Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused
by motor neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic
(SALS), but about 5–10% are familial (FALS). Mutations in superoxide dismutase 1 (SOD1)
1,2
, TAR DNA-binding protein (TDP43)
3,4
and fused in sarcoma/translated in liposarcoma (FUS/TLS)
5,6
account for approximately 30% of classic FALS. Mutations in several other genes have
also been reported as rare causes of ALS or ALS-like syndromes
7–15
. The causes for the rest of familial ALS and the vast majority of sporadic ALS are
unknown. Despite extensive studies of previously identified ALS-causing genes, the
pathogenic mechanism underlying motor neuron degeneration in ALS remains largely obscure.
Dementia, usually of the frontotemporal lobar type (FTD), may occur in some ALS cases.
It is unclear if ALS and dementia share common etiology and pathogenesis in ALS/dementia.
Here, we show that mutations in UBQLN2, which encodes a ubiquitin-like protein, ubiquilin2,
cause dominantly inherited chromosome X-linked ALS and ALS/dementia. We describe novel
ubiquilin2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia
cases with or without UBQLN2 mutations. Ubiquilin2 is a member of the ubiquilin family
(ubiquilins), which regulate degradation of ubiquitinated proteins. Functional analysis
showed that mutations in UBQLN2 lead to an impairment of protein degradation. Our
findings, therefore, link abnormalities in ubiquilin2 to defects in the protein degradation
pathway, abnormal protein aggregation and neurodegeneration, implying a common pathogenic
mechanism that can be exploited for therapeutic intervention.
We identified a five-generation family (Family #186) with ALS, including 19 affected
individuals (Supplementary information). The disease is transmitted in a dominant
fashion with reduced penetrance in females. Mutations in the known ALS-linked genes
were excluded. No evidence of genetic linkage was found with genome-wide set of autosomal
microsatellite markers. There was no evidence of male-to-male transmission of the
disease, so we screened the family with markers from the X chromosome. Linkage was
established with several X chromosome microsatellite markers, with the highest two-point
LOD score of 5.0 with marker DXS9736 at θ=0 (Supplementary Table 1). Detailed mapping
with dense microsatellite markers and Illumina’s Sentrix HumanHap300 Genotyping BeadChip
defined the disease-causing gene in a 21.3Mb minimum candidate region (MCR) between
markers rs6417786 and DXS1275, which is located in the pericentric region from Xp11.23
to Xq13.1.
No additional large ALS families without male to male transmission were available
to us to narrow down the MCR. We therefore focused on finding the causative gene in
Family #186. Of the 206 genes in this MCR, 191 genes were protein coding. Genes in
this MCR were analyzed based on their expression profile, function, structure and
potential relevance of their encoded proteins to disease. Forty-one genes were sequenced
and a unique mutation in UBQLN2 was identified. This mutation, c.1490C>A, is predicted
to result in an amino acid substitution of proline by histidine at codon 497 (P497H)
(Fig. 1a). The c.1490C>A mutation co-segregated with the disease in this large X-ALS
pedigree (Fig. 1a). This mutation was not present in the SNP database nor was it present
in 928 ethnically matched control samples (representing 1332 X chromosomes).
UBQLN2 is an intronless gene. To test if mutations of UBQLN2 are causative for other
ALS patients, we analyzed 188 probands from families with ALS or ALS/dementia, but
without male-to-male transmission. Mutations in SOD1, TDP43 and FUS were excluded
in this cohort. The sequenced region covered the entire coding sequence (Materials
and Methods). We found four other UBQLN2 mutations in four unrelated families, including
c.1489C>T (p.P497S), c.1516C>A (p.P506T), c.1525C>T (p.P509S) and c.1573C>T (p.P525S)
(Fig. 1 and Supplementary Fig.1). All the amino acids residues at the mutated sites
are conserved (Fig. 1c). None of these mutations were present in the SNP database
and 928 control samples. Remarkably, all the five ALS-linked UBQLN2 mutations identified
in this study involved proline residues within a unique PXX repeat region (Fig. 1c
and 1d).
Clinical data were obtained from 40 individuals in the five families with UBQLN2 mutations,
including 35 patients and five obligate carriers. We estimated a penetrance of approximately
90% by the age of 70 years. The age of onset of the disease ranged from 16 to 71 years.
