found additionally in the horizontal basal cells; (iii) neurotrophin immunoreactivity required a fixative that included parabenzoquinone (not used in previous studies of olfactory tissue); (iv) TrkB and TrkC are restricted to the globose basal cell and neuron layers while TrkA is found in the horizontal basal cells and in the supporting cells where it co-localizes with the low affinity receptor for NGF (p75NTR). These findings confirm that neurotrophins are produced within the olfactory epithelium, suggesting autocrine and paracrine regulation of olfactory neurogenesis.

Developmental vitamin D deficiency alters the expression of genes encoding mitochondrial, cytoskeletal and synaptic proteins in the adult rat brain.

Epidemiology has highlighted the links between season of birth, latitude and the prevalence of brain disorders such as multiple sclerosis and schizophrenia. In line with these data, we have hypothesized that "imprinting" with low prenatal vitamin D could contribute to the risk of these two brain disorders. Previously, we have shown that transient developmental hypovitaminosis D induces permanent changes in adult nervous system. The aim of this study was to examine the impact of prenatal hypovitaminosis D on gene expression in the adult rat brain. Vitamin D deficient female rats were mated with undeprived males and the offspring were fed with a control diet after birth. At Week 10, gene expression in the progeny's brain was compared with control animals using Affymetrix gene microarrays. Prenatal hypovitaminosis D causes a dramatic dysregulation of several biological pathways including oxidative phosphorylation, redox balance, cytoskeleton maintenance, calcium homeostasis, chaperoning, post-translational modifications, synaptic plasticity and neurotransmission. A computational analysis of these data suggests that impaired synaptic network may be a consequence of mitochondrial dysfunction. Since disruptions of mitochondrial metabolism have been associated with both multiple sclerosis and schizophrenia, developmental vitamin D deficiency may be a heuristic animal model for the study of these two brain diseases.

Autologous olfactory ensheathing cell transplantation in human spinal cord injury.

Olfactory ensheathing cells transplanted into the injured spinal cord in animals promote regeneration and remyelination of descending motor pathways through the site of injury and the return of motor functions. In a single-blind, Phase I clinical trial, we aimed to test the feasibility and safety of transplantation of autologous olfactory ensheathing cells into the injured spinal cord in human paraplegia. Participants were three male paraplegics, 18-55 years of age, with stable, complete thoracic injuries 6-32 months previously, with stable spinal column, no implanted prostheses, and no syrinx. Olfactory ensheathing cells were grown and purified in vitro from nasal biopsies and injected into the region of damaged spinal cord. The trial design includes a matched injury group as a control for the assessors, who are blind to treatment status. Assessments, made before transplantation and at regular intervals subsequently, include MRI, medical, neurological and psychosocial assessments, and standard American Spinal Injury Association and Functional Independence Measure assessments. One year after cell implantation, there were no medical, surgical or other complications to indicate that the procedure is unsafe. There is no evidence of spinal cord damage nor of cyst, syrinx or tumour formation. There was no neuropathic pain reported by the participants, no change in psychosocial status and no evidence of deterioration in neurological status. Participants will be followed for 3 years to confirm long-term safety and to compare neurological, functional and psychosocial outcomes with the control group. We conclude transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to one year post-implantation.

Rotenone Susceptibility Phenotype in Olfactory Derived Patient Cells as a Model of Idiopathic Parkinson's Disease.

Parkinson's disease is a complex age-related neurodegenerative disorder. Approximately 90% of Parkinson's disease cases are idiopathic, of unknown origin. The aetiology of Parkinson's disease is not fully understood but increasing evidence implies a failure in fundamental cellular processes including mitochondrial dysfunction and increased oxidative stress. To dissect the cellular events underlying idiopathic Parkinson's disease, we use primary cell lines established from the olfactory mucosa of Parkinson's disease patients. Previous metabolic and transcriptomic analyses identified deficiencies in stress response pathways in patient-derived cell lines. The aim of this study was to investigate whether these deficiencies manifested as increased susceptibility, as measured by cell viability, to a range of extrinsic stressors. We identified that patient-derived cells are more sensitive to mitochondrial complex I inhibition and hydrogen peroxide induced oxidative stress, than controls. Exposure to low levels (50 nM) of rotenone led to increased apoptosis in patient-derived cells. We identified an endogenous deficit in mitochondrial complex I in patient-derived cells, but this did not directly correlate with rotenone-sensitivity. We further characterized the sensitivity to rotenone and identified that it was partly associated with heat shock protein 27 levels. Finally, transcriptomic analysis following rotenone exposure revealed that patient-derived cells express a diminished response to rotenone-induced stress compared with cells from healthy controls. Our cellular model of idiopathic Parkinson's disease displays a clear susceptibility phenotype to mitochondrial stress. The determination of molecular mechanisms underpinning this susceptibility may lead to the identification of biomarkers for either disease onset or progression.

