Deposition of mutant ubiquitin in parkinsonism-dementia complex of Guam.

Guam parkinsonism–dementia complex (G-PDC) is an enigmatic neurodegenerative disease that affects the Chamorro residents of the Pacific island of Guam. G-PDC is clinically characterized by progressive cognitive impairment with extrapyramidal signs. Pronounced loss of neurons and abundant neurofibrillary tangles (NFTs) are observed throughout the brain of G-PDC patients [6, 7]. Although several hypotheses have been suggested for the cause of G-PDC, notably genetic predisposition and exposure to neurotoxins (e.g., β-N-methylamino-L-alanine (BMAA)), the etiology and pathogenesis remain elusive [10].

A frameshift mutant of ubiquitin, known as ubiquitin-B+1 (UBB+1), was previously found to accumulate in the neuropathological hallmarks of Alzheimer’s disease and several other disorders, including tauopathies and polyglutamine diseases [1, 3, 12] (Fig. 1a-b). UBB+1 is a dose-dependent inhibitor of the ubiquitin-proteasome system (UPS) and its accumulation in cells an indicator of protein quality control failure. Impaired protein homeostasis is a frequent feature of neurodegenerative diseases and we hypothesized that accumulation of UBB+1 might also be observed in G-PDC. To test whether UBB+1 is detectable in G-PDC brains, immunohistochemical analyses were performed on G-PDC post-mortem brain tissue (Table 1). Immunohistochemistry confirmed the presence of numerous NFTs in G-PDC brains [5] (not shown), as well as other pathology that has been described to occur in G-PDC, i.e., TAR DNA-binding protein 43 (TDP-43)-positive inclusions [5] (Fig. 1f-h). Importantly, our results show that UBB+1 is present in G-PDC brains. UBB+1 deposits were found specifically in cytoplasm of pyramidal neurons and glia (astrocytes in the alveus and stratum oriens) in Ammon’s horn, showing a granular and tangle-like pattern of distribution (Fig. 1c-e). UBB+1 was not detected in young control brains (n = 2, non-Guamanian cases, ages: 52 and 59 years old) [8]. Aggregate structures containing distinct components of the UPS, i.e., the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase L1 (UCH-L1) [9] (Fig. 1i-k) and the proteasomal ATPase subunit Rpt3/S6b [13] (Fig. 1l-n), were also present in these brains.

Fig. 1

Mutant ubiquitin (UBB+1) is deposited in Guam parkinsonism–dementia complex (G-PDC) brains. a UBB+1 is generated through “molecular misreading”, a type of transcriptional mutagenesis. The resulting unfaithful RNA messengers can generate abnormal proteins with cytotoxic properties. b UBB+1 contains an extended C-terminal domain, which can be recognized by anti-UBB+1 antibodies. Deubiquitating enzymes (DUBs) can hydrolyze this extended C-terminus. However, inhibition of these DUBs, e.g., by oxidative stress conditions, prevents this cleavage, preserving the epitope [2]. c-e Immunostaining for UBB+1 (Ubi2A, 1:400, Dr. F.W. van Leeuwen [3]) reveals many cytoplasmic structures in neurons and glial cells (i.e., astrocytes in the alveus and stratum oriens) of the hippocampus. f-h Additionally, cytoplasmic TAR DNA-binding protein 43 (TDP-43) aggregates can be observed in the same cell types (mouse anti-TDP-43, 1:1000, Abnova). i-k Aggregates containing ubiquitin C-terminal hydrolase L1 (UCH-L1) (rabbit anti-UCH-L1, 1:500, Biomol), a DUB, and l-n Rpt3/S6b (rabbit anti-Rpt3, 1:400, Biomol), a proteasomal subunit [13], are also found in G-PDC. Several immunoreactive structures show a granular staining pattern (arrowheads). All immunostainings were carried out on 6 μm thick formalin-fixed, paraffin-embedded sections. Panels c-n all show representative images of G-PDC hippocampi (adjacent sections from subject #2, Table 1). Scale bars 200 μm (c, f, i, l), 100 μm (d, g, j, m), and 50 μm (e, h, k, n)

Table 1

Description of the subjects

Subject	Sex	Age of death (years)	Age of onset (years)	Disease duration (months)	Brain weight (g)	Post-mortem delay	Cause of death	UBB+1
1	F	51	42	116	850	3 h	perforated gastric ulcer	++
2	M	64	58	72	1275	7 h	pulmonary atelectasis	++++
3	M	52	42	126	1025	4 h	bronchopneumonia	++
4	M	56	46	126	1235	< 10 h	bronchopneumonia	+++
5	F	51	46	59	1135	8 h	bronchopneumonia	++
6	M	84	80	50	1100	14 h	bronchopneumonia	++

This demonstration of UBB+1-immunoreactivity and accumulation of particular UPS components in G-PDC brains (n = 6) might have important implications for understanding of the pathological mechanisms underlying the disease. UBB+1 has previously been shown to induce neuronal defects in in vitro and in vivo experimental models: long-term UPS inhibition due to UBB+1 expression causes memory deficits and central breathing dysfunction in mice [4, 8, 11]. In addition, UBB+1 might act as a modifier of other pathology in G-PDC. For example, UBB+1 may enhance the aggregation and cellular toxicity of the RNA-binding protein TDP-43 through interfering with its degradation. It is striking that UBB+1 accumulates in glial cells in G-PDC, because similar glial inclusions have been reported in progressive supranuclear palsy (PSP) [3], a disease that displays some similar topography of neurofibrillary degeneration [10]. Recognition of common mechanistic themes shared by neurodegenerative disorders, such as dysfunctional (ubiquitin-dependent) protein degradation and proteotoxic stress, may help in identifying therapeutic targets that prevent neurodegeneration. It will be interesting to investigate the potential contribution of disrupted proteostasis and UBB+1 to G-PDC in more detail in future studies.