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Altered Volatile Organic Compounds Profile in Transgenic Rats Bearing A53T Mutation of Human Alpha-Synuclein: Comparison with Dopaminergic and Serotonergic Denervation John P. M. Finberg, Yuval Aluf, Yelena Loboda, Morad K. Nakhleh, Raneen Jeries, Manal Aboud, Salman Zubedat, Avi Avital, soliman khatib, Jacob Vaya, and Hossam Haick ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00318 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017
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ACS Chemical Neuroscience
Altered Volatile Organic Compounds Profile in Transgenic Rats Bearing A53T Mutation of Human Alpha-Synuclein: Comparison with Dopaminergic and Serotonergic Denervation
John P. M. Finberg1*, Yuval Aluf1, Yelena Loboda1, Morad K. Nakhleh2, Raneen Jeries2, Manal Aboud2, Salman Zubedat1, Avi Avital1, Soliman Khatib3, Jacob Vaya3, and Hossam Haick2* 1
Neuroscience Department, Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 3200003, Israel
2
Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa 3200003, Israel
3
Laboratory of Natural Medicinal Compounds, Migal-Galilee Research Institute, Kiryat Shmona and Tel Hai College, Israel
Abstract Early diagnosis of Parkinson’s disease (PD) is of great importance due its progressive phenotype. Neuroprotective drugs could potentially slow down disease progression if used at early stages. Previously, we have reported an altered content of volatile organic compounds (VOCs) in the breath of rats following a 50% reduction in striatal dopamine (DA) content induced by 6hydroxydopamine. We now report on the difference in the breath-print and content of VOCs between rats with mild and severe lesions of DA neurons, serotonergic neuronal lesions, and transgenic (Tg) rats carrying the PD-producing A53T mutation of the SNCA (alpha-synuclein) gene. The Tg rats had an increased content of 3-octen-1-ol and 4-chloro-3-methyl phenol in blood, while in brain tissue, hexanal, hexanol and 2,3-octanedione were present in controls but absent in Tg rats. Levels of 1-heptyl-2-methyl cyclopropane were increased in brain tissue of Tg rats. The data confirm the potential of breath analysis for detection of human- idiosyncratic as well as autosomal dominant PD.
Keywords: Parkinson; Diagnosis; Breath; Volatile Organic Compound; Sensor; 6Hydroxydopamine; 5,7-Dihydroxytryptamine; Transgenic
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Introduction Early diagnosis is important in many disease conditions, cheap and non-invasive techniques that are readily-available which can be applied to large numbers of subjects are much needed.1-4 Analysis of exhaled breath by a sensitive sensor array is a rapidly-advancing technique,5 which is being developed for wide-scale use in early detection of a number of disease conditions, including gastric, pulmonary and other types of cancer,5-21 renal and hepatic disease22-25 as well as infectious diseases such as tuberculosis.26,27 Bioinspired, this system relies on detection of the collective alteration in the profile of exhaled Volatile Organic Compounds (VOCs) which is known to alter during human disease conditions.28 Early detection of Parkinson’s disease (PD) is particularly important, since clinical motor symptoms of the disease, on which the diagnosis is mainly based, only appear after the loss of 50-70% of the target dopaminergic neurons in the substantia nigra pars compacta.29,30 Up to this extent of loss, the brain compensates by increasing the release of dopamine (DA) from the remaining neurons,31 which increases the level of oxidative stress in these neurons.32 Early detection could promote early neuroprotective therapy, potentially slowing down disease progression and improving the clinical outcomes. Although only one neuroprotective agent, rasagiline, has been found to reduce the rate of disease progression in Parkinsonian patients,33 yet much research is being directed to the development of additional therapies. We previously reported that an artificial olfactory system, based on organically functionalized34,35, random networks of single-walled carbon nanotubes (CNT) and/or gold nanoparticle (GNP) sensors could be trained to detect 6-hydroxydopamine-induced (6-OHDA) CNS dopamine (DA) depletion in rats by exhaled breath analysis.36 The system successfully discriminated between rats with 50% degeneration of CNS dopaminergic neurons and controls, with an accuracy of 90%.36 Moreover, in one clinical study, the examination of breath samples from PD patients and control subjects resulted in a sensitivity of 70 %, specificity of 100 % and accuracy of 79 %, based on the pattern response of the sensors,37 and a more recent study38 has shown the ability of this system to distinguish between patients with idiopathic Parkinson’s disease (IPD) and parkinsonism (a disease condition which resembles IPD). A complementary quantitative analysis of breath samples, in both clinical studies, revealed that a number of exhaled VOCs were significantly altered, thus producing a specific and distinctive volatolomic pattern, that is recognized by the sensor array.37,38
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Current theories on the etiology of Parkinson’s disease stress the role of toxic oligomers of the protein α-synuclein (SNCA) in producing the neurodegeneration.39 This protein is a normal constituent of neuronal tissue which has been shown to adopt a misfolded conformation following exposure to a variety of triggers, including increased oxidative stress, and in certain mutated forms.40 One of the known toxic mutations of SNCA is Ala53Thre (A53T), which is linked to certain genetically-inherited autosomal dominant forms of the disease.41 Transgenic rats (Tg) bearing the human A53T SNCA mutation are therefore a suitable animal model for investigation of PD, and for elucidation of potential disease-related biomarker molecules. We now describe the results of further research with the 6-OHDA rat model, and recent studies with the Tg rat model, aimed to obtain additional support for the use of our nanomaterial-based sensor array in diagnosis of human PD, and to extend the search for VOCs specific to this disease.