Developmental Vitamin D3 deficiency alters the adult rat brain.

There is growing evidence that Vitamin D(3) (1,25-dihydroxyvitamin D(3)) is involved in brain development. We have recently shown that the brains of newborn rats from Vitamin D(3) deficient dams were larger than controls, had increased cell proliferation, larger lateral ventricles, and reduced cortical thickness. Brains from these animals also had reduced expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor. The aim of the current study was to examine if there were any permanent outcomes into adulthood when the offspring of Vitamin D(3) deficient dams were restored to a normal diet. The brains of adult rats were examined at 10 weeks of age after Vitamin D(3) deficiency until birth or weaning. Compared to controls animals that were exposed to transient early Vitamin D(3) deficiency had larger lateral ventricles, reduced NGF protein content, and reduced expression of a number genes involved in neuronal structure, i.e. neurofilament or MAP-2 or neurotransmission, i.e. GABA-A(alpha4). We conclude that transient early life hypovitaminosis D(3) not only disrupts brain development but leads to persistent changes in the adult brain. In light of the high incidence of hypovitaminosis D(3) in women of child-bearing age, the public health implications of these findings warrant attention.

Reduced protein synthesis in schizophrenia patient-derived olfactory cells.

Human olfactory neurosphere-derived (ONS) cells have the potential to provide novel insights into the cellular pathology of schizophrenia. We used discovery-based proteomics and targeted functional analyses to reveal reductions in 17 ribosomal proteins, with an 18% decrease in the total ribosomal signal intensity in schizophrenia-patient-derived ONS cells. We quantified the rates of global protein synthesis in vitro and found a significant reduction in the rate of protein synthesis in schizophrenia patient-derived ONS cells compared with control-derived cells. Protein synthesis rates in fibroblast cell lines from the same patients did not differ, suggesting cell type-specific effects. Pathway analysis of dysregulated proteomic and transcriptomic data sets from these ONS cells converged to highlight perturbation of the eIF2α, eIF4 and mammalian target of rapamycin (mTOR) translational control pathways, and these pathways were also implicated in an independent induced pluripotent stem cell-derived neural stem model, and cohort, of schizophrenia patients. Analysis in schizophrenia genome-wide association data from the Psychiatric Genetics Consortium specifically implicated eIF2α regulatory kinase EIF2AK2, and confirmed the importance of the eIF2α, eIF4 and mTOR translational control pathways at the level of the genome. Thus, we integrated data from proteomic, transcriptomic, and functional assays from schizophrenia patient-derived ONS cells with genomics data to implicate dysregulated protein synthesis for the first time in schizophrenia.

Increased abundance of translation machinery in stem cell-derived neural progenitor cells from four schizophrenia patients.

The genetic and epigenetic factors contributing to risk for schizophrenia (SZ) remain unresolved. Here we demonstrate, for the first time, perturbed global protein translation in human-induced pluripotent stem cell (hiPSC)-derived forebrain neural progenitor cells (NPCs) from four SZ patients relative to six unaffected controls. We report increased total protein levels and protein synthesis, together with two independent sets of quantitative mass spectrometry evidence indicating markedly increased levels of ribosomal and translation initiation and elongation factor proteins, in SZ hiPSC NPCs. We posit that perturbed levels of global protein synthesis in SZ hiPSC NPCs represent a novel post-transcriptional mechanism that might contribute to disease progression.

On the Life Span of Olfactory Receptor Neurons.