A significant difference in age at onset was noted between male and female patients,
with male patients having earlier age of onset (33.9 ± 14.0 vs. 47.3 ± 10.8 years,
p=0.003, two-tailed Student t-test) (Supplementary Table 2). However, differences
in the duration of the disease were not statistically significant (43.1 ± 42.1 vs.
48.5 ± 19.9 months, p=0.61). Eight patients with both ALS and dementia were identified.
Dementia in these patients was similar to FTD type, including abnormalities in both
behavior and executive function. The dementia was progressive, and eventually global
in most ALS/dementia patients. In some cases the dementia preceded motor symptoms,
but all patients eventually developed motor disability. Pathological analysis of spinal
cord autopsy samples from two patients with either P497H or P506T mutation revealed
axonal loss in the corticospinal tract, loss of anterior horn cells, and astrocytosis
in the anterior horn of the spinal cord (Supplementary Fig. 2).
Protein aggregates/inclusions have been recognized as a pathological hallmark in several
neurodegenerative disorders, such as extracellular amyloid-beta plaques and intracellular
tau neurofibrillary tangles in Alzheimer disease (AD), and α-synuclein-containing
Lewy bodies in Parkinson disease (PD)
16
. In ALS protein aggregates/inclusions are most common in spinal motor neurons, and
are typically skein-like in morphology. These ubiquitin-positive inclusions among
others are considered to be a hallmark of ALS pathology. Notably, several proteins
that are mutated in a small subset of ALS, such as SOD1, TDP43, FUS and optineurin
(OPTN) are prominent components of these inclusions
6,12,17–20
. To test if ubiquilin2 is present in the characteristic skein-like inclusions, we
performed immunohistochemical analysis of the postmortem spinal cord sections from
two patients with a P497H or P506T mutation. Two different ubiquilin2 antibodies were
used. One was a commercially available mouse monoclonal antibody raised with a polypeptide
of 71 amino acids (aa) at the C-terminus (554aa-624aa, ubiquilin2-C). The other was
a rabbit polyclonal antibody that we generated using a polypeptide of 17aa at the
N-terminus (8aa-24aa, ubiquilin2-N). The polypeptide of 17aa is unique to ubiquilin2
and not present in other members of the ubiquilin family or any known protein. The
ubiquilin2-N antibody recognized human and mouse ubiquilin2 (Supplementary Fig. 3).
We also detected a single band of the expected size by Western blots using ubiquilin2-N
and ubiquilin2-C antibodies with human spinal cord autopsy tissues (Supplementary
Fig. 3). Using immunohistochemistry, we observed skein-like inclusions that were immunoreactive
with both ubiquilin2-C and ubiquilin2-N antibodies (Supplementary Fig. 4), suggesting
that ubiquilin2 is involved in inclusion formation in X-ALS. We then examined if the
inclusions in X-ALS cases were also immunoreactive with antibodies against other proteins
that are known to be involved in the formation of the inclusions in other types of
ALS. We found that the skein-like inclusions in the X-ALS patients were also immunoreactive
with antibodies to ubiquitin, p62, TDP43, FUS and OPTN (Fig. 2a-c and Supplementary
Figures 4 and 5), but not SOD1.
Mutations in TDP43, FUS or optineurin occur in a small fraction of FALS, but these
proteins have been found in the inclusions of a wide spectrum of ALS
6,12,17,18,20
. To test if ubiquilin2 is involved in inclusion formation of other types of ALS,
we examined 47 post-mortem spinal cord samples, including SALS (n=23), FALS without
mutations in SOD1, TDP43 and FUS (n=5), ALS with dementia (n=5), FALS with SOD1 mutations
[n=7, (A4V, n=4; G85R, n=2; E100G, n=1)], FALS with a G298S mutation in TDP43 (n=1),
and controls without ALS (n=6). We observed ubiquilin2-positive and skein-like inclusions
in all ALS cases (Supplementary Figures 6 and 7), suggesting that ubiquilin2 is a
common component in the skein-like inclusions of a wide variety of ALS as well.
