Optineurin pathology in the spinal cord of amyotrophic lateral sclerosis/parkinsonism‐dementia complex patients in Kii Peninsula, Japan

Dear Sir,

Amyotrophic lateral sclerosis/parkinsonism‐dementia complex (ALS/PDC) is an endemic neurodegenerative disease reported on Guam and the Kii Peninsula of Japan (Kii ALS/PDC) 1, 4. The clinical features of Kii ALS/PDC include unique combinations of parkinsonism, dementia, muscular weakness, amyotrophy, hyperreflexia and spasticity as symptoms of damage to the upper and lower motor neurons. Neuropathological findings in ALS/PDC show numerous neurofibrillary tangles (NFTs) associated with nerve cell loss in the cerebral cortex and brainstem 6. These are in addition to the typical pathological findings of ALS, which includes transactive response DNA‐binding protein (TDP, TARDBP)‐43 pathology 3.

The optineurin gene (OPTN) has been identified as a causative gene for normal‐tension glaucoma and primary open‐angle glaucoma. Optineurin (OPTN) is an adaptor protein that interacts with various proteins. It is involved in regulating many cellular functions that include vesicular trafficking from the Golgi to plasma membrane, endocytic trafficking and signaling leading to NF‐κB activation 10. The exon 5 deletion, p.Q398* nonsense and p.E478G missense mutations of OPTN have been reported in patients with familial ALS 5. Various intracytoplasmic inclusions, such as skein‐like, eosinophilic round hyaline, and large spherical with central hyaline core, have been immunohistochemically identified using anti‐OPTN antibodies in spinal cord anterior horn cells (AHCs) of ALS with OPTN mutations. These are also found in sporadic ALS and familial ALS (FALS) caused by superoxide dismutase 1 (SOD1) mutations. Remarkably, OPTN‐positive intracytoplasmic structures co‐localize with anti‐ubiquitin and anti‐TDP‐43 reactivity, suggesting that OPTN might be associated with common pathomechanisms in sporadic ALS (SALS), familial FALS with SOD1 mutations, and FALS with OPTN mutations 5. Here, we measured the number and size of AHCs by choline acetyltransferase (ChAT) staining in the spinal cords of seven patients with Kii ALS/PDC and eight age‐matched controls (five males, mean ± SD age = 73 ± 9.0 years; three females, mean age = 67 ± 2.7 years, recruited from the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology) and examined OPTN, and phosphorylated TDP‐43 (pTDP‐43) immunoreactivity in the spinal cords of 10 patients with Kii ALS/PDC (mean ± SD, age 69 ± 5.1 years). We performed mutational analysis of OPTN in six patients with Kii ALS/PDC to reveal whether OPTN is associated with the pathomechanisms of Kii ALS/PDC.

Informed consent was obtained from the families of all patients who participated in this study. This study was approved by the Ethics Committee of Mie University Hospital, Mie, Japan and Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan. The clinical and neuropathological profiles of the patients are shown in Table 1. Of the 10 patients, six patients had ALS features and four patients did not. All patients had numerous NFTs, with no or minimal senile plaques, and nerve cell loss chiefly in the temporal cortex, frontal cortex, and nuclei of the brainstem. Loss of AHCs and degeneration of the pyramidal tract of the spinal cord were common features 4.

Table 1.

Clinical profiles and optineurin (OPTN)/TAR DNA‐binding protein 43 kDa (TDP‐43) pathology in the anterior horn cells of patients with amyotrophic lateral sclerosis/parkinsonism‐dementia complex of the Kii Peninsula (Kii ALS/PDC). Abbreviations: DOI = duration of illness; A = amyotrophy; P = parkinsonism; D = dementia; N/E = not examined; AHCs = anterior horn cells.

Patients	Age	Gender	DOI (years)	Phenotype			AHCs		OPTN mutation
				A	P	D	OPTN	pTDP‐43
1	63	F	5	+	–	–	–	– *	No mutation
2	66	F	3	+	–	–	+	+	No mutation
3	70	F	13	+	–	–	–	+	N/E
4	70	F	13	+	–	–	–	+	N/E
5	77	M	7	+	–	+	–	+	No mutation
6	70	F	14	–	+	+	+	+	No mutation
7	74	M	7	–	+	+	–	+	N/E
8	70	F	12	–	+	+	–	–*	No mutation
9	70	F	8	–	+	+	+	+	No mutation
10	60	F	9	+	+	+	–	–*	No mutation

*Neuropil threads.

