Neuronal biomarkers from patients with mental illnesses: a novel method through nasal biopsy combined with laser-captured microdissection

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A major objective of molecular psychiatry is to identify molecular biomarkers relevant to disease-associated phenotypes and endophenotypes. Until now, many studies have used non-neuronal tissues and cells, such as lymphocytes and fibroblasts, to address this question. A major criticism of this approach is that the information obtained from these tissues/cells may not reflect disease-associated ‘neuronal’ changes. Olfactory epithelium (OE) is a unique tissue in the olfactory system. In OE, dividing neuronal stem and basal precursor cells continuously give rise to immature neurons that migrate through the epithelium and differentiate into mature olfactory receptor neurons.1 Thus, OE represents, in part, developmental processes of the nervous system, even in adulthood. As deficits associated with neurodevelopment are involved in the pathology of schizophrenia,2 the biological information from OE may reflect this pathology-associated disturbance more directly. Furthermore, OE is an accessible tissue that can be reached without significant invasive procedures. Thus, at least two groups have made a pioneered contribution in using nasal biopsy and OE in schizophrenia research.3, 4 Nonetheless, one major drawback in using OE-containing tissue has been its substantial contamination with non-neuronal cells and poor enrichment of neuronal cells. To overcome this limitation, we applied a laser-captured microdissection of neuronal OE layers from biopsy samples for the first time (Figure 1a). After the microdissection, a relative expression level of olfactory marker protein, a marker for olfactory receptor neurons, was nearly 30-fold higher in the microdissected tissue compared with that in the whole OE. Tubulin beta 3 (TUBB3), an immature olfactory receptor neuronal marker, was expressed about three times higher in the microdissected tissue than in the whole OE. In contrast, relative expression levels of a nasal submucosal marker, aldehyde dehydrogenase 1A3, were decreased in the microdissected tissue. Hence, the results clearly show neuronal enrichment by laser-captured microdissection (Figure 1b). To address whether this neuronal tissue is a good resource to identify molecular profile changes related to endophenotypes/phenotypes of schizophrenia, we examined the association of gene expression with performance on an odor identification test, which is known to be impaired in patients with schizophrenia.5, 6, 7, 8 Correlation between the University of Pennsylvania Smell Identification Test score9 and gene expression was calculated across all microarray probes that surpassed the empirical expression level cutoff. The full list of genes used for correlation analysis is shown in the Supplementary Table. The correlation for several top genes was confirmed by quantitative real-time PCR. In this report, we describe a novel strategy to study disease-associated molecular profiles in a neuronal cell population by combining nasal biopsy and laser-captured microdissection to enrich OE neuronal layers. We also show that the enriched OE neuronal layer is a good resource for identifying molecular profile changes that could facilitate the discovery of biomarkers. We note that non-neuronal sustentacular cells may still be present with this method of dissection. This limitation could be easily overcome by combining histological approaches, such as immunohistochemistry and in situ hybridization. Odor identification deficits examined in this study might reflect abnormalities of both peripheral and central olfactory circuitry. Instead, odor-specific detection threshold sensitivity, which reflects abnormalities in the peripheral components of the olfactory system, especially OE, could be a more useful index of odor function to correlate with gene expression profiles in future studies.10 One caution that usually applies to all research in the field of mental illnesses, especially schizophrenia, is the use of samples from patients with chronic medication. Thus, it will then be important to study first-onset patients and extend the research to their non-affected first-degree relatives to overcome the effect of medications on gene expression. This methodology may be useful to follow up patients in order to identify biomarkers of treatment response. This can also be applied to individuals at risk in order to predict passage to a full-blown psychosis. Finally, we hope to develop various biomarkers that can be tested during a simple clinical examination and reflect etiology-based mechanisms of disease susceptibility and pathology.

Figure 1
figure1

Enrichment of neuronal cells from nasal biopsy samples by laser-captured microdissection. (a) Laser-captured microdissection of olfactory epithelium (OE) neuronal layers. Black arrowheads indicate the microdissected OE. Red arrowhead indicates the submucosa. (b) Quantitative real-time PCR for marker genes, such as olfactory marker protein (OMP, a marker for olfactory receptor neurons); tubulin beta 3 (TUBB3, a marker for immature olfactory receptor neurons); hairy and enhancer of split homolog-1 (HES1, a marker for sustentacular cells); aldehyde dehydrogenase 1A3 (ALDH1A3, a marker for nasal submucosa); and regenerating islet-derived 3-gamma (REG3G, a marker for respiratory epithelium). The color reproduction of the figure is available on the online html full text version of the figure.

Conflict of interest

This work is supported, in part, by Astellas Pharma Inc.

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Acknowledgements

This work was supported by USPHS grants of MH-084018 Silvo O Conte center and MH-069853 (AS), and grants from Stanley, CHDI, HighQ, RUSK foundations, NARSAD and S-R foundations (AS).

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Correspondence to A Sawa or N G Cascella.

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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

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Tajinda, K., Ishizuka, K., Colantuoni, C. et al. Neuronal biomarkers from patients with mental illnesses: a novel method through nasal biopsy combined with laser-captured microdissection. Mol Psychiatry 15, 231–232 (2010) doi:10.1038/mp.2009.73

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