Dementia was a prominent feature in eight UBQLN2-linked cases. To examine if ubiquilin2-immunoreactive
inclusions are present in brain, and to explore the potential link between ubiquilin2
inclusions and dementia, we analyzed brain autopsy samples from two patients with
the P506T mutation. We observed ubiquilin2 pathology, which was most prominent in
the hippocampus (Fig. 2d–g and Supplementary Fig. 8). Small ubiqulin2 inclusions were
predominantly situated in the neuropil (1–5 μm in diameter). The fascia dentata presented
with a band of radially oriented dendritic and neuropil inclusions in the intermediate
region of the molecular layer (Supplementary Fig. 8). In addition to the small neuropil
inclusions, large inclusions (up to 20 μm in diameter) were observed in some pyramidal
neurons, especially those in the CA3 and CA1 (Fig. 2f and 2g, and Supplementary Fig.
8). Co-localization of ubiquilin2 and ubiquitin in these inclusions were confirmed
with confocal microscopy (Supplementary Fig. 8). This type of hippocampal pathology
has not been previously observed in any other neurodegenerative disorder. The ubiquilin2/ubiquitin
positive inclusions did not appear to be co-localized with major glial markers (Supplementary
Fig. 9). In addition, we also observed a novel membrane-bound perikaryal structure,
which contained eosinophilic granules of varying sizes in some hippocampal pyramidal
neurons. These structures were strongly immunoreactive for ubiquilin2 (Supplementary
Fig. 10).
To test if ubiquilin2 pathology is present in the hippocampus of ALS/dementia cases
without UBQLN2 mutations, and to explore the correlation of ubiquilin2 pathology with
dementia in ALS, we further examined hippocampal sections of 15 pathologically characterized
ALS cases without UBQLN2 mutation, including five cases of ALS/dementia with pathological
signatures corresponding to frontotemporal lobar degeneration of motor neuron disease
type (FTLD-MND/FTLD-U). We found prominent ubiquilin2 pathology in the hippocampus
of all five cases with ALS/dementia (Supplementary Fig. 11). Similar to the ubiquilin2
inclusions in UBQLN2-linked ALS/dementia cases, the ubiquilin2 inclusions in these
non-UBQLN2-linked ALS/dementia cases were also positive with ubiquitin and p62 (Supplementary
Fig. 11), but negative with FUS. Although there was no apparent TDP43 neuritic pathology
in the dentate molecular layer, we observed variable numbers of cytoplasmic TDP43
inclusions in dentate granule cells, which have been previously shown in ALS/dementia
18
(Supplementary Fig. 11). However, we noted that a significant number of the ubiquilin2/ubiquitin/p62
inclusions were negative for TDP43 (Supplementary Figures 11 and 12). The presence
of TDP43-negative inclusions was further confirmed with an antibody specific to phosphorylated
TDP43 that is only present in the cytoplasmic inclusions
18
(Supplementary Fig. 13). We also observed that the ubiquilin2/ubiquitin/p62 inclusions
were largely negative for TDP43 in the CA regions in the non-UBQLN2-linked ALS/dementia
cases (Supplementary Fig. 12). We did not observe the ubiquilin2 pathology in the
hippocampus of the 10 ALS cases without dementia. The correlation of the hippocampal
ubiquilin2 pathology and dementia in ALS cases with or without UBQLN2 mutations suggest
that ubiquilin2 is widely involved in ALS-related dementia, even without UBQLN2 mutations.
TDP43 inclusions have been observed in dentate granule cells of the hippocampus in
most of the cases with FTLD
18
. FUS inclusions have been shown in most of the TDP43-negative FTLD cases
21,22
. To test if ubiquilin2 co-aggregates with these two known ALS- and dementia-linked
proteins in vitro, we generated 10 expression constructs (Supplementary information)
and co-transfected Neuro2a cells with different combinations. Both wild-type and mutant
ubiquilin2 were largely distributed in the cytosol. We did not observe obvious differences
in the distribution between the wild type and mutant ubiquilin2. The wtFUS and wtTDP43
were located almost exclusively in the nuclei (Fig. 3 and Supplementary Fig. 14);
whereas, mutant FUS showed prominent cytoplasmic distribution (Supplementary Fig.
