Acta Neuropathologica

, Volume 121, Issue 4, pp 555–557

Optineurin is co-localized with FUS in basophilic inclusions of ALS with FUS mutation and in basophilic inclusion body disease

Authors

    • Department of NeurologyKansai Medical University
    • Department of NeurologyKyoto University Graduate School of Medicine
  • Kengo Fujita
    • Department of NeurologyKansai Medical University
  • Masataka Nakamura
    • Department of NeurologyKansai Medical University
  • Reika Wate
    • Department of NeurologyKansai Medical University
  • Satoshi Kaneko
    • Department of NeurologyKansai Medical University
  • Shoichi Sasaki
    • Department of NeurologyTokyo Women’s Medical University
  • Kiyomi Yamane
    • Department of NeurologyOhta-Atami Hospital
  • Naoki Suzuki
    • Department of NeurologyTohoku University School of Medicine
  • Masashi Aoki
    • Department of NeurologyTohoku University School of Medicine
  • Noriyuki Shibata
    • Department of PathologyTokyo Women’s Medical University
  • Shinji Togashi
    • Department of NeurologyKofu City Hospital
  • Akihiro Kawata
    • Department of Neurology and PathologyTokyo Metropolitan Neurological Hospital
  • Yoko Mochizuki
    • Department of Neurology and PathologyTokyo Metropolitan Neurological Hospital
  • Toshio Mizutani
    • Department of Neurology and PathologyTokyo Metropolitan Neurological Hospital
  • Hirofumi Maruyama
    • Department of Epidemiology, Research Institute for Radiation Biology and MedicineHiroshima University
  • Asao Hirano
    • Division of NeuropathologyMontefiore Medical Center
  • Ryosuke Takahashi
    • Department of NeurologyKyoto University Graduate School of Medicine
  • Hideshi Kawakami
    • Department of Epidemiology, Research Institute for Radiation Biology and MedicineHiroshima University
  • Hirofumi Kusaka
    • Department of NeurologyKansai Medical University
Correspondence

DOI: 10.1007/s00401-011-0809-z

Cite this article as:
Ito, H., Fujita, K., Nakamura, M. et al. Acta Neuropathol (2011) 121: 555. doi:10.1007/s00401-011-0809-z

We recently reported that mutations in the gene encoding optineurin (OPTN) cause amyotrophic lateral sclerosis (ALS) [2]. In that report, we demonstrated the co-localization of OPTN with TAR DNA-binding protein of 43 kDa (TDP-43) or Cu/Zn superoxide dismutase (SOD1) in the pathognomonic inclusions of sporadic (SALS) or familial ALS (FALS) with mutated SOD1, respectively [2].

Fused in sarcoma (FUS) is another causative gene of ALS [1, 7]. FUS-immunoreactivity is identifiable in basophilic inclusions (BIs) from patients with sporadic basophilic inclusion body disease (BIBD) [4] and in those from ‘FALS with FUS mutation’ patients. The fact that both FUS and OPTN cause ALS when mutated prompted us to investigate the correlation between these proteins.

We analyzed postmortem material from three patients with sporadic BIBD and from three with FALS with FUS mutation. All the patients manifested upper and lower motor neuron signs, but no cognitive impairment was noted. Their demographic and clinical features are given in Online Resource 1. The ‘FALS with FUS mutation’ patients had missense mutations R514S, R521C, and P525L in their respective FUS gene. Genetic analysis of the sporadic BIBD patients for FUS and OPTN was unsuccessful, probably because of deterioration of the genomic DNA in the formalin-fixed material. No frozen tissue was available.

Paraffin-embedded lumbar cord, frontal cortex, and brainstem were uniformly investigated immunohistochemically (Online Resource 2). The primary antibodies used are listed in Online Resource 3. To confirm the co-localization of OPTN and FUS, we employed a staining procedure on consecutive sections and double immunofluorescence staining (Online Resource 2).

In the sections from controls, the OPTN and myosin VI immunoreactivities were faintly recognizable in the neuronal cytoplasm; and the anti-FUS antibodies showed essentially no immunoreactivity when they were titrated in a way that did not recognize physiologic FUS (data not shown).