The life span of olfactory receptor neurons was investigated after injection of a retrograde tracer into the olfactory bulb. Mice were injected unilaterally with colloidal gold conjugated with Concanavalin A and their olfactory epithelia were examined after 7, 14, 30, 60, and 90 days. Gold particles could be seen in the epithelia at all survival periods after silver intensification. There was no gold in the epithelia on the uninjected side. In order to test whether gold could be recycled within the epithelium upon the death of receptor neurons, the olfactory bulbs of some mice were ablated 4 days after colloidal gold injection. None of the receptor neurons in these epithelia contained gold at any survival period. To investigate whether gold was continuously available at the injection site, olfactory bulbs were examined by electron microscopy. By 7 days after injection all gold was sequestered intracellularly and was presumably unavailable for uptake by the olfactory axons. These results indicate that olfactory receptor neurons live for at least three times the commonly accepted life span of 30 days. A long life span challenges the widely held view that olfactory receptor neurons are regularly replaced.

Development of voltage-dependent currents in taste receptor cells.

Taste buds, the specialized end organs of gustation, comprise a renewing sensory epithelium. Undifferentiated basal cells become taste receptor cells by elongating and extending processes apically toward the taste pore. Mature taste cells are electrically excitable and express voltage-dependent Na+ Ca2+, and K+ currents, whereas basal stem cells exhibit only slowly activating K+ currents. The question we have addressed in the present study is whether contact with the taste pore is required for expression of voltage-dependent inward currents in Necturus taste cells. Mature taste cells were distinguished from developing cells by labeling the apical surface of the cells with fluorescein-isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), while the tissue was still intact. Elongate cells lacking FITC-WGA staining were interpreted as developing taste cells that had not yet reached the taste pore. Giga-seal whole-cell recording revealed that most developing taste cells lacked inward currents. Although some developing cells expressed inward currents, they were much smaller than those of mature cells, and the activation kinetics of the K+ currents were slower than in mature cells. Electron microscopy confirmed the identity of labeled and unlabeled cells. All FITC-WGA-labeled cells exhibited the ultrastructural characteristics of mature taste receptor cells, whereas most unlabeled taste cells had a characteristic morphology that was markedly different from mature taste receptor cells or basal stem cells. These data suggest that contact with the taste pore is required for the development of inward currents in taste cells.

Growth factor regulation of neurogenesis in adult olfactory epithelium.

Neurogenesis continues throughout adult life in the mammalian olfactory epithelium. This process is a dynamic state of proliferation, differentiation and cell death, probably regulated by autocrine and paracrine signals such as peptide growth factors. Previous investigations have demonstrated roles for some growth factors in olfactory neurogenesis in vitro, but the assay systems used make it difficult to be certain of their effects (proliferation, differentiation, enhanced survival) or their target cells. The present study investigated the effects of growth factors in cultures of purified olfactory epithelium comprising only basal cells and supporting cells in serum-free media. The advantage of this culture system is that proliferation, differentiation and survival of the basal cells and neurons can be examined separately. Under these conditions, three growth factors exerted well-defined effects: (i) fibroblast growth factor-2 stimulated proliferation of the globose basal cells; (ii) transforming growth factor-beta2 induced these cells to differentiate into neurons; and (iii) platelet-derived growth factor promoted survival of the differentiated neurons. We conclude that fibroblast growth factor-2, transforming growth factor-beta2 and platelet-derived growth factor act sequentially on precursor cells and immature neurons during neurogenesis in the adult olfactory epithelium.

Vitamin D3 and brain development.

Evidence for the presence of the vitamin D receptor in brain implies this vitamin may have some function in this organ. This study investigates whether vitamin D(3) acts during brain development. We demonstrate that rats born to vitamin D(3)-deficient mothers had profound alterations in the brain at birth. The cortex was longer but not wider, the lateral ventricles were enlarged, the cortex was proportionally thinner and there was more cell proliferation throughout the brain. There were reductions in brain content of nerve growth factor and glial cell line-derived neurotrophic factor and reduced expression of p75(NTR), the low-affinity neurotrophin receptor. Our findings would suggest that low maternal vitamin D(3) has important ramifications for the developing brain.