13) and the C-terminal fragment (218–414, C-TDP43) of TDP43 that has been linked to
ALS and FTLD
18,23
was almost exclusively located in the cytosol (Fig. 3). We did not observe cytoplasmic
inclusions in cells transfected with wtFUS and mutant FUS (Supplementary Fig. 14)
or wtTDP43 (Fig. 3). However, cytoplasmic inclusions were observed in cells expressing
either wild-type or mutant ubiquilin2. Notably, the C-TDP43 was co-localized with
either wild-type or mutant ubiquilin2 in the cytoplasmic inclusions (Fig. 3). We obtained
consistent data using two expression systems, either tagged ubiquilin2 or tag-free
ubiquilin2 (Fig. 3 and Supplementary Figures 14 and 15). These data suggest that both
ALS- and dementia-linked ubiquilin2 and TDP43 are prone to co-aggregation. We also
noted that inclusion formation was apparently dose-dependent, because the cells with
low expression of wild-type and mutant ubiquilin2, or C-TDP43 did not show cytoplasmic
inclusions. Moreover, ubiquilin2-positive, but C-TDP43-negative inclusions were frequently
observed in cells with relatively lower levels of expression (Fig. 3). This phenomenon
suggests that ubiquilin2 may be more prone to aggregation than TDP43. This notion
is consistent with the pathology observed in ALS/dementia cases, where the ubiquilin2-containing
inclusions in the molecular layer and in some dentate granule cells were TDP43-negative.
Ubiquilin2 is a member of the ubiquitin-like protein family (ubiquilins). Humans have
four ubiquilin genes, each encoding a separate protein. Ubiquilins are characterized
by the presence of a N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated
(UBA) domain (Fig. 1d). The middle part of ubiquilins is highly variable. This structural
organization is characteristic of proteins that function to deliver ubiquitinated
proteins to the proteasome for degradation. In accordance with this function, the
UBL domain of the ubiquilins binds subunits of the proteasome, and its UBA domain
binds to polyubiquitin chains that are typically conjugated onto proteins marked for
degradation by the proteasome
24
. In addition to the UBL and UBA domains that are shared by all ubiquilins, ubiquilin2
has a unique repeat region containing 12 PXX tandem repeats (Fig.1d). Remarkably,
all the five ALS-linked mutations identified in this study involve proline residues
within this short PXX repeat region (Fig. 1c and 1d), suggesting that these mutations
may confer on ubiquilin2 a common property that may be related to the pathogenic mechanism
of the disease.
Based on the involvement of ubiquilin2 in the protein degradation pathway, we then
investigated the functional consequences of mutant ubiquilin2 in protein degradation
through the UPS. We used the UPS reporter substrate UbiquitinG76V-Green Fluorescent
Protein (UbG76V-GFP)
25
to test the effects of mutant ubiquilin2 on ubiquitin-mediated protein degradation.
Two mutations at two different sites were tested (P497H and P506T) using the UbG76V-GFP
reporter system. The G76V substitution prevents removal of the N-terminal-fused ubiquitin
by cellular de-ubiquitinating enzymes, leading to efficient proteasomal degradation
of the UbG76V-GFP reporter
25
. We first tested the transfection efficiency of wild-type and mutant ubiquilin2 constructs,
and observed similar levels of exogenous ubiquilin2 expression (Supplementary Fig.
16). We also tested the functionality of the UbG76V-GFP reporter system using proteasomal
inhibitor MG-132 in transiently transfected cells. As expected, incubation with MG-132
resulted in remarkable accumulation of the UbG76V-GFP signal (Supplementary Figure
17). We then examined the accumulation of UbG76V-GFP reporter in Neuro2a cells transiently
transfected with either wild-type (WT) or mutant ubiquilin2 constructs. Expression
of mutant ubiquilin2 resulted in significantly higher accumulation of UbG76V-GFP than
WT-ubiquilin2 (Fig. 4a). Similar data were obtained using SH-SY5Y cells (Supplementary
Fig. 18).
We further analyzed the dynamics of UbG76V-GFP reporter degradation after new protein
synthesis was blocked with cycloheximide for 0, 2, 4, and 6 hours in Neuro2a cells.
We found that the rates of reporter degradation were significantly slower in both
ubiquilin2-P497H and ubiquilin2-P506T mutants when compared to wild-type ubiquilin2
at 4 hours (p <0.05) and 6 hours (p <0.001) (Fig. 4b), further supporting the notion
that the ubiquilin2 mutants impair the protein degradation pathway.