We chose to use the spinal cords for immunohistological evaluation based on a previous report in which OPTN‐positive structures were found in spinal AHCs 5. Formalin‐fixed, paraffin‐embedded specimens of the spinal cords were cut into 9‐µm‐thick sections for hematoxylin and eosin, Klüver‐Barrera and Gallyas‐Braak staining. Sections (6‐µm thick) were used for immunohistochemical studies using the avidin–biotin peroxidase complex (ABC) method with a Vectastain ABC kit (Vector, Burlingame, CA, USA). We used an automated immunostainer (Ventana Medical Systems Inc., Tucson, AZ, USA) for the immunostaining. The antibodies used were anti‐OPTN‐C antibody (polyclonal, 1:100; Cayman Chemical, MI, USA), anti‐OPTN‐I antibody (polyclonal, 1:100; Cayman Chemical, MI, USA), anti‐pTDP‐43 antibody (PSer409/410; monoclonal, a gift from Dr M. Hasegawa, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), antiphosphorylated tau antibody (AT8; monoclonal, 1:100; Innogenetics, Ghent, Belgium) and anti‐ChAT antibody (monoclonal, 1:200; Atlas Antibodies, Bromma, Sweden). For anti‐ChAT antibody immunostaining, sections in target retrieval solution, citrate pH 6 (×10) (Dako A/S, Glostrup, Denmark), were autoclaved (21°C, 10 min) for antigen retrieval and Can Get Signal^® immunostain, solution A (TOYOBO, Osaka, Japan) was used to dilute the primary antibody.

Double labeling immunohistochemistry and immunofluorescence were performed on selected sections of the spinal cord. Deparaffinized sections were incubated simultaneously with polyclonal anti‐OPTN (OPTN‐C) and monoclonal anti‐pTDP‐43 (PSer409/410) antibodies. Primary antibodies were visualized with Vectastain ABC kit (Vector, Burlingame, CA, USA) and DISCOVERY RedMap Kit (RUO) (Roche, Basel, Switzerland) for immunohistochemistry, and anti‐rabbit Alexa 546 Fluor and anti‐mouse immunoglobulin G Alexa 488 (Molecular Probes, Eugene, OR, USA) were visualized under a confocal laser microscope (LSM5, PASCAL; Carl Zeiss, Jena, Germany) for the immunofluorescence study.

Mutational analyses of OPTN and TARDBP were performed using whole genome sequence analysis (six patients) and whole exome analysis (one patient). Exome analysis was performed using SureSelect V5+UTRs (Agilent Technologies, Santa Clara, CA, USA). Sequence analysis was performed using a HiSeq2000 sequencer (Illumina, San Diego, CA, USA). Short reads were aligned to the reference genome using Burrows–Wheeler Alignment (BWA) tool with default settings. Base calls were made using SAM tools. Statistical analyses were performed using the JMP 11 software (SAS Institute Inc., Cary, NC, USA) to perform Dunnett's test for parametric analysis.

Of the 10 Kii ALS/PDC patients investigated, three (30%) patients had a few OPTN‐positive neuronal cytoplasmic inclusions (NCIs), glial cytoplasmic inclusions (GCIs) and/or dystrophic neurites in the anterior horn (Figure 1A–I). Few positive structures were seen in the cytoplasm of large AHCs. Skein‐like inclusions were mainly observed as NCIs (also in axons), while round hyaline inclusions and large spherical inclusions with a central hyaline core were rare. Seven (70%) Kii ALS/PDC patients had pTDP‐43 pathology in AHCs, and few pTDP‐43 inclusions were also positive for OPTN (Figure 2) in this study. Double immunofluorescent labeling with OPTN and pTDP‐43 antibodies revealed occasional co‐localization (Figure 2G,H), and the majority of pTDP‐43 inclusions did not co‐localize with OPTN.

Figure 1.

Various shapes of OPTN‐positive structures in (A) an astrocyte, (B) a small neuron, (C) intra‐nucleus and (D–I) AHCs of spinal cords from Kii ALS/PDC patients. Allows show the inclusions.

Figure 2.

Structures immunoreactive with anti‐OPTN‐C antibody (A‐1: cluster of skein‐like inclusions, A‐2: skein‐like inclusions and A‐3: granular inclusions) and anti‐phosphorylated TDP‐43 (pTDP‐43) antibody (B‐1: cluster of skein‐like inclusions, B‐2: skein‐like inclusions and B‐3: granular inclusions). Co‐localization of OPTN and pTDP‐43 (C‐1, 2, 3, D, E and f). Immunoreactive structures with OPTN were visualized with 3,3′‐diaminobenzidine (DAB) (brown), and immunoreactive structures with pTDP‐43 were visualized with alkaline phosphatase red (red). In the immunofluorescence study (G‐1, 2 and 3: round inclusion and H‐1, 2 and 3: skein like inclusions), immunoreactive structures with OPTN were visualized with Alexa 546 Fluor (red) and immunoreactive structures with pTDP‐43 were visualized with Alexa 488 Fluor (green). The OPTN‐ and pTDP‐43‐positive structures co‐localized. Arrows show the inclusions. Scale bar = 20 µm.

Furthermore, we measured quantitatively the number and area of ChAT‐immunoreactive AHCs in the cervical and lumbar enlargement of the spinal cord of Kii ALS/PDC patients and age‐matched controls by using immunostaining for anti‐ChAT antibody and Image Scope (Leica Biosystems, Nussloch, Germany). We used five cases (cases 3, 4, 5, 7 and 9) and eight age‐matched controls for cervical enlargement and four cases (cases 2, 4, 6 and 7) and the same eight controls for lumbar enlargement based on cases where the enlargement was identified precisely and positively stained by anti‐ChAT antibody. Regarding the other cases, we estimated neuronal loss in the anterior horn neurons qualitatively by KB staining.

