Regular ArticleHuman nasal olfactory epithelium as a dynamic marker for CNS therapy development
Highlights
► Use of nasal biopsies as a surrogate marker for drug effects in the CNS. ► Astrocytic proteins expressed in human peripheral olfactory epithelial tissue. ► Glutamate transporter upregulating agents increase transporter mRNA in human olfactory epithelial tissue samples. ► Nasal biopsies can be reliably and safely employed in human clinical trials.
Introduction
A biomarker is a characteristic that is objectively measured as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention. The discovery of new CNS acting therapeutics has been slowed down by the lack of useful relevant biomarkers and/or pharmacodynamic measures of disease or drug action. The difficulty for biomarkers in the vast majority of neurologic and psychiatric diseases lies in part in the lack of availability of appropriate tissue samples. Therefore, finding peripheral markers or easily accessible central nervous system (CNS) markers that accurately reflect biology in the CNS is a major goal and critical endeavor in clinical pharmacology of CNS drug development (Collins, 2011). Recently, non-neuronal cells, such as astrocytes, have been reported to play a highly significant role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, Huntington's disease, as well as psychiatric disease, such as depression (Lobsiger and Cleveland, 2007; Yamanaka et al., 2008). For example in ALS, Huntington's disease, and multiple sclerosis astrocytes are characterized by a loss of excitatory amino acid transporter 2 (EAAT2; rodent nomenclature: glutamate transporter 1 (GLT-1)) protein and mRNA expression (Edwards, 2009; Ronnett et al., 2003; Rothstein et al., 1995; Werner et al., 2001; Yang et al., 2009). The loss of this transporter results in inefficient clearance of extracellular glutamate, which in the case of ALS leads to glutamate-mediated motor neuron cell death (excitotoxicity), one pathway implicated in ALS pathogenesis (Rothstein et al., 1996; Rothstein et al., 1995). At the same time, manipulation of synaptic glutamate by altering transporter efficacy has been proposed as a novel approach to behavioral disorders such as depression (Banasr et al., 2010; Mineur et al., 2007).
While drugs are increasingly considered for targeting various glial cell types, astroglia are restricted to the central nervous system and thus not readily accessible to assess phenotypic or functional changes in response to targeted drug therapy. Cultured embryonic human or rodent astroglia show some of the characteristics of in vivo astroglia cells (e.g. expression of minor subtypes of glutamate transporter proteins) but are not reliable predictors of functional responses similar to those observed in vivo and in adult tissues. Therefore it is crucial to develop a method that allows us to follow the biological activity of a drug in vivo using an effective and cell specific but minimally invasive approach.
Here we report the development of a pharmacodynamic marker employing neural olfactory epithelial (OE) tissue as a drug validation tool for expression of astroglial proteins, including EAAT2/GLT-1 and glial fibrillary acidic protein (GFAP). EAAT2/GLT-1 is an astrocytic protein that is downregulated in diseased states, plays a significant role in triggering excitotoxic neuronal cell death during disease progression, and is a potential target for drug development for both neurological and psychiatric disease. GFAP is an astroglial specific intermediated filament protein.
It was recently shown that rodent olfactory epithelial mucosa does express specific markers of CNS cell types including astrocytic marker GFAP, as well as neuronal markers such as glutamate receptor subtypes, nerve growth factor receptors and neurotransmitters GABA and glutamate (Au and Roskams, 2002; Au and Roskams, 2003; Priest and Puche, 2004; Thukral et al., 1997). Therefore, nasal olfactory tissue has been used to study CNS disease mechanisms either by performing histological pathology analyses or by culturing OE cells in vitro (Hahn et al., 2005; Seal and Edwards, 2006; Voglmaier and Edwards, 2007; Voglmaier et al., 2006). However, to our knowledge, no studies have used OE tissue samples as a surrogate pharmacodynamic marker for astroglial drug targets.
Based on our interest in glutamate transporter function and dysfunction, we chose to test drugs that upregulate EAAT2/GLT-1 protein and mRNA. Upregulation of astroglial glutamate transporter gene expression has been suggested to protect against glutamate-mediated neuronal cell death by removing excess extracellular glutamate (Rothstein et al., 1996). The neuroprotective properties of glutamate transporter upregulating compounds has since been shown in a number of animal models of neurodegenerative diseases including ALS, HD, ischemia and psychiatric disease such as depression (Hota et al., 2008; Lipski et al., 2007; Miller et al., 2008; Rothstein et al., 2005; Thone-Reineke et al., 2008).
The drugs used in our study, ceftriaxone and thiamphenicol, were discovered from a screen of FDA approved compounds (Rothstein et al., 2005) and were subsequently tested for CNS penetration. Therefore, they were ideal candidates to use for the validation of an assay which monitors EAAT2/GLT-1 levels in peripheral easily accessible neural tissue which we discovered serves as an accurate pharmacodynamic reporter of drug action in the CNS.
To access olfactory tissue mice and human volunteers underwent a biopsy of neural olfactory epithelial tissue. Treatment of mice with EAAT2/GLT-1-upregulating compound thiamphenicol (TAP) showed increased EAAT2/GLT-1 gene expression in nasal neuro-epithelial rodent tissue samples that was similar to the increase in rodent brain tissue. These observations were then validated in human tissue with a human clinical trial measuring EAAT2/GLT-1 levels in OE tissue samples before and after drug treatment with the same EAAT2/GLT-1 upregulating compound in healthy volunteers. The results suggest that the nasal olfactory biopsy, a simple, repeatable and safe procedure, is a valuable approach to evaluate the biological activity of drugs targeted for astrocytes or neurons in patients during the course of clinical drug development.