It is interesting to note that all the five ALS-linked UBQLN2 mutations identified
in the present study involve four proline residues in the PXX region. Proline is a
unique amino acid in that it has a side-chain cyclized onto the backbone nitrogen
atom, leading to sterical restriction of its rotation, and thus, hindering the formation
of major known secondary structures. Moreover, among the primary structures of many
ligands for protein-protein interactions, a proline residue is often critical
26
. Some protein-protein interaction domains, such as SH3, prefer ligand sequences containing
tandem PXXP motifs, as noted in the PXX domain of ubiquilin2, for high affinity and
selectivity of such interactions
27
. Further studies of the consequences of proline mutations in ubiquilin2 may shed
light on the understanding of the pathogenic mechanism.
The exact function of ubiquilin2 is not well understood. However, increasing lines
of evidence have shown that ubiquilins, together with their interactions with other
proteins, are involved in a broad spectrum of neurodegenerative disorders. Ubiquilin1,
another member of the ubiquilins, is associated with AD and it interacts with presenilins
1 and 2
28
, and TDP43
29
. We observed that ubiquilin2 formed cytoplasmic inclusions with ALS- and FTLD-linked
TDP43, implicating that an interaction of TDP43 and ubiquilin2 underlies the pathogenesis
of ALS and ALS/dementia, and possibly other neurodegenerative disorders as well.
The removal of misfolded or damaged proteins is critical for optimal cell functioning.
In the cytosol and the nucleus, a major proteolytic pathway to recycle misfolded or
damaged proteins is the UPS. Though impaired UPS is thought to be associated with
the formation of proteinaceous inclusions in many neurodegenerative disorders, direct
evidence of mutations in the UPS pathway has been limited
30
. In this study, we show mutations of ubiquilin2, a ubiquitin-like protein in five
families with ALS and ALS/dementia. We also show that ubiquilin2-containing inclusions
are a common pathological feature in a wide spectrum of ALS and ALS/dementia. Functional
studies indicate an impairment of ubiquitin-mediated proteasomal degradation in cells
expressing mutant ubiquilin2. These data provide robust lines of evidence for an impairment
of protein turnover in the pathogenesis of ALS and ALS/dementia, and possibly for
other neurodegenerative disorders as well. Further elucidation of these processes
may be central to the understanding of pathogenic pathways. These pathways should
provide novel molecular targets for designing rational therapies for these disorders.
Methods Summary
Genomic DNA was PCR-amplified and Sanger-sequenced using a CEQ 8000 Genetic Analysis
System (Beckman Coulter, Fullerton, CA). Western blot, immunohistochemistry and confocal
microscopy were performed using previously established methods
17
. Construction of expression vectors, cell culture and flow cytometry were performed
according to standard protocols. For statistical analysis, all graphs show mean ±
s.e.m. Significance was calculated using Student t-test (*, p <0.05; **, p <0.01;
***, p <0.001).
Full methods and associated references are available in the online version of the
paper at www.nature.com/nature.
Materials and Methods
Patients and samples
This study was approved by the local institutional review boards. ALS patients met
the diagnosis of probable or definite ALS as defined in the revised EL-Escorial
31
. Patients with dementia met the criteria for FTD or FTLD proposed by Neary et al
32
or Cairns et al
33
. The dementia was similar to FTD on inception and was progressive, and eventually
global in most patients. One patient had mild mental retardation before onset of dementia.
There were eight patients with both ALS and dementia. Dementia preceded motor symptoms
in some patients, but no patient remained free of motor involvement. The FTLD symptoms
included abnormalities in both behavior and executive function, although the degree
of severity varied depending on the individuals in different stages of the disease.
Pedigrees and clinical data were collected by specialists in neuromuscular medicine
and were verified by medical records or decent examination to establish diagnosis
(Supplementary Table 2). DNA and other samples were taken after obtaining written
informed consent. Overall, DNA from over 200 cases with ALS and 928 controls were
used for genetic analysis. Spinal cord autopsy samples from two X-linked ALS cases
(P497H or P506T each), 41 cases with ALS or ALS dementia and six non-ALS controls
were studied. In addition, available autopsy samples from the motor cortex region
of a patient with the P497H mutation (F#186, IV-7), brain regions (including hippocampus,
cerebellum, optic nerve, visual cortex, pons and midbrain) of two patients with the
P506T mutation (F#6316, II-3 and III-4), and the hippocampal region of 10 ALS and
five ALS/dementia cases were also used for pathological and immunohistological studies.
