Olfactory Neuronal Precursors Research Articles

Olfactory neuronal precursors as a model to analyze the effects of melatonin in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder of great importance affecting millions globally and its prevalence will triple in the following decades. Therefore, analysis and identification of substances which can effectively reduce the pathological process of this disease in different study models are crucial. Melatonin works as a multitasking substance and some of its activities could be used to target the neurodegenerative process of AD. These include, but not limited to, its potent antioxidant activity, regulation of sleep-wake rhythms (important for the consolidation of memory and cognition) and its action as a neurotrophic growth factor that promotes differentiation and neuronal proliferation. To evaluate the effects of melatonin at cellular level in AD, it is essential to have a study model that reflects the pathological process occurring in the CNS. In this, review we summarize the potential use of olfactory neuronal precursors derived from olfactory neuroepithelium directly obtained from patients for such purposes.

Antidepressant Low Doses of Ketamine and Melatonin in Combination Produce Additive Neurogenesis in Human Olfactory Neuronal Precursors.

Melatonin (MEL), an indolamine with diverse functions in the brain, has been shown to produce antidepressant-like effects, presumably through stimulating neurogenesis. We recently showed that the combination of MEL with ketamine (KET), an NMDA receptor antagonist, has robust antidepressant-like effects in mice, at doses that, by themselves, are non-effective and have no adverse effects. Here, we show that the KET/MEL combination increases neurogenesis in a clone derived from human olfactory neuronal precursors, a translational pre-clinical model for effects in the human CNS. Neurogenesis was assessed by the formation of cell clusters > 50 µm in diameter, positively stained for nestin, doublecortin, BrdU and Ki67, markers of progenitor cells, neurogenesis, and proliferation. FGF, EGF and BDNF growth factors increased the number of cell clusters in cultured, cloned ONPs. Similarly, KET or MEL increased the number of clusters in a dose-dependent manner. The KET/MEL combination further increased the formation of clusters, with a maximal effect obtained after a triple administration schedule. Our results show that the combination of KET/MEL, at subeffective doses that do not produce adverse effects, stimulate neurogenesis in human neuronal precursors. Moreover, the mechanism by which the combination elicits neurogenesis is meditated by melatonin receptors, CaM Kinase II and CaM antagonism. This could have clinical advantages for the fast treatment of depression.

Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer’s Disease

Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer’s disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.

Potential Use of Exfoliated and Cultured Olfactory Neuronal Precursors for In Vivo Alzheimer's Disease Diagnosis: A Pilot Study.

Histopathological hallmarks of dementia have been described postmortem in the brain of patients with Alzheimer's disease (AD). Tau, a microtubule associated protein, is abnormally arranged in neurofibrillary tangles. In living AD patients, total tau (t-tau) and hyperphosphorylated tau (p-tau) levels are increased in the cerebrospinal fluid obtained by lumbar puncture. Herein, we studied the t-tau and p-tau levels as well as the subcellular distribution of t-tau in olfactory neuronal precursors obtained by exfoliation of the nasal cavity of AD patients and control participants. Data showed that t-tau and p-tau levels were increased in cell homogenates from AD patients. Also, t-tau immunoreactivity was arranged in a punctate pattern in olfactory neuronal precursors derived from an AD participant with 5years of evolution and in the oldest participants, either control subjects or those with Alzheimer's disease. Results support that exfoliated neuronal precursors have tau alterations demonstrated in postmortem brain and in the cerebrospinal fluid. This evidence and because the obtainment of olfactory neuronal precursors is a noninvasive procedure, detection of tau alterations shown here might be useful for an early diagnosis of AD-type dementia.

Olfactory neuroepithelium as a model for the studies of molecular mechanisms of schizophrenia

Olfactory neuroepithelium (OE) is shown to be a suitable experimental model to study neuronal biomarkers of psychiatric diseases including schizophrenia. Olfactory neuronal precursors can be useful for studying neurodevelopmental stages, neuronal markers, pharmacological screening. However, a limited number of research groups have used this cell model in a small number of patients and healthy people that can be explained by several factors. Based on literature reports and own research, the authors analyze the advantages and limitations of OE-derived cell/tissue models. The main limitations of these models are decreased rate of harvesting and culturing OE-derived cell and low percentage of patients who agreed to participate in the study. Our results showed that only 10% of patients with schizophrenia signed informed consent for nasal biopsy, 80% of them underwent biopsy.