Section snippets
Ethics
This study was conducted according to the principles expressed in the Declaration of Helsinki. This study was approved by the Institutional Review Board of the Johns Hopkins University, USA (Protocol Approval NA_00016223) and SGS Life Science Services, Belgium (EC Approval #2987), which conducted the nasal biopsies in drug-treated healthy volunteers. All patients provided written informed consent for the collection of samples and subsequent analysis. For animal experiments, the full study was
Expression of EAAT2/GLT-1 in olfactory epithelial tissue
To study the expression of astroglial EAAT2/GLT-1 in the olfactory epithelium, we first analyzed mouse and human nasal tissue for EAAT2/GLT-1 protein levels using standard immunoblotting techniques. Human olfactory mucosa was obtained via outpatient nasal biopsy, a low-risk procedure that is well established with no discernible adverse effects including on olfactory function (Lanza et al., 1994; Lovell et al., 1982). Lysates of mouse and human nasal tissue samples were separated on SDS gels and
Discussion
These studies provide both pre-clinical and human validation of a new relatively simple approach to measuring biological activity of CNS targeted pharmaceuticals in peripheral tissue samples. The development of CNS relevant pharmacodynamic and biomarker tissue tools is a serious unmet need in the development of CNS acting pharmaceuticals. Biomarkers provide the opportunity to relate long-term clinical outcome measures to immediate validated biochemical changes caused by drug therapy. A
Conflict of interest
J.D.R. and R.C. have an equity interest in Psyadon Pharmaceuticals; J.D.R. and R.C. are on the Psyadon Board of Directors. D. B. has equity interest and serves on the Board of Directors of Gliknik Pharma. R.S. and J.D.R. are on a pending patent for the use of thiamphenicol in various neurological and psychiatric disorders. No other authors have competing interests to declare.
Author contributions
A.Y. and L.C. performed animal and human qPCR; A.Y. performed all immunohistochemistry; L.C. performed immunoblotting assays; R.S., D.B., R.C., J.D.R. designed rodent and human experiments; A.S. developed biopsy method; A.S., D.B. and R.C. edited the manuscript; R.S. and J.D.R. wrote the manuscript.
Acknowledgment
The authors thank Drs. Jeffrey Wolf and Rodney Taylor, University of Maryland and Dr. Sandra Lin, Johns Hopkins University for performing the surgical biopsies; Dr. Yongjie Yang, Johns Hopkins University for assistance in quantitative PCR data analysis; Rebecca Michaud and Jackie Gutenkunst for technical assistance, Drs. Randy Reed, Johns Hopkins University and Frank Margolis, University of Maryland with isolation and analysis of rodent nasal tissue. This work was supported by NIH NS33958,
References (62)
- et al.
Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes
Neuroscience
(1982) - et al.
Identification and molecular regulation of neural stem cells in the olfactory epithelium
Exp. Cell Res.
(2005) Glutamate uptake
Prog. Neurobiol.
(2001)- et al.
Application of real-time radiation dosimetry using a new silicon LET sensor
Mutat. Res.
(1999) What the neuron tells glia
Neuron
(2009)- et al.
Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia
Schizophr. Res.
(1999) - et al.
Adenosine reduces cortical neuronal injury induced by oxygen or glucose deprivation in vitro
Neurosci. Lett.
(1988) Cell dynamics in the olfactory mucosa
Tissue Cell
(1973)- et al.
Ceftriaxone rescues hippocampal neurons from excitotoxicity and enhances memory retrieval in chronic hypobaric hypoxia
Neurobiol. Learn. Mem.
(2008) - et al.
An essential role for dendritic cells in human and experimental allergic rhinitis
J. Allergy Clin. Immunol.
(2006)
Mechanism of ceftriaxone induction of excitatory amino acid transporter-2 expression and glutamate uptake in primary human astrocytes
J. Biol. Chem.
Harmine, a natural beta-carboline alkaloid, upregulates astroglial glutamate transporter expression
Neuropharmacology
Neuroprotective potential of ceftriaxone in in vitro models of stroke
Neuroscience
Cell cycle alterations in biopsied olfactory neuroepithelium in schizophrenia and bipolar I disorder using cell culture and gene expression analyses
Schizophr. Res.
Up-regulation of GLT1 expression increases glutamate uptake and attenuates the Huntington's disease phenotype in the R6/2 mouse
Neuroscience
Antidepressant-like effects of ceftriaxone in male C57BL/6J mice
Biol. Psychiatry
Localization of neuronal and glial glutamate transporters
Neuron
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate
Neuron
Expression of non-N-methyl-d-aspartate glutamate receptor subunits in the olfactory epithelium
Neuroscience
Do different endocytic pathways make different synaptic vesicles?
Curr. Opin. Neurobiol.
Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling
Neuron
Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia
Neuroscience
Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1
Neuron
Culturing olfactory ensheathing glia from the mouse olfactory epithelium
Methods Mol. Biol.
Olfactory ensheathing cells of the lamina propria in vivo and in vitro
Glia
Thiamphenicol excretion in subjects with renal insufficiency
Int. Z. Klin. Pharmakol. Ther. Toxikol.
Glial pathology in an animal model of depression: reversal of stress-induced cellular, metabolic and behavioral deficits by the glutamate-modulating drug riluzole
Mol. Psychiatry
Distribution of the glutamate transporters GLT-1 (SLC1A2) and GLAST (SLC1A3) in peripheral organs
Anat. Embryol. (Berl.)
Topological relations between the dendrites of olfactory sensory cells and sustentacular cells in different vertebrates. An ultrastructural study
J. Anat.
Reengineering translational science: the time is right
Sci. Transl. Med.
Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport
Nat. Neurosci.
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