These five ALS/dementia cases were classified as having frontotemporal lobar degeneration
of motor neuron disease type (FTLD-MND/FTLD-U), including four cases with pathological
type 3 and one case with pathological type 2 according to the classification system
proposed by Mackenzie et al.
34
. These cases were evaluated by a neuropathologist (E.H.B).
Genetic analysis
Genomic DNA was extracted from whole peripheral blood, transformed lymphoblastoid
cell lines or available tissues by standard methods (QIAGEN, Valencia, CA). Intronic
primers flanking exons were designed at least 50 nucleotides away from the intron/exon
boundary. When a PCR product was over 500 bp, multiple overlapping primers were designed
with an average of 50 bp overlap. Forty nanograms of genomic DNA were used for PCR
amplification. The amplification protocol consisted of the following steps: incubation
at 95°C for 1 min, 32 cycles of 95°C (30s), 55°C (30s) and 72°C (1 min) and a final
5 min extension at 72°C, with modifications when necessary. Unconsumed dNTPs and primers
were digested with Exonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT, USB,
Santa Clara, CA). When nonspecific PCR amplification occurred, the PCR products were
separated by 1.5% agarose gel and the specific PCR product was cut out from the gel
and purified using QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). For sequencing
of a PCR product, fluorescent dye labeled single-strand DNA was amplified using Beckman
Coulter sequencing reagents (GenomeLab DTCS Quick Start Kit) followed by single-pass
bidirectional sequencing with a CEQ 8000 Genetic Analysis System (Beckman Coulter,
Fullerton, CA). We sequenced the entire protein-coding exons and 30–50 bp intronic
sequences flanking the exons. UBQLN2 is an intronless gene. We divided the UBQLN2
gene into five overlapping PCR fragments to carry out sequencing analysis. These five
overlapping PCR fragments cover the entire coding sequence (1,872bp), 125bp of the
5′ UTR and 293bp of the 3′UTR.
Ubqln2-1F: 5′-cttcatcacagaggtaccgtg-3′; Ubqln2-1R: 5′-gtgtggagttactcctgggag-3′
Ubqln2-2F: 5′-catgatgggctgactgttcac-3′; Ubqln2-2R: 5′-ctcttgtgcggcattcagcatc-3′
Ubqln2-3F: 5′-gacctggctcttagcaatctag-3′; Ubqln2-3R: 5′-gtgtctggattctgcatctgc-3′
Ubqln2-4F: 5′-cacagatgatgctgaatagcc-3′; Ubqln2-4R: 5′-gctgaatgaactgctggttgg-3′
Ubqln2-5F: 5′-ctgcacctagtgaaaccacgag-3′; Ubqln2-5R: 5′-aacagcattgattcccaccac-3′
For fragments 4 and 5, the PCR protocol consisted of the following steps: incubation
at 96°C for 2 min, 32 cycles of 96°C (30s), 56°C (30s) and 72°C (1min) and a final
5 min extension at 72°C. The PCR products were separated on a 1.5% agarose gel and
purified with QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) before sequencing.
Antibodies
Two anti-ubiquilin2 antibodies were used. One was a mouse monoclonal antibody (5F5,
Cat# H00029978-M03, Novus Biologicals Inc. Littleton, CO). We made the other, which
was raised in rabbit using a polypeptide of human ubiquilin2 (8aa-24 aa, NH2-SGPPRPSRGPAAAQGSA-COOH).
The antiserum was affinity purified. Other polyclonal and monoclonal antibodies that
were used in this study included those against: ubiquitin (PRB-268C, Covance, Emeryville,
CA; 10R-U101b, Fitzgerald Industries International, Concord, MA; Ub (N-19): sc-6085,
Santa Cruz Biotechnology, Santa Cruz, CA), p62 (H00008878-M01, Abnova, Taipei, Taiwan;
NB110-74805, Novus Biologicals, Inc., Littleton, CO), TDP43 (TIP-PTD-P01, Cosmo Bio
Co, Tokyo, Japan; 10782-2-AP, ProteinTech Group, Chicago, IL; 60019-2-Ig; ProteinTech
Group, Chicago, IL; WH0023435M1-100UG, Sigma-Aldrich, St. Louis, MO), FUS (11570-1-AP,
ProteinTech Group, Chicago, IL), OPTN (100000, Cayman, Ann Arbor, MI) and SOD1
35
, c-myc (MMS-150P, Covance, Emeryville), GFAP (Z0334, Dako North America, Carpinteria,
CA; G3893, Sigma-Aldrich, St. Louis, MO), Iba1 (019-19741, Wako Pure Chemical Industries,
Osaka, Japan), and CNPase (MAB326R, Millipore, Temecula CA).