Results and Discussion: In our previous results, we showed a clear difference in exhaled VOC patterns between 6-OHDA and control rats using the nanosensor array.36 Here we examined an additional rat denervation model (serotonergic, produced by injection of the neurotoxin 5,7-dihydroxytryptamine to the raphe nucleus) by comparison with 50 and 70% dopaminergic denervation, produced by intracerebroventricularly-injected (icv) 6-OHDA (Figure 1). We also examined the exhaled breath of the Sprague-Dawley (SD) transgenic rat (Tg) model carrying the human A53T SNCA mutation as an additional parkinsonian model. The use of a model that is based on a human PDcausing mutation permits a more direct connection of the breath result obtained here from the animal model with those potentially obtained from human breath samples. These rats show a pronounced decrease in general motor activity above age 6 months, as well as loss of substantia nigra DA neurons and alterations in expression of mGluR1 glutamate receptors at age 16 months.42 However, at the age of the rats used in this study (4 months) the behavioral changes were minimal.
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Figure 1 - Striatal DA levels in 6-OHDA- and 5,7-DHT-lesioned rats. Striatal DA content is expressed as ng per mg wet weight of tissue following 250 or 325 µg 6-OHDA injected intra-cerebroventricularly which produced 50% or 70% respectively loss of striatal DA content. ** PWT*
RT
Compound name
Control WT
Tg
8.7
Unidentified
Present
Present>WT*
8.9
3-octen-1-ol
Present
Present >WT*
11.7
Unidentified
Present
Present >WT*
17.2
Phenol, 4-chloro-3-methyl
Present
Present >WT**
Blood
To explain the differences in the sensor responses to different groups of breath samples, a complementary quantitative analysis was carried out, using gas chromatography linked with mass spectrometry (GC-MS). Results of this analysis in 6-OHDA rats were reported previously.36,44 Alterations in abundance of VOCs in blood and brain tissue between Tg rats and their controls using GC-MS methodology are shown in Figures 5 and 6. Four VOCs obtained at retention time (RT) of 8.66, 8.9, 17.2 and 21.2 min were found to be significantly increased in the blood of the Tg rats versus controls. The peak at 8.9 min. was identified as 3-octen-1-ol and the peak at 17.2 was identified as 4-chloro-3-methyl phenol (Figure 5, Table 1). The quantity of VOCs in peaks at RT of 8.66 and 21.2 min was too small to permit determination of their identities. In the brain tissue homogenates, compounds at RT of 3.02, 4.84 and 9.11 min, which were identified as hexanal, hexanol and 2,3-octanedione respectively, were present only in the controls, and totally absent in Tg rats (Figure 6, Table 1). The compound at RT of 9.17 was
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detected in Tg rats only but could not be identified due to its small amount. Compounds at RT of 12.83, 13.79 and 21.03 were significantly increased in the Tg rat brain homogenate versus controls. The compound at RT 12.83, was identified as 1-heptyl-2-methyl, cyclopropane, but the others were in too small an amount to permit identification. Unfortunately, due to a technical failure we were unable to obtain VOC data in breath samples from the Tg group. Therefore, there were difficulties in correlating between the blood, brain and exhaled breath samples, and this topic will need to be further addressed in future studies.
Figure 5 – GC-MS chromatogram of VOCs obtained from striatal homogenate of Tg rats and WT controls following extraction by SPME. *P
