Movement Disorders Focus: Thinking Outside the Brain

Alpha-synuclein is largely located in the brain, specifically at the tips of neurons in presynaptic terminals. It has attracted the interest of Parkinson’s researchers because it is a major constituent of Lewy bodies, and due to compelling evidence from studies that it may have a part in the development of both familial and sporadic cases of Parkinson’s disease.

Interestingly, alpha-synuclein pathology has been found to exist outside the body systems traditionally associated with PD, as well as in patients who did not experience any clinical features of the disease. For this reason, study of alpha-synuclein has been given focus by organizations like the Michael J. Fox Foundation to test the emerging hypothesis that Parkinson’s affects many areas of the central nervous system beyond the substantia nigra, and even extends beyond the boundaries of the central nervous system. Here, we take a look at approaches that seek to expand the horizon of treatment.

RETINAL BIOMARKERS

Thomas Beach, MD, PhD

Banner Sun Health Research Institute

A number of structural and functional eye and vision abnormalities have been reported in subjects with PD, and much attention has been paid to the possibility of an ophthalmological biomarker or diagnostic test for PD. Perhaps the most activity has come from the field of optical coherence tomography (OCT), which has enabled very high resolution structural imaging of the retina. Several groups have used OCT to measure the thickness of retinal layers and generally concur that the retina is thinner in PD subjects as compared to healthy controls. It is unclear, however, whether the differences are sufficient and consistent enough to serve as a diagnostic marker and whether they are specific to PD or are a change held in common with other neurodegenerative conditions. Retinal thinning has also been reported, for example, in Alzheimer’s disease, which often co-exists with PD.

The protein alpha-synuclein accumulates in brain cells to form the characteristic Lewy bodies of PD and is normally present in several types of retinal cells, so there is a possibility that it may accumulate in PD retinas and serve as a biomarker. A very recent study, however, reported no pathological alpha-synuclein in the retinas of PD subjects. We are currently seeking to replicate or refute this finding, using a different staining method and a different way of preparing the retina (retinal wholemounts rather than paraffin-embedded retinal cross-sections), reasoning that these changes might offer greater sensitivity and specificity.

The great promise of retinal biomarkers for PD is that an accurate diagnosis of PD might ultimately be made in the ophthalmologist’s office. Currently, the discovery of new drugs that might act in early-stage PD to slow or stop disease progression is hampered by a low clinical diagnostic accuracy rate for these patients. Published studies indicate that for PD patients who have had disease symptoms for less than five years, when neurologists diagnose PD they are wrong up to 50 percent of the time, and the patients actually have a different cause of their parkinsonism, such as progressive supranuclear palsy. This means that in any clinical trial of such early-stage patients, the drug being trialed may not work in up to 50 percent of patients and the clinical trial may fail as a result.

SUBMANDIBULAR GLAND BIOMARKERS

Charles Adler, MD, PhD

Mayo Clinic College of Medicine

In collaboration with Dr. Thomas Beach, we have found that in 28 autopsied patients with advanced PD, all 28 had evidence of phosphorylated alpha-synuclein binding in neurologic tissue in the submandibular gland when looking at slides from whole mount sections of tissue. As a proof-of-concept Dr. Beach then used a needle to extract tissue from 19 of these submandibular glands and found that in 17 of them he could find staining for the alpha-synuclein, meaning that a needle biopsy might miss a few affected cases. All of this was done postmortem. We then biopsied 15 living patients with PD (disease duration more than five years) and 12 of them on needle biopsy had submandibular gland tissue present, and of those 12, nine had alpha-synuclein staining of neurologic tissue elements.

In our biopsy study of 15 living PD patients we also did minor salivary gland biopsies of all the cases and only 1/15 was positive for phosphorylated alpha-synuclein binding. Thus, we do not think that the minor salivary glands are a good biopsy site for PD. (Of note: Recent results did not support the use of minor salivary glands biopsy for the detection of Lewy pathology in living subjects.)

Neurologists should now be aware that first and foremost PD is not a disease that only involves the central nervous system, rather it also involves the peripheral nervous system. Therefore, it may now be feasible to perform a tissue biopsy to confirm the diagnosis. Even in advanced PD, only 85-90 percent of cases that come to autopsy actually have pathologic findings of PD, so a tissue biopsy could be very important prior to sending patients for invasive or high-risk procedures and also could be important to validate other biomarker, genetic, and epidemiological studies.

I think that we need to have more data on the sensitivity and specificity of the biopsy for the submandibular gland to be a sufficient diagnostic tool. Therefore we need to do longitudinal studies where patients are biopsied and then followed to autopsy to confirm they had PD. Additionally, subjects with other forms of parkinsonism will need to be compared to those with PD and controls.

GASTROINTESTINAL TRACT BIOMARKERS

Kathleen Shannon, MD

Rush Medical College

Alpha-synuclein aggregation is widespread in the submucosa of the entire gastrointestinal tract, and some (but not all) postmortem studies suggest this is an early finding in the evolution of alpha-synuclein pathology. Published studies in living subjects suggest that alpha-synuclein immunostaining can be seen in most (69-100 percent) of Parkinson’s disease (PD) subjects, but is not commonly seen in multiple system atrophy, another disorder of abnormal synuclein aggregation. Some studies also suggest alpha-synuclein is detectable before the affected person becomes aware of PD motor symptoms. We hope our study will demonstrate that alpha-synuclein immunohistochemistry will be useful in distinguishing PD from multiple system atrophy and healthy subjects.