Immunohistochemistry and Confocal Microscopy
The basic protocols for immunohistochemistry and confocal microscopy have been described
in detail in a previous study
17
. In brief, 6 μm sections were cut from formalin-fixed, paraffin-embedded spinal cord
and brain regions containing the frontal lobe or the hippocampus. The sections were
deparaffinized with xylene and rehydrated with a descending series of diluted ethanol
and water. Antigens in the sections were retrieved using a high pressure decloaking
chamber. For immunohistochemistry, endogenous peroxidase activity was blocked with
2% hydrogen peroxide. Non-specific background was blocked with 1% bovine serum albumin.
The titers of the antibodies were determined based on preliminary studies using serial
dilution of the antibodies. Various antibodies against ubiquilin2 or other proteins
were used as primary antibodies. These antibodies included rabbit polyclonal anti-ubiquilin2
(ubiquilin2-N; 0.5 μg/ml; generated by us), mouse monoclonal anti-ubiquilin2 antibody
(ubiquilin2-C; 0.2 μg/ml; H00029978-M03; Novus Biologicals, Littleton, CO), rabbit
polyclonal anti-FUS antibody (3 μg/ml; 11570-1-AP; ProteinTech Group, Chicago, IL),
mouse monoclonal anti-TDP43 antibody (1 μg/ml; 60019-2-Ig; ProteinTech Group, Chicago,
IL), rabbit polyclonal anti-TDP43 antibody (0.1 μg/ml; 10782-2-AP; ProteinTech Group),
mouse monoclonal anti-ubiquitin antibody (0.5 μg/ml; 10R-U101B; Fitzgerald Industries
International, Concord, MA), rabbit polyclonal anti-ubiquitin antibody (0.5 μg/ml;
PRB-268C, Covance, Emeryville, CA), goat polyclonal anti-ubiquitin (0.5 μg/ml; Ub
(N-19):sc-6085; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-p62
antibody (1 μg/ml; H00008878-M01; Abnova Coporation, Taipei, Taiwan), rabbit polyclonal
anti-optineurin antibodies (C-term, 0.2 μg/ml, 100000, Cayman Chemical, Ann Arbor,
Michigan), Biotinylated goat anti-rabbit and anti-mouse IgG, biotinylated mouse anti-goat
IgG or biotinylated rabbit anti-mouse IgG were used as the secondary antibodies. Immunoreactive
signals were detected with peroxidase-conjugated streptavidin (BioGenex, San Ramon,
CA) using 3-amino-9-ethylcarbazole as a chromogen. The slides were counterstained
with hematoxylin and sealed with Aqua Poly/Mount (Polyscience, Warrington, PA).
For confocal microscopy, antibodies generated in different species were used in various
combinations. These antibodies included those against ubiquilin2, FUS, TDP43, p62,
optineurin, ubiquitin, c-myc, GFAP, Iba1 and CNPase. Fluorescence signals were detected
with appropriate secondary anti-rabbit, anti-mouse or anti-goat IgG conjugated with
rhodamine or fluorescein isothiocyanate (Invitrogen, Carlsbad, CA; Thermo Scientific,
Rockford, IL) using an LSM 510 META Laser Scanning Confocal Microscope with the multi-tracking
setting
17
. The same pinhole diameter was used to acquire each channel.
Western Blot
Western blotting was performed using the protocol previously described
17
. Briefly, spinal cord tissues from lumbar segments were processed and homogenized.
Cell lysates or the supernatants of tissue homogenates were subjected to total protein
quantification, gel electrophoresis and blotted on PVDF membranes. Ubiquilin2 was
detected using ubiquilin2-N or ubiquilin2-C antibody. The membranes were stripped
and blotted with an antibody against β-actin, (A5441, Sigma-Aldrich, St. Louis, MO).
