The Journey From Metabolic Profiling to Biomarkers: The Potential of NMR Spectroscopy Based Metabolomics in Neurodegenerative Disease Research

Neurodegenerative diseases have become a “hot” topic in recent years. A major factor for this is that as life expectancy of the population in developed countries increases, so does the probability of developing neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), to name the two most well-known. In many cases, however, neurological and mental diseases are poorly understood. In particular, there is a lack of specific biomarkers which would allow early unambiguous identification of a neurodegenerative disease, distinguishing between e.g. AD; PD; PD with dementia; and Dementia with Lewy bodies, or indicating therapeutic effects. Ultimately, this complicates the search for effective treatments. Thus, there is a high demand for preclinical work to elucidate underlying disease mechanisms and pave the way for disease management. In terms of biomarker research, hope has been set on small molecules that participate in metabolism, since they provide a closer link between cellular mechanisms (with genetic as well as environmental inputs) and the disease phenotype. More specifically, it is expected that not one but a combination of several metabolites may serve as an indicator for disease onset and progression, given that neurodegenerative diseases, whilst often described as “idiopathic”, are understood to arise from complex pathologies expressing themselves with a broad spectrum of phenotypes. Therefore, non-targeted metabolic profiling appears to offer great potential for biomarker discovery in this area. One of the major technical platforms for non-targeted metabolic profiling is high resolution nuclear magnetic resonance (NMR) spectroscopy, a technique that is also available for the non-invasive application in vivo. Hence, in theory, biomarker discovery research using NMR spectroscopy based metabolomics provides a promising means for translation from in vitro/ex vivo research to eventual clinical use. This review will therefore discuss the potential for NMR spectroscopy based metabolomics to be applied to biomarker discovery in the field of neurodegenerative disease. Keywords: Brain, metabolites, metabolomics, metabonomics, metabolic profiling, NMR spectroscopy, neurodegenerative diseases. INTRODUCTION Metabolic Profiling of Brain and its Diseases Numerous publications in recent years demonstrate the successful application of metabolic profiling techniques to biofluids and tissues for the purposes of characterising metabolic perturbations associated with diseased states. These include cardiovascular conditions e.g. [1‐4], disorders associated with metabolic syndrome [5-8] and cancer [9] to name but a few. In most cases, multifactorial metabolic changes were identified in those disorders suggestive of the human biocomplexity and indicating that more than just one singular marker is necessary for their comprehension. The application of metabolic profiling techniques to brain diseases has consequently been proposed as a novel approach to shed light onto cellular mechanisms preceding, underlying and following complex neurological and mental conditions. Most of these disorders are incompletely *Address corresponence to this author at the MetaboConsult UK, Heanor, Derbyshire, DE75 7UY, UK; Tel: +44 (0)1773530326; E-mail: clare.daykin@metaboconsult.co.uk understood, resulting in poor diagnostic accuracy and unsatisfactory disease management [10, 11]. Although often explained as “idiopathic” (of unknown origin), genetic predisposition as well as environmental inputs are believed to contribute to a number of neurodegenerative diseases and psychiatric illnesses expressing themselves in altered gene expressions and proteinaceous states [12, 13]. Given that the metabolic state of a biological system, at least in principle, incorporates both genetic (including transcriptional and proteinaceous) and environmental influences, metabolic profiling techniques, with their power to capture information relating to a great number of metabolites in a single analytical run, should be well placed to aid our understanding of the biochemical perturbations associated with these diseases. It is therefore hoped that this relatively novel approach aids the metabolic characterisation of central nervous system (CNS) diseases and hence, the search for valid biomarkers of brain disease onset, progression or therapeutic effects. Further, given the recent late-development stage failure of several AD drugs from pharmaceutical companies, Pfizer, Johnson & Johnson (J&J) and Elan, in Phase III trials, the potential value of a metabolomics approach within this field is clear [14]. 2213-23 /13 $58.00+. 