Stress induces neurogenesis in non-neuronal cell cultures of adult olfactory epithelium.

Among the basal cells of the olfactory epithelium is a stem cell which divides and whose progeny differentiate into new sensory neurons throughout adult life. Olfactory neurogenesis is highly regulated, for example it is stimulated by epithelial damage. Previous reports implicate several growth factors in progenitor cell proliferation and neuronal differentiation in vitro but these studies differ in growth conditions and age of donors making it difficult to determine precisely the roles of neurogenic stimuli and their sites of action. The aims of the present study were to develop purified basal cell cultures from adult olfactory epithelium and to stimulate neurogenesis in defined growth conditions in order to elucidate the cellular mechanisms by which neurogenesis is stimulated after epithelial damage. We show here that differentiated olfactory sensory neurons arise after biochemical or mechanical stress of rat and mouse olfactory epithelial cell cultures in the absence of growth factors, complex media (e.g., serum, conditioned media, pituitary and hypothalamic extracts), or other cells (e.g., explants, feeder layers of glia, or other non-epithelial cells). Prior to the stress, these cultures contained basal cells and supporting cells but not neurons. After the stress, some cells differentiated into bipolar neurons expressing a number of neuronal proteins including olfactory marker protein. Bromodeoxyuridine experiments show that the differentiated neurons arose from recently divided cells which did not divide again before differentiating. We conclude that stress disrupts cell surface contacts to induce the immediate neuronal precursors to undergo final differentiation into olfactory sensory neurons. This may be a mechanism for enhanced neurogenesis after epithelial damage.

Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia.

The causes of schizophrenia are unknown, but there is evidence linking subtle deviations in neural development with schizophrenia. Embryonic brain development cannot be studied in an adult with schizophrenia, but neurogenesis and early events in neuronal differentiation can be investigated throughout adult life in the human olfactory epithelium. Our past research has demonstrated that neuronal cultures can be derived from biopsy of the human adult olfactory epithelium. In the present study, we examined mechanisms related to neurogenesis and neuronal differentiation in adults with schizophrenia versus well controls. Forty biopsies were collected under local anaesthesia from ten individuals with DSM III-R schizophrenia and ten age- and sex-matched well controls. All patients, except one, were receiving antipsychotic medication at the time of the biopsy. Immunostaining for neuronal markers indicated that neurogenesis occurred in the biopsies from both patients and controls since all contained cells expressing tubulin and/or olfactory marker protein. The major findings of this study are: 1. biopsies from patients with schizophrenia showed a significantly reduced ability to attach to the culture slide: 29.9% of patient biopsies attached compared to 73.5% of control biopsies; 2. biopsies from patients with schizophrenia had a significantly greater proportion of cells undergoing mitosis: 0.69% in the patients compared to 0.29% in the controls; and 3. dopamine (10 microM) significantly increased the proportion of apoptotic cells in the control cultures but significantly decreased the proportion in patients' cultures.

Differentiation in an olfactory cell line. Analysis via differential display.

The olfactory epithelium is a unique system, in which new neurons are continually generated throughout adult life. Olfactory neurons are derived from stem cells that lie adjacent to the basal lamina of the olfactory epithelium; these stem cells divide several times and their progeny differentiate into mature sensory neurons. In our laboratory immortalized cell lines have been derived from these dividing cells. The morphology of these cell lines and their expression of neuronal markers varies with culture conditions. When grown in low serum medium one of these cells lines, OLF 442, differentiates by extending long neurites and increasing its expression of neurofilament and B50/GAP43 proteins at the same time reducing expression of glial fibrillary acidic protein (GFAP). Identification of differentially expressed mRNA in cell lines has previously relied on both screening for known markers, and the use of subtractive techniques for identification of unique mRNA species. The differential display technique allows simultaneous detection of differentially expressed mRNA at different time periods and growth conditions. A modified Liang and Pardee differential display technique was used to screen OLF 442 over a number of time intervals in serum-depleted media, and compared with OLF 442 grown in complete media. The differentially displayed fragments were cloned and sequenced, leading to the identification of a number of sequences, both known and unknown. The known sequences include SPARC (encoding a Ca2+ binding secreted Protein which is Acidic and Rich in Cysteine), which is reported to function as a modulator of the cell matrix, and RHAMM, the receptor for hyaluronan-mediated motility. Both the known and the unknown sequences are being studied further to provide insight into the differentiation of olfactory neurons.

