Optineurin is colocalized with ubiquitin in Marinesco bodies

Abstract

Marinesco bodies (MBs) were originally described in 1902 [11]. They are small, spherical intranuclear inclusions that are observed in neurons of the substantia nigra (SN) and locus coeruleus (LC). They have traditionally been considered to be benign. However, there is increasing evidence that they may be an early signal of neuronal dysfunction. They appear in neurons with a high content of melanin, suggesting their association with particular oxidative processes. They are ubiquitinated [1], indicating contents targeted for degradation through the ubiquitin-proteasomal pathway. Their numbers increase with age in both humans [2, 19] and monkeys [10, 17]. This contrasts with losses during aging of SN and LC neurons. More than 40% of SN neurons can be lost without onset of extrapyramidal signs [4, 13]. Why these neurons drop out with aging is unknown but oxidative stress from high levels of catecholamine turnover is a possibility. A relationship between catecholamine turnover and the appearance of MBs has been indicated by the report of Beach et al. [2] of an inverse correlation of dopaminergic markers in the basal ganglia with the number of MBs in SN. They found MB density to be highest in diffuse Lewy body disease cases. This correlates with the finding of Kanaan et al. [10] who found decreased immunoreactivity for tyrosine hydroxylase in MB containing neurons in monkey SN. Here we report that an antibody to the C terminus of optineurin labels MBs. Optineurin (for optic neuropathy inducing protein) came into prominence when mutations in the gene were found to be a cause of adult-onset primary open-angle glaucoma [15]. Later, mutations were found to be a cause of some cases of familial ALS [8, 9, 12]. The protein has been found to interact with huntingtin (htt), transcription factor IIIA [3] and Rab8 [6]. Osawa et al. [14] and Hortobagyi et al. [7] reported a varying extent of optineurin immunoreactivity in cytoplasmic inclusions of Alzheimer disease, Parkinson disease, Creutzfeldt-Jakob disease, multiple system atrophy, and Pick disease. Since mutated optineurin can be a causative factor in some degenerative neurological diseases, and the normal protein can be a significant participant in the pathology of others, the appearance of optineurin in MBs may be a strong signal that they are pathological entities. Several other proteins have been found in association with MBs, among them ataxin-3, which in its mutated form causes spinocerebellar ataxia type 3 [5]. In addition to optineurin, we investigated MBs for the presence of several proteins reported to be associated with other types of intranuclear inclusions, including phosphoTDP-43, phospho-alpha synuclein, huntingtin, fused in sarcoma (FUS), beta tubulin and neurofilament protein. It has been suggested that imbalance between cytoplasmic and nuclear localization of some of these proteins may be part of the pathogenic process by interfering with normal function [18]. We examined the SN of five elderly control cases, five cases with Parkinson’s disease and five cases with Alzheimer’s disease. These cases had previously been found to contain numerous MBs in their SN neurons. Brain tissues were lightly fixed in 4% paraformaldehyde in phosphate buffer. Immunohistochemistry was performed as described previously [16] with the following antibodies: optineurin C-terminal (Cayman #100000, 1:500), ubiquitin (Dako Z0458, 1:1,000 and Zymed, 13-1600, 1:500), phosphorylated C. Schwab (&) S. Yu P. L. McGeer Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada e-mail: claudia.schwab@ubc.ca