00 © 2013 Bentham Science Publishers NMR Spectroscopy Based Metabolomics in Neurodegenerative Disease Research Current Metabolomics, 2013, Vol. 1, No. 2 161 Investigations of metabolism in the CNS have proven to be complex, given the inaccessibility of the brain. The use of peripheral biofluids as diagnostic media may be questioned to some extent, because direct communication between the CNS and the periphery is hampered by the blood-brain barrier. Nevertheless, the use of blood plasma, serum and urine samples have been reported to determine altered metabolic profiles in schizophrenia [15], Parkinson’s disease (PD) [1618] and Alzheimer’s disease (AD) [19]. Alternatively, cerebrospinal fluid (CSF) is in direct contact with the brain and its samples may therefore reflect the extracellular state of the CNS [20, 21]. Metabolic profiles of CSF have been established for multiple sclerosis [22] and degenerative dementias [23] including AD [24], PD [25] and Huntington’s disease [26]. However, the collection of this central biofluid is not trivial and associated with some health risks. It is not collected routinely during clinical checks, therefore samples are not universally available. Access to samples from controls, healthy individuals is also problematic due to ethical issues associated with sample collection where pain and health risks are involved. Additionally, CSF collection is usually carried out via lumbar puncture and given its slow flow rate, may not necessarily provide an accurate representation of the metabolic processes in the brain [27]. This is reflected in the notion that lumbar and ventricular cerebrospinal fluids are not identical [11]. A further complicating factor is that the brain is a complex, multicompartmental organ, and neurological diseases often affect only particular regions directly. However, metabolic profiles of the cerebrospinal fluid are not region-specific. Therefore, sampling tissues of specific brain areas are perhaps the most suitable matrix for investigating cerebral diseases in order to obtain localised metabolic information. NMR Spectroscopy in Brain Diseases The development of in vivo magnetic resonance spectroscopy (MRS) has enabled the non-invasive recording of the metabolic contents of the whole brain or targeted regions within it. However, it suffers from poor sensitivity and spectral resolution given relatively low magnetic field strengths and the semi-solid texture of the tissue, and can therefore only provide information about abundant metabolites (e.g. Fig. (1). In vivo A) and ex vivo B), C) spectra of rat brain extracts, where A) and B) show the frontal cortex, whilst C) shows the hippocampus. The in vivo spectrum was acquired at 7T using a wide bore magnet. The ex vivo spectrum was obtained from a methanol extract using a 5 mm probe at 9.7 T. KEY: Lact lactate, Ala alanine, GABA γ-aminobutyric acid, NAA N-acetylaspartic acid, Glx glutamate (Glu) and glutamine (Gln), Asp aspartate, Cr creatine, Chol choline, PChol phosphocholine GPChol glycerophosphocholine, Tau taurine, mIns μ-inositol, Gly glycine. 162 Current Metabolomics, 2013, Vol. 1, No 2 Geiszler et al. glutamate) or those whose chemical structures give rise to prominent NMR spectra, such as N-acetylaspartate (NAA). Despite these shortcomings, MRS has still proven to be useful in terms of both research and clinical application of brain disorders such as AD [28, 29], disorders related to PD [30], brain tumours and abscesses [31, 32], cerebral ischemia [3335], schizophrenia [36] and anxiety [37]. The application of high resolution NMR spectroscopy to excised brain tissues or their extracts provides a picture of cerebral metabolism ex vivo. Of course, one disadvantage of ex vivo NMR spectroscopy of brain tissue is that collecting these samples is maximally invasive, hence, unethical in humans except where there is clinical necessity to remove tissue e.g. due to tumour growth. This clearly prohibits longitudinal investigations, which are only possible using biofluids or in vivo MRS. On the other hand, NMR spectroscopy is less expensive and more sensitive and is therefore capable of informing in vivo studies. The spectral difference between an in vivo (brain tissue of a living rat) and an ex vivo (rat brain extract) acquisition is illustrated in (Fig 1). Post-mortem investigations of brain tissues using ex vivo NMR spectroscopy have been applied to e.g. samples from demented patients [38-40] or schizophrenic subjects [41]. However, human post-mortem samples are representative of the late stages of progressive diseases and therefore information relating to the early stages of disease is inaccessible. A second issue is that post-mortem intervals (the time between death and storage of the tissue during which degradation processes occur and the metabolic profile of the tissue consequently changes) are difficult to control, although the introduction of “brain banks” may have improved post-mortem procedures [11]. In contrast to human samples, brain tissues can be excised from animal models of CNS diseases. These models can prove useful for investigation because they, (i) display particular characteristics of disease mechanisms and phenotypes, and (ii) their pathological signs may progress with time, which allows evaluation of differe