In the BIBD and ‘FALS with FUS mutation’ cases, H&E staining invariably demonstrated neuronal intracytoplasmic BIs in all the regions examined. Immunohistochemically, virtually all the BIs were positive for OPTN, FUS, and myosin VI (Fig. 1a–c, respectively). In contrast, the antibodies against TDP-43 and SOD1 did not react with the BIs (Fig. 1d, e, respectively). Noticeably, staining of the consecutive sections by H&E and immunohistochemistry for FUS and OPTN, as well as the double immunofluorescence staining for FUS and OPTN, evidently demonstrated that the distribution of OPTN immunoreactivity faithfully matched that of FUS within the BIs (Fig. 1f–l). OPTN immunoreactivity in FUS-positive glial inclusions was indiscernible. Further studies are warranted to clarify whether or not OPTN would co-localize with FUS within structures other than the BIs.
https://static-content.springer.com/image/art%3A10.1007%2Fs00401-011-0809-z/MediaObjects/401_2011_809_Fig1_HTML.jpg
Fig. 1

Representative photomicrographs of basophilic inclusions (BIs). ae BIs (arrows) within cortical neurons from BIBD patients are evidently immunopositive for OPTN (a), FUS (b), and myosin VI (c) throughout their entire structure. In contrast, no immunoreactivity indicating pTDP-43 (d) or SOD1 (e) is recognizable within the BIs (arrows). f–h Three consecutive sections from BIBD patient No. 1, stained with H&E (f) or subjected to immunohistochemistry for FUS (g) and OPTN (h), demonstrate that the 2 cortical BIs (arrows) seen are noticeably immunopositive for both FUS and OPTN. i–l Double immunofluorescence staining of neurons in the lateral cuneate nucleus in the medulla oblongata of ‘FALS with FUS mutation’ patient No. 1 evidently demonstrates that FUS (i; green) and OPTN (j; red) are faithfully co-localized (k; merge) in the BI (l; H&E). Scale bars 10 μm

We recently showed that OPTN is co-localized with TDP-43 or SOD1 [2]. However, as shown here, the BIs in the above patients showed no immunoreactivity for TDP-43 or SOD1, but were positive for FUS as well as OPTN. Therefore, our present and earlier results provide evidence that OPTN associates with each of 3 major ALS-related proteins, i.e., TDP-43, SOD1, and FUS.

The pathomechanism of involvement of OPTN in the BIs and that of neurodegeneration in BIBD and ‘FALS with FUS mutation’ patients remain to be elucidated. Since OPTN and FUS share roles in intracellular trafficking in collaboration with myosin VI, it is likely, at least under pathologic conditions, that these proteins would encounter each other when delivering cargos, and could conceivably form a complex through myosin VI within the BIs. Thereby, OPTN and FUS would be sequestered from the cytoplasm.

FUS is known to act as a co-activator of NF-κB [6]. On the contrary, OPTN negatively regulates NF-κB activation [2]. Therefore, it is plausible that sequestration of both OPTN and FUS would induce dysregulation of NF-κB activation, leading to neurodegeneration.

Another promising hypothesis concerns a dysfunctional Golgi apparatus. OPTN and myosin VI play a role in the maintenance of Golgi organization [5]. When OPTN is depleted from cells via RNA interference, the Golgi becomes fragmented [6]. This observation is noteworthy because Golgi fragmentation has been observed in the anterior horn cells in ALS [3]. Further investigations are warranted to determine whether dysfunctional OPTN could be essential for the underlying pathomechanism at play in ALS.

Acknowledgments

This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (No. 21500336) and from the Japan Science and Technology Agency (AS2211173G). We thank Dr. Toshio Oyama (Yamanashi Prefectural Central Hospital) for providing materials from ‘FALS with FUS mutation’ patient No. 1, and Drs. Makoto Arai and Haruhiko Akiyama (Tokyo Institute of Psychiatry) for genetic analysis of ‘FALS with FUS mutation’ patient No. 3.

Conflict of interest

The authors report no conflicts of interest.

Supplementary material

401_2011_809_MOESM1_ESM.pdf (109 kb)
Supplementary material 1 (PDF 108 kb)

Copyright information

© Springer-Verlag 2011