Primary cilia formation is diminished in schizophrenia and bipolar disorder: A possible marker for these psychiatric diseases

Primary cilium (PC) is a microtubule-rich organelle that protrudes from the plasma membrane and acts as a cellular antenna sensing extracellular signals during brain development. DISC1 (Disrupted-in-Schizophrenia-1) is involved in PC formation and is considered a risk factor for neuropsychiatric disorders. We have previously described altered subcellular distribution of DISC1 and an aberrant microtubule organization in olfactory neuronal precursors (ONP) obtained from schizophrenia (SCZ) and bipolar disorder (BD) patients. Herein, we analyzed in vitro PC formation in healthy control subjects, SCZ and BD patients. The results indicated that 66.73±4.33% of ONP from control subjects showed immunostaining for the PC marker, acetylated α-tubulin. By contrast, only a small percentage of cells in culture from paranoid SCZ and BD patients showed PC staining (SCZ, 12.8±4.43%; BD, 12.32±5.86%). However, cells from an affected proband with disorganized SCZ and a subject with BD displayed a higher percentage of cells with cilia (SCZ, 42.20%; BD, 38.59%). Additionally, cilia elongation was observed in lithium-treated ONP derived from all groups, with a more evident response in cells from the BD group. The present study provides novel evidence that the molecular pathways involved in PC formation are defective in SCZ and BD, and impairment in these processes may be involved in the physiopathology of both diseases. Our observations also suggest that ONP is a patient-derived cell model with a potential use for diagnosis and high-throughput drug screening for brain diseases.

Abnormally Increased Secretion in Olfactory Neuronal Precursors from a Case of Schizophrenia Is Modulated by Melatonin: A Pilot Study.

The alterations that underlie the pathophysiology of schizophrenia (SCZ) include the dysregulation of structural and functional properties of neurons. Among these, the secretion of neurotransmitters and hormones, which plays a key role for neuronal communication and development, is altered. Neuronal precursors from the human olfactory epithelium have been recently characterized as a reliable model for studying the etiopathogenesis of neuropsychiatric diseases. Our previous work has shown that melatonin enhances the development of morphological and functional features of cloned olfactory neuronal precursors (ONPs) from a healthy subject. In this work we found that primary cultures of ONPs obtained from a schizophrenic patient display an increased potassium-evoked secretion, when compared with ONPs from an age- and gender-matched healthy control subject (HCS). Secretion was evaluated by FM1-43 fluorescence cumulative changes in response to depolarization. Interestingly, a 12 h-melatonin treatment modulated the abnormally increased secretion in SCZ ONPs and brought it to levels similar to those found in the HCS ONPs. Our results suggest that the actin cytoskeleton might be a target for melatonin effects, since it induces the thickening of actin microfilament bundles. Further research will address the mechanisms by which melatonin modulates neurochemical secretion from ONPs.

The role of melatonin in the neurodevelopmental etiology of schizophrenia: A study in human olfactory neuronal precursors.

Dim light exposure of the mother during pregnancy has been proposed as one of the environmental factors that affect the fetal brain development in schizophrenia. Melatonin circulating levels are regulated by the environmental light/dark cycle. This hormone stimulates neuronal differentiation in the adult brain. However, little is known about its role in the fetal human brain development. Olfactory neuronal precursors (ONPs) are useful for studying the physiopathology of neuropsychiatric diseases because they mimic all the stages of neurodevelopment in culture. Here, we first characterized whether melatonin stimulates neuronal differentiation in cloned ONPs obtained from a healthy control subject (HCS). Then, melatonin effects were evaluated in primary cultures of ONPs derived from a patient diagnosed with schizophrenia (SZ) and an age- and gender-matched HCS. Axonal formation was evidenced morphologically by tau immunostaining and by GSK3β phosphorylated state. Potassium-evoked secretion was assessed as a functional feature of differentiated neurons. As well, we report the expression of MT1/2 receptors in human ONPs for the first time. Melatonin stimulated axonal formation and ramification in cloned ONPs through a receptor-mediated mechanism and enhanced the amount and velocity of axonal and somatic secretion. SZ ONPs displayed reduced axogenesis associated with lower levels of pGSK3β and less expression of melatonergic receptors regarding the HCS ONPs. Melatonin counteracted this reduction in SZ cells. Altogether, our results show that melatonin signaling is crucial for functional differentiation of human ONPs, strongly suggesting that a deficit of this indoleamine may lead to an impaired neurodevelopment which has been associated with the etiology of schizophrenia.