As a result, all cases had mild‐to‐severe neuronal loss in the anterior horn neurons including the cases with few pTDP‐43 in the anterior horn of Kii ALS/PDC patients. Neuronal loss in the AHCs with an ALS phenotype was more severe than in controls (in measurable cases: controls vs. cases with ALS phenotype; P = 0.0004 in the cervical enlargement, P = 0.0091 in the lumbar enlargement; Figure 3A). The number of cholinergic anterior horn neurons in patients without an ALS phenotype tended to be less than that in controls, and the number of cholinergic anterior horn neurons in patients with an ALS phenotype tended to be less than that in patients without an ALS phenotype. In addition, the percentage of number of AHCs in small size (less than 800 μm²) was increased in patients with an ALS phenotype. Furthermore, AHCs in patients without an ALS phenotype tended to enlarge (Figure 3B). Furthermore, OPTN‐positive findings did not relate to the degree of anterior horn cell loss.

Figure 3.

The number and size of cholinergic neurons in the spinal cord anterior horn of controls and Kii ALS/PDC patients. A. The number of cholinergic anterior horn neurons in cervical enlargement and lumbar enlargement of patients with an ALS phenotype were significantly lower than that in controls. The number of cholinergic anterior horn neurons in cervical enlargement and lumbar enlargement of patients with a PDC phenotype tended to be lower than in controls. Figures in the graph show the case number. Error bars show standard error. **P < 0.001. B. The percentage of number of AHCs in small size (less than 800 μm²) was increased in the patients with an ALS phenotype. Y‐axis shows the percentage of total number of AHCs per 200 μm².

The clinical phenotypes of OPTN‐positive patients were one with ALS (10%), one with PDC (10%), and one with PDC and ALS (10%). There were no differences in the distribution and the number of positive structures among these clinical phenotypes. Mutational analysis of all exon regions of OPTN and TARDBP in six patients did not reveal any pathogenic mutations (Table 1) 9.

Motor neuron cell death in the spinal cord and motor cortex is a key neuropathological feature of Kii ALS/PDC, although the pathomechanism of this loss is unclear. Endogenous OPTN has a diffuse cytoplasmic distribution and co‐localizes partially with the Golgi complex, although it is not a membrane protein. OPTN is prone to oligomerization and aggregation when mutated or overexpressed in cells. It also forms complexes with the transcription factors Rab8, huntingtin and myosin IV, which, along with OPTN are involved in the membrane trafficking of proteins. Overexpression and primary open angle glaucoma‐related mutation (E50K) of OPTN impair protein trafficking, which might be a pathomechanism that contributes to ALS with OPTN mutation. However, OPTN levels are influenced by several factors including proinflammatory cytokine levels, which are upregulated in ALS 7. Homozygous OPTN deletion and truncation mutations are expected to decrease OPTN protein levels and motor neuron degeneration.

OPTN immunopositive inclusions have occasionally been observed in other neurodegenerative diseases in addition to ALS. For example, Hortobágyi et al reported that Alzheimer's disease (AD) patients had OPTN‐positive neuronal and glial cytoplasmic inclusions in the cerebrum 2. There was no co‐localization of OPTN and phosphorylated tau, or of OPTN and pTDP‐43 in these AD patients. In Huntington's disease, increased granular, predominantly perinuclear, cytoplasmic labeling with no evidence of OPTN labeling of intranuclear inclusions was observed. No abnormal staining or inclusions were detected in the brains of patients with frontotemporal lobar degeneration (FTLD), fused in sarcoma (FUS), FTLD‐tau (Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and FTLD with microtubule‐associated protein tau mutation), dementia with Lewy bodies or spinocerebellar degeneration 2.

In this study, OPTN‐positive cytoplasmic inclusions were detected in the spinal cord of 30% of patients with Kii ALS/PDC, which is similar to that in patients with SALS (34%) 2. However, few motor neurons with OPTN‐positive inclusions were detected and the accumulation of OPTN in large motor neurons was rare. Additionally, the majority of pTDP‐43 inclusions were OPTN negative.

These results suggest that OPTN may be a secondary accumulation rather than a causative factor for the pathogenesis of Kii ALS/PDC, with TDP‐43 playing a more important role. However, the OPTN‐positive findings implied the involvement of neuronal inflammation including the NF‐κB pathway in the pathomechanism of Kii ALS/PDC. We will pursue the role of neuronal inflammation in Kii ALS/PDC in the future.

To the best of our knowledge, this is the first report of OPTN‐positive inclusions in patients with Kii ALS/PDC. OPTN‐positive inclusions were observed in patients with Kii ALS, Kii PDC and Kii PDC with ALS, supporting the idea that Kii ALS and Kii PDC are part of the same disease spectrum 6, 8.