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July 2011

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Is spit the future in diagnosis of preclinical Parkinson's?

By Emily Zhou
July 2011

Leroy Worth, Ph.D.

NIEHS Scientific Review Administrator Leroy Worth, Ph.D., hosted the talk. NIEHS has made several training and research grants to Zhang, who joked, "EHS is my host institution," because of its long-term support for his research. (Photo courtesy of Steve McCaw)

Jing Zhang, M.D., Ph.D.

Early in his talk, Zhang said, "Many [researchers] get discouraged with omics studies, because of low reproducibility." However, he said, "[Although] the overlapping of precise targets from omics studies is low, the overlap of pathways [such as mitochondrial dysfunction, oxidative stress, and inflammation] is highly consistent." (Photo courtesy of Steve McCaw)

Honglei Chen, Ph.D.

Along with staff from the NIEHS Division of Extramural Research and Training, scientists from other divisions also wanted to hear Zhang's talk. Shown above, NIEHS Division of Intramural Research epidemiologist Honglei Chen, Ph.D., above, who studies risk factor associations for PD, ponders the potential of non-invasive early detection. (Photo courtesy of Steve McCaw)

NIEHS grantee( Jing Zhang, M.D., Ph.D., is a scientist on a journey, searching for the Holy Grail of biomarkers for early detection of Parkinson's disease (PD). Zhang, a Shaw Endowed Chair in Neuropathology at the University of Washington School of Medicine, discussed his work in a talk June 3 at NIEHS.

The seminar described Zhang's first-of-its-kind research utilizing proteomics to dissect PD by detecting protein markers in human saliva for early diagnosis and tracking of disease progression. Understanding how to translate bench science discoveries in PD biomarkers to the bedside, where easy and non-invasive sampling is essential, has become a major focus of Zhang's research.

In the clinic of the future, Zhang said, "Spitting could be a standard routine" for sample collection to analyze biomarkers for several neurodegenerative diseases, including PD. He emphasized that mass spectrometry (MS) analysis of proteins can be useful in identifying novel biomarkers, because proteins are functional units, and he argued that saliva maybe the future for early diagnosis because it is easy to collect non-invasively, compared to blood or cerebrospinal fluid (CSF).

Biomarker discovery for PD using proteomics by MS

Due to the limitations of dopamine (DA) imaging (see text box), Zhang turned to genomics and metabolomics approaches in his quest for the ideal PD biomarkers. He found, however, that results from these studies were difficult to replicate, partly because of the heterogeneity of human samples and technical difficulties.

Instead, Zhang's laboratory now uses a proteomics platform utilizing MS and refined quantification methods. As he pointed out, "The take home message of MS is that the absence of a protein in an MS analysis only means that it is not detected, not that it's absent."

Zhang picked isotope-labeling technology to better quantify proteins from an MS peak due to its advantage in detection with less abundant proteins and higher quantitative precision. In addition, he and his colleagues utilized a subproteome analysis to examine proteins in discrete neuronal populations and cellular constituents, such as substantia nigra pars compacta, middle frontal cortex, and Lewy bodies. As a result, the group discovered 400 proteins that were altered in abundance in response to the presence and severity of PD.

Validation - the critical test for biomarkers

To verify candidate biomarkers, Zhang utilized Luminex technology to analyze multiple proteins. This technology allows the coating of beads with different fluorescent tags. The coating of the captured antibody pinpoints the identity of the analyte, while a second laser/detector records abundance. Using a combination of different markers for PD diagnosis, progression, and severity, Zhang said he was able to achieve 95 percent specificity and sensitivity. Two major issues he emphasized during the seminar were the utilization of independent cohorts and inclusion of disease control groups for validation studies.

Saliva as the source for preclinical diagnosis of PD

According to Zhang, although most researchers consider CSF the most informative specimen, collection involves extraction of fluid from the spine, a procedure most patients are reluctant to undergo for screening purposes. Blood-based biomarkers have major limitations, especially when a proteomics approach is used, and targeted searches have had limited success to date.

Therefore, Zhang turned to saliva, most of which is produced by the submandibular gland. This gland is regulated by the sympathetic and parasympathetic nervous systems, and, Zhang said, Lewy body pathology in PD patients can be detected in saliva even before patients manifest symptoms.

"The future of preclinical diagnosis [of PD] could be in saliva," concluded Zhang. "Saliva could be useful for [screening] vulnerable populations at high risk for PD, such as those with autonomic failure, heart denervation, constipation, depression, and disorders of olfaction."

(Emily Zhou, Ph.D., is a research fellow in the NIEHS Laboratory of Signal Transduction Inositol Signaling Group.)

PD and current methods of diagnosis

PD is the most serious movement disorder afflicting millions of Americans. Pathological hallmarks of PD are loss of dopaminergic neurons in the substantia nigra with resultant depletion of DA and the presence of Lewy bodies in the remaining neurons. Currently, DA imaging is the gold standard biomarker for PD diagnosis, and it could also provide early diagnosis of parkinsonism. "Even at early clinical stages, a majority of DA is already lost," said Zhang.

However, differentiating PD from other parkinsonian disorders, including multiple system atrophy and progressive supra-nuclear palsy, cannot be readily achieved by DA imaging. DA imaging is also subject to the influence of medications, which makes it difficult to monitor disease progression with this method.

As Zhang explained, "DA imaging, in addition to cost and accessibility, is only a surrogate biomarker, which does not point to any pathological mechanism of PD." According to Zhang, quite a few other movement disorders mimic PD clinically, making an accurate diagnosis of PD often difficult even in the best hands. Finally, the natural course of PD varies substantially, with most patients developing first mild cognitive impairment and then dementia as the disease progresses.

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