The microtubular cytoskeleton of olfactory neurons derived from patients with schizophrenia or with bipolar disorder: Implications for biomarker characterization, neuronal physiology and pharmacological screening.

Schizophrenia (SZ) and Bipolar Disorder (BD) are highly inheritable chronic mental disorders with a worldwide prevalence of around 1%. Despite that many efforts had been made to characterize biomarkers in order to allow for biological testing for their diagnoses, these disorders are currently detected and classified only by clinical appraisal based on the Diagnostic and Statistical Manual of Mental Disorders. Olfactory neuroepithelium-derived neuronal precursors have been recently proposed as a model for biomarker characterization. Because of their peripheral localization, they are amenable to collection and suitable for being cultured and propagated in vitro. Olfactory neuroepithelial cells can be obtained by a non-invasive brush-exfoliation technique from neuropsychiatric patients and healthy subjects. Neuronal precursors isolated from these samples undergo in vitro the cytoskeletal reorganization inherent to the neurodevelopment process which has been described as one important feature in the etiology of both diseases. In this paper, we will review the current knowledge on microtubular organization in olfactory neurons of patients with SZ and with BD that may constitute specific cytoskeletal endophenotypes and their relation with alterations in L-type voltage-activated Ca(2+) currents. Finally, the potential usefulness of neuronal precursors for pharmacological screening will be discussed.

Contrasting effects of basic fibroblast growth factor and epidermal growth factor on mouse neonatal olfactory mucosa cells

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) affect proliferation and survival of many cell types, but their role in the maintenance of olfactory mucosa cells remains unclear. In the neonatal mouse olfactory mucosa, cell proliferation mainly occurs in the neuroepithelium and, to a lesser extent, in the lamina propria. To establish whether bFGF and EGF affect proliferation and/or survival of these cells, we isolated olfactory mucosa cells from the neonatal mouse and cultured them as free-floating spheres under bFGF or EGF stimulation. Our data demonstrate that bFGF is a mitogen for the rapidly dividing cells (olfactory neuronal precursors and olfactory ensheathing cells), and also a survival factor for both slowly and rapidly dividing cells of the olfactory mucosa. In contrast, EGF appears to be primarily a survival factor for both the olfactory stem and precursor cells.

Progressive and Inhibitory Cell Cycle Proteins Act Simultaneously to Regulate Neurotrophin-mediated Proliferation and Maturation of Neuronal Precursors

Neuronal stem cell expansion and differentiation is a process involving stages of proliferation and maturation governed by the sequential and combinatorial exposure of cells to extrinsic factors. The olfactory epithelium is an excellent model to investigate regulation of this process, as it undergoes neuronal replacement post-natally. We have shown that the neurotrophins NGF and BDNF sequentially promote proliferation of developing olfactory sensory neuronal precursors, although their kinetics of proliferation and cell fate outcomes differ. Interestingly, CNP inhibits this neurotrophin-induced proliferation and promotes the maturation of these precursors to their next developmental stage. Here, we investigate the mechanisms behind these actions. Both NGF and BDNF increase the expression of cyclin D1 and cyclin-dependent kinase 4 (cdk4), with temporal expression patterns that parallel the proliferation kinetics of their cellular targets. The timing of cyclin D1 expression reflects differences in the need for transcription and translation in early and late stage precursors. CNP inhibits neurotrophin-induced cyclin D1 expression, and induces the expression of different profiles of inhibitory cell cycle proteins, which are neurotrophin-specific and correlate with the attainment of different maturational cell fates. Inhibition of protein degradation reverses the effects of neurotrophins and CNP on cyclin D1 and inhibitor expression levels, respectively. These results suggest a model for cell cycle regulation that involves the simultaneous expression of progressive and inhibitory cell cycle regulatory proteins in response to both proliferation and differentiation agents, followed by selective degradation of these proteins, providing a mechanism for rapid and exquisite control of the cell cycle.