Expression constructs
A full length human cDNA clone (Homo sapiens ubiquilin 2, IMAGE:4543266) was used
as a template for construction of the expression constructs. Two primers anchored
with an XhoI (ubiquilin2-TP1, 5′-ttctcgagggccgccatggctgagaat-3′) and BamHI (ubiquilin2-TP2,
5′-catggatcctgtatgtctgtattacc-3′) were used to amplify the full length coding sequence.
The amplified fragment was cloned into plasmid vector pBluescript M13. The ubiquilin2
sequence was verified by direct sequencing. Each of the P497H and P506T mutations
was introduced into the plasmid vector by site-directed mutagenesis using a primer
containing each respective mutation. The XhoI/BamHI fragment containing wild-type
UBQLN2, UBQLN2P497H or UBQLN2P506T was released from the pBluescript M13 vector and
cloned into the XhoI and BamHI sites of dual expression vectors pIRES2-DsRed2 or pIRES2-ZsGreen1,
to create such constructs as wtUBQLN2, ZsGreen1; mutant ubiquilin2 (P497H, or P506T)
(mUBQLN2, ZsGreen1) (Clontech, Mountain View, CA).
In addition, we generated seven other expression constructs, including wild-type ubiquilin2
tagged with GFP (wtUBQLN2-GFP), mutant ubiquilin2 (P497H or P506T) tagged with GFP
(mUBQLN2-GFP), wild-type TDP43 tagged with mCherry (wtTDP43-mCherry), an ALS- and
dementia-linked C-terminal fragment of TDP43 (amino acids 218–414, C-TDP43)
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tagged with mCherry (C-TDP43-mCherry), wild-type FUS tagged with myc (myc-wtFUS) and
mutant FUS (R495X) tagged with myc (myc-mFUS).
Expression of wild-type and mutant ubiquilin2
Cells of SH-SY5Y, Neuro2a and HEK293 lines were grown on collagen-coated plates in
Dulbecco’s modified Eagle’s medium containing 10% (v/v) human serum, 2 mM L-glutamine,
2 U/ml penicillin, and 2 mg/ml streptomycin at 37°C in a humidity-controlled incubator
with 5% CO2. The cells were transiently transfected with different combinations of
expression vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to
manufacturer’s instructions.
UPS Reporter Assay
SH-SY5Y and Neuro2a cells were grown in 24-well plates and double-transfected with
a UPS reporter vector containing UbG76V-GFP
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(Addgene plasmid #11941), and a dual expression vector containing DsRed2 with either
wild-type or mutant ubiquilin2. Forty-eight hours post-transfection, cells were harvested
and resuspended in PBS. UbG76V-GFP transfected cells were used for control experiments
to test the functionality of the UPS reporter. In these control experiments, media
was changed 24 hours post-transfection to that containing 5 μM of the proteasomal
inhibitor MG-132 (A.G. Scientific, Inc, San Diego, CA). Cells were incubated in this
media for 24 hours and then harvested and resuspended in PBS. For cycloheximide chase
of UBG76V-GFP, transiently transfected Neuro2a cells were used. Twenty-four hours
post-transfection, the cells were transferred to medium containing 5 μM MG-132. After
incubation with MG-132 for 16 hours to accumulate the UbG76V-GFP reporter, cells were
washed in sterile PBS and incubated with medium containing 100 μg/ml cycloheximide
(Sigma, St. Louis, MO) for 0, 2, 4, and 6 hours. At each time point, cells were washed,
harvested and resuspended in ice-cold PBS supplemented with 100 μg/ml cycloheximide.
The fluorescence intensities at each time point were measured by FACS. The fluorescence
intensity at time=0 hours was taken to be maximal fluorescence (100%). All flow cytometric
data were collected and analyzed using a MoFlo cell sorter and Summit software (DakoCytomation,
Fort Collins, CO). Argon-ion (488 nm) and yellow (565 nm) lasers were used for excitation.
The GFP and DsRed2 signals were collected using 530/40 nm and 600/30 nm bandpass filters,
respectively. In all experiments data were gated on GFP/DsRed2 dual-labeled cells.
At least 500–1,000 such events were recorded in each experiment. The DsRed2 expression
levels and profiles were similar across experiments. Data were collected from three
independent experiments. Two-tailed unpaired Student t-test (p <0.05) was used for
statistical analysis.
Supplementary Material
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