The main advantages of colon biomarker research are: the colon, especially the distal colon, is easily accessible. We use unprepped flexible sigmoidoscopy, which requires neither prep nor anesthesia, thus can be used repeatedly in a prospective study. In addition, current medical practice dictates screening colonoscopy in persons over 50, so were the technique to prove useful in identifying premotor synuclein pathology, it might be possible to screen large populations by simply obtaining tissue during routine colonoscopy.

The studies to date have been small, and not all subjects show pathology. There are several explanations for this, including variation of involvement of tissue, a change in the pathology over time, or unsuitability of this measure as a biomarker. Further development must include: (a) study of larger populations and inclusion of more atypical parkinsonism subjects (multiple system atrophy, vascular parkinsonism, drug-induced parkinsonism, progressive supranuclear palsy, etc.) to assess sensitivity/specificity/positive and negative predictive value as a diagnostic biomarker; (b) prospective study of biomarkers over time to derive information about whether it can be used as a marker of progression in PD.

CUTANEOUS ALPHA-SYNUCLEIN DEPOSITION

Roy Freeman, MD

Harvard Medical School

Previous Parkinson’s research supports our interest in cutaneous alpha-synuclein deposition. First, investigators hypothesize that alpha-synuclein pathology may begin outside of the brain and migrate to the brainstem; this theory is based in part on the appearance of non-motor symptoms, including autonomic symptoms before the cardinal motor complications of Parkinson’s disease. Second, several studies suggest that the peripheral nervous system is involved in Parkinson’s disease. For example, in a report published in 2008 in Brain, evidence of cutaneous denervation in skin biopsies of PD patients and healthy controls was examined. People with PD showed a significant increase in tactile and thermal thresholds, a significant reduction in mechanical pain perception, and significant loss of epidermal nerve fibers and Meissner corpuscles (MC). Disease severity correlated with loss of MCs, and reduction in cold and pain perception. These results suggest peripheral sensory nerve degeneration in PD.

The skin is a remarkable tissue for research studies in that it contains many nervous system components. There are, of course, sensory nerves but there are also several kinds of autonomic nerves: those that innervate the sweat glands, those that innervate the blood vessels and those that innervate the piloerector muscles. Many non-motor symptoms of Parkinson’s disease—such as those affecting blood pressure, bladder and bowel control—involve autonomic territories so those autonomic nerves are of interest. Also, the skin is easily accessible. One can access these nerves by doing a minimally invasive skin biopsy, easily repeated time and time again. This allows us to examine disease pathology in living humans as opposed to in models, a great benefit when studying any disease. So the combination of autonomic involvement in PD, the presence of autonomic nerves in the skin and the accessibility of the skin make this area a great prospect for biomarker study.

A study published in 2008 in the Journal of Neuropathology and Experimental Neurology suggested that the skin may be a useful biopsy site for the pathological diagnosis of Lewy body diseases. Seventy percent of PD cases and 40 percent of those with PD dementia showed positive skin alpha-synuclein immunoreactivity, while skin immunoreactivity was absent in cases of multiple-system atrophy, progressive nuclear palsy and corticobasal degeneration. These and other studies provide support for the examination of cutaneous tissue in Parkinson’s research, and the investigation of the disease’s hallmark pathology, alpha-synuclein within cutaneous autonomic nerves.

In our study of 20 PD patients and 14 controls, we found greater alpha-synuclein deposition within the autonomic nerves innervating the sweat glands and piloerector muscles in skin biopsies of PD patients but not in the sensory nerves. We found a relationship between the amount of alpha-synuclein deposition in autonomic nerves and PD severity measured using the Hoehn and Yahr scale scores. There is also a relationship between alpha-synuclein deposition and physiological measures of autonomic function (e.g. blood pressure and heart rate autonomic control). While these results are promising, we need to look at subjects across a wide array of disease stages, including very early in the course of illness. If we measure the disease longitudinally we could see whether these measures can predict the rate of progression.

We are currently engaged in several follow-up studies. In one of these, we will look at the relationship between what we see in the skin in living subjects with clinically diagnosed PD and similar measures in individuals who have a confirmed autopsy diagnosis of Parkinson’s. This will allow us to determine the relationship between central nervous system and cutaneous alpha-synuclein deposition in confirmed Parkinson’s patients. In another study, we will measure phosphorylated alpha-synuclein deposition. The antibody that we used in our study measured total alpha-synuclein, and we want to see what proportion of that total is phosphorylated. This may help increase the specificity of the skin biopsy test for Parkinson’s and validate our methods and results.

This article is published through an editorial partnership with The Michael J. Fox Foundation for Parkinson’s Research. While the outlined research was funded by the Foundation, the content is authored by the researchers as indicated.

1. Nolano M, Provitera V, Estraneo A, et al. Sensory deficit in Parkinson’s disease: evidence of a cutaneous denervation. Brain. 2008 Jul;131(Pt 7):1903-11.
2. Ikemura M, Saito Y, Sengoku R, et al. Lewy body pathology involves cutaneous nerves. J Neuropathol Exp Neurol. 2008 Oct;67(10):945-53.
3. Wang N, Gibbons CH, Lafo J, Freeman R. a-Synuclein in cutaneous autonomic nerves. Neurology. 2013 Oct 29;81(18):1604-10.