GSK3β and PKCζ function in centrosome localization and process stabilization during Slit-mediated neuronal repolarization

In comparison with other migratory cells, neurons exhibit a unique, highly polarized morphology and a distinctive pattern of movement. This migration consists of a repeating of three distinct phases: neurite outgrowth, movement of the centrosome into the leading process, and translocation of the nucleus towards the centrosome. The direction of movement is under the control of extracellular guidance cues, but mechanisms by which these determine neuronal polarity, centrosome position, and neuronal movement are not well understood. We found that in primary olfactory bulb neuronal precursors, Slit-mediated repolarization consisted of growth of a new process from the previous trailing edge, then reorientation of the centrosome followed by nuclear translocation in the reverse direction. Inhibition of cell polarity factors GSK3β or PKCζ resulted in impaired centrosome reorientation and process stabilization. Our findings suggest that activation of cell polarity signaling and positioning of the centrosome ahead of the nucleus are important steps in repolarization in response to guidance cues.

Atrial natriuretic peptide type C induces a cell-cycle switch from proliferation to differentiation in brain-derived neurotrophic factor- or nerve growth factor-primed olfactory receptor neurons.

With the discovery of postnatal stem cells within the brain, it has become important to understand how extracellular factors might affect the maturation of neuronal precursors in the postnatal brain. Neurotrophic factors are known to play a role in neuronal development but display pleiotrophic effects, in part because of their physiological interactions with other factors. One factor positioned to interact with neurotrophins in the brains of postnatal animals is atrial C-type natriuretic peptide (CNP). In this study, we used olfactory receptor neurons (ORNs) as a model, because their precursors demonstrate the most robust and functional postnatal neurogenesis of those systems thus far described. We examined the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and the interactions of these neurotrophins and CNP in postnatal olfactory neuronal precursors. Results obtained using mice with targeted deletion of the gene for BDNF indicated that BDNF is a neuroproliferation-inducing and survival factor for ORN precursors. These roles were confirmed in vitro using primary cultures of ORNs. NGF was found to be a proliferation-inducing factor but not a survival factor. The addition of CNP to either BDNF- or NGF-treated neuronal precursors resulted in an inhibition of proliferation and the promotion of maturation. These effects were accompanied by changes in cell-cycle proteins that suggest possible mechanisms for these effects. Thus, CNP may function in the postnatal brain to regulate the exit from the cell cycle in neuronal precursor cells.

Atrial Natriuretic Peptide Type C Induces a Cell-Cycle Switch from Proliferation to Differentiation in Brain-Derived Neurotrophic Factor- or Nerve Growth Factor-Primed Olfactory Receptor Neurons

With the discovery of postnatal stem cells within the brain, it has become important to understand how extracellular factors might affect the maturation of neuronal precursors in the postnatal brain. Neurotrophic factors are known to play a role in neuronal development but display pleiotrophic effects, in part because of their physiological interactions with other factors. One factor positioned to interact with neurotrophins in the brains of postnatal animals is atrial C-type natriuretic peptide (CNP). In this study, we used olfactory receptor neurons (ORNs) as a model, because their precursors demonstrate the most robust and functional postnatal neurogenesis of those systems thus far described. We examined the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and the interactions of these neurotrophins and CNP in postnatal olfactory neuronal precursors. Results obtained using mice with targeted deletion of the gene for BDNF indicated that BDNF is a neuroproliferation-inducing and survival factor for ORN precursors. These roles were confirmed in vitro using primary cultures of ORNs. NGF was found to be a proliferation-inducing factor but not a survival factor. The addition of CNP to either BDNF- or NGF-treated neuronal precursors resulted in an inhibition of proliferation and the promotion of maturation. These effects were accompanied by changes in cell-cycle proteins that suggest possible mechanisms for these effects. Thus, CNP may function in the postnatal brain to regulate the exit from the cell cycle in neuronal precursor cells.