Generation of neurons from a nonneuronal precursor in adult olfactory epithelium in vitro.

Within the olfactory epithelium is a stem cell which can divide and differentiate to produce new sensory neurons. The identity of the neuronal stem cell is unknown but one candidate is the horizontal basal cell which lies adjacent to the basement membrane and expresses keratin. Previous attempts to generate mature sensory neurons from purified horizontal basal cells in vitro were unsuccessful. We show here for the first time that olfactory neurogenesis can be reproduced in vitro from partially-purified cultures of adult rat precursor cells cultivated in a serum-free medium. Rat olfactory epithelium was dissected from the nasal septum and separated from the underlying lamina propria, and its cells were dissociated and grown in a medium containing epidermal growth factor for 5 days. Immunochemistry showed that only supporting cells (SUS1-positive) and horizontal basal cells (keratin-positive) survived for this period. At day 6, the cells were stressed either by passaging them or by a simple mechanical stress. In each case, a morphological and immunological differentiation was observed within 24-48 hr. Newly formed bipolar cells were found to be S100-, glial fibrillary acidic protein (GFAP-), neural cell adhesion molecule (N-CAM+), and/or microtubule-associated protein 5 (MAP-5+). After passaging 14% of the surviving cells were immature neurons (MAP-5+) and 4% were mature olfactory neurons (MAP-5+) and olfactory marker protein (OMP+)). In addition the same experiment was conducted on transgenic mice in which the lacZ gene was linked to the OMP promoter. Using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining we showed that OMP+ cells disappeared before day 5 in culture but reappeared after passaging. These results suggest that olfactory sensory neurons can arise from a non-neuronal precursor, probably the keratin-positive horizontal basal cell.

Neurogenesis in adult human.

This report describes neurogenesis in the adult human olfactory epithelium in vitro. Olfactory epithelium was collected at autopsy and by biopsy, and grown in serum-free medium. Basic fibroblast growth factor induced the differentiation of bipolar cells which were immunopositive for several neuronal proteins but not glial proteins. [3H]thymidine autoradiography confirmed that these neurones were born in vitro. The results demonstrate that the adult human olfactory epithelium retains the capacity for neurogenesis and neuronal differentiation, at least until the age of 72 years. It is now possible to examine neurones and neurogenesis in biopsies from patients with disorders that may involve a neurodevelopmental or neurodegenerative aetiology such as schizophrenia, bipolar disorder and Alzheimer's disease.

Loss of sense of smell in adult, hypothyroid mice.

Adult hypothyroid humans can lose their sense of smell. The present study was designed to investigate whether anosmia follows hypothyroidism in mice. If so, this would provide an animal model in which to study the mechanism for this effect. Adult mice were made hypothyroid with propylthiouracil (PTU) in their drinking water. Their sense of smell was tested before and after treatment by measuring the amount of time they spent sniffing food vs water odours, after a 24-h fast. Thyroid function was assessed histologically or by radioimmunoassay of blood serum for free thyroxine or free triiodothyronine. In Expt. 1 mice treated for 50 days with PTU were hypothyroid and anosmic. Control, euthyroid mice maintained their sense of smell, as did PTU-treated mice which also received daily thyroxine injections. PTU-induced anosmia was reversible: 50 days after removal of treatment previously anosmic mice were euthyroid and had regained their sense of smell (Expt. 3). It was possible that hypothyroidism induced non-specific effects which indirectly affected the olfactory function tests. However, short-term treatment with PTU caused severe hypothyroidism but no anosmia (Expt. 2). Additionally, non-specific effects of hypothyroidism were examined in open-field activity tests after short- and long-term hypothyroidism: euthyroid and hypothyroid animals were similarly active (Expts. 1 and 2). We conclude that chronic hypothyroidism produces anosmia in mice, as it does in humans. This anosmia is prevented by daily injections of thyroxine, and the sense of smell can recover to normal once thyroid function is restored.