Alpha-synuclein immunoreactivity in minor salivary glands: A potential pathological biomarker for Parkinson?s disease?

MARIA GRACIELA CERSÓSIMO; PERANDONES CLAUDIA; FEDERICO EDUARDO MICHELI; GABRIELA BEATRIZ RAINA; RADRIZZANI MARTIN; ANA MARIA BERON; GUSTAVO NASSWETTER; EDUARDO ELIAS BENARROCH

Buenos Aires

Congreso; 14th International Congress of Parkinson?s Disease and Movement Disorders; 2010

Movement Disorders

Hyposialorrhea occurs at early stages of Parkinson?s disease (PD)and probably reflects early involvement of the autonomic fibers innervating the salivary glands by a-synuclein(a-SYN) pathology. This is supported by a recent study on autopsy material showing accumulation of a-SYN immunoreactive inclusions in the submandibular glands in both incidental Lewy body disease and in PD cases. Labial minor salivary gland biopsy, which is a standard procedure for ̈diagnosis of Sjogren disease, allows the identification ofautonomic fibers innervating both the acini and blood vessels. Thus, it could provide a relatively simple approach to detect a-SYN aggregates in the salivary glands. In this pilot study, we sought to determine whether a-SYN pathology can be detected in labial minor salivary gland biopsy of PD patients.We studied three PD cases (2 men, age: 60.3 6 11.2years old) and three age matched controls (3 women, age:60.3 6 10.5 years old). The study was approved by the local ethics committee of the Hospital de Clinicas, University of Buenos Aires. Written informed consent was obtained from all subjects. PD diagnosis was made according with the United Kingdom Parkinson?s Disease Brain Bank criteria.4 PD patients were evaluated with the Hoehn and Yahr stage and the Unified Parkinson?s Disease Rating Scale motor scale.5 We also assessed the presence of symptoms of dry mouth and constipation (defined as three or less bowel movements per week). Exclusion criteria included history of autonomic or other disorders affecting the salivary glands. Controls were patients evaluated for ̈possible Sjogren syndrome. In these cases, salivary glands were histologically normal.Minor salivary gland biopsy was performed under local anesthesia according to standard procedures.3 Samples were fixed in formalin and embedded in paraffin; 5 um thick sections were obtained and mounted on silanized glass (Silane, Sigma, St. Louis, MO). The slices were then deparaffinized and processed for antigen retrieval by incubation in buffer citrate for 20 min at 948C. The sections were then incubated overnight at 48C for immunohistochemical staining for either a-SYN (1:100, rabbit polyclonal antibody, catalogue number 18-272-196445, GeneWay Biotech., SanDiego, CA) or human neurofilament (NF) protein (1:300,mouse monoclonal antibody; clone 2F11, code: M 0762,Dako, Denmark). After incubation, the sections were washed in PBS to remove antibody excess and then developed with alkaline phosphatase system. Specificity of theimmunostaining was determined by preincubating during 3hours the a-SYN antiserum with a blocking recombinant protein (1 lg/lL) (GeneWay Biotech, number10-663-45667, Swiss Prot acc P37840) or by omission of the primary antibody. Immunofluorescence and colocalization assays were performed using secondary antibodies coupled to either FITC (for a-SYN) or rhodamine (for NF protein)(Sigma). Images were acquired using IX-71 microscope and digitized with a camera DP72 (Olimpus, Japan). Merged images were obtained using the Image-J-Pro 4.0 and Adobe PhotoShop programs. Abundant a-SYN immunoreactive profiles were detected in the labial salivary gland in two of the three PD cases but in none of the controls (Fig. 1A,B). The a-SYN profiles were concentrated in the periacinar space, which contains the autonomic nerves supplying the gland. Preincubation with the specific blocking protein eliminated a-SYN immunoreactivity (Fig. 1C). Double labeling studies indicated that the a-SYN inclusions colocalized with NF protein (Fig. 1F). The clinical characteristics of the PD patients with and without a-SYN inclusions are shown in Table 1. Our results are consistent with recent findings showing the presence of a-SYN inclusions in the submandibular glands of both PD cases and cases with incidental Lewy body disease, which is considered a preclinical stage of PD.2 Thus,minor salivary gland biopsy could be useful to detect a-SYN pathology in subjects with early premotor symptoms of PD or in individuals at risk for development of the disease, such as carriers of LRRK2 or GBA mutations. The results of our pilot study have to be confirmed in larger populations of PD and control cases. This will help to determine whether minor salivary gland biopsy could be used as a simple and safe tool for early detection of a-SYN pathology in PD patients during life.