Extracellular Signals That Regulate the Tangential Migration of Olfactory Bulb Neuronal Precursors: Inducers, Inhibitors, and Repellents

Neuronal migration is an essential developmental step in the construction of the vertebrate nervous system, but the extracellular signals involved in initiating and regulating neuronal movement remain unclear. Here we report the identification of a novel astrocyte-derived migration-inducing activity (MIA). Using an in vitro assay, we show that MIA induces the migration of olfactory bulb interneuron precursors, increasing the number of migrating cells and the distance they move. We established quantitative criteria to distinguish between the biological effects of inducers, inhibitors, repellents, and attractants on migrating cells and used them to compare the effects of MIA with those of Slit, a putative repulsive guidance cue. Our analysis demonstrates that, by themselves, MIA induces and Slit inhibits migration from subventricular zone explants. However, when presented together with MIA, Slit acts as a repellent. This study shows that glial cells play a critical role in initiating and modulating the movement of neuronal precursors through the release of a diffusible protein. Moreover, this study provides evidence that the guidance of migrating neuronal precursors is an integrative process, resulting from the cooperation of distinct extracellular factors, and that the function of Slit is context dependent.

BFGF Induces Differentiation and Death of Olfactory Neuroblastoma Cells

Olfactory neuroblastoma (ONB) is a highly vascularized and malignant tumor arising in olfactory neuronal precursors from the paranasal sinuses. Previously, we showed that treatment of JFEN cells with transforming growth factor (TGF)-α caused them to differentiate and respond to chemical odorants, whereas basic fibroblast growth factor (bFGF) treated cells differentiated and died. In the present study we show that established ONB tumors treated with bFGF upregulate the bFGF receptor (FGFR1) prior to differentiation. This cellular differentiation was evidenced by bFGF-induced expression of the human runt homologue AML1 (PEBP2αB, CBFA-2) that is highly expressed in developing olfactory neuroepithelium and TrkA, a preferred nerve growth factor receptor. Since TrkA is expressed in supporting cells, but not in mature olfactory neurons, we hypothesize that the expression of AML1 and TrkA in bFGF-treated JFEN cells induced supporting cell differentiation. Collectively, these results have implications for the treatment of patients afflicted with ONB.

Basic fibroblast growth factor supports human olfactory neurogenesis by autocrine/paracrine mechanisms

Throughout life, olfactory sensory neurons are renewed from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of olfactory neuronal precursors; however, evidence indicates that soluble mediators may play a central role in olfactory neurogenesis. To identify molecules that regulate olfactory self-renewal and differentiation, we have recently established, cloned and propagated in vitro primary long-term cell cultures from the human fetal olfactory neuroepithelium. Here, we show that primary human olfactory neuroblasts synthesize and release biologically active basic fibroblast growth factor which, in turn, supports neuroblast growth by autocrine/paracrine mechanisms. The growth-promoting activity of basic fibroblast growth factor is dose dependent, and is accompanied by morphological changes of the cells and by an increase in the expression of neuronal-related genes. These observations indicate that endogenous basic fibroblast growth factor participates in controlling olfactory self-renewal and suggest that this cytokine represents a key regulatory element of olfactory neurogenesis.

Focal Ventricular Origin and Migration of Oligodendrocyte Precursors into the Chick Optic Nerve

During central nervous system (CNS) development, oligodendrocyte precursors originate in specific locations and subsequently migrate to all regions of the CNS. Here, we demonstrate that the chick optic nerve is populated by oligodendrocyte precursors, which initially appeared in a focal region at the ventral midline of the third ventricle at stage 26–27. Oligodendrocyte precursors migrated into the chiasmal end of the nerve by stage 29 and became uniformly distributed by stage 35. Migrating precursors were restricted to the anterior region of the nerve, closely apposed to axons, and had a bipolar morphology. In contrast to the polysialic acid (PSA)–dependent cooperative streaming migration of olfactory neuronal precursors, the migration of oligodendrocyte precursors along the optic nerve appeared axophilic and unaffected by removal of neural cell adhesion molecule (N-CAM)–associated PSA. These data indicate that during development, defined domains of the ventricular zone give rise to distinct cell types that utilize discrete mechanisms to navigate specific migrational pathways.