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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: J Neurochem. 2016 Oct;139(2):285–293. doi: 10.1111/jnc.13748

Increased Ubqln2 Expression Causes Neuron Death in Transgenic Rats

Bo Huang 1, Qinxue Wu 1, Hongxia Zhou 2, Cao Huang 1,3, Xu-Gang Xia 1,3
PMCID: PMC5117623  NIHMSID: NIHMS805998  PMID: 27456931

Abstract

Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While overexpression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats overexpressing wildtype human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats overexpressing rat own Ubqln2. When overexpressed in rats, both human and rat wildtype Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Overexpressed wildtype UBQLN2 formed protein inclusions attracting the autophagy substrate p62 and the proteasome component Rpt1. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis.

Keywords: frontotemporal lobar degeneration, FTLD, amyotrophic lateral sclerosis, ALS, Ubqln2, p62, rats, Rpt1, protein aggregation

Summarizing schematic

Pathogenic mutation in Ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wildtype UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that overexpression of human or rat wildtype Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis.

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Introduction

Protein aggregation is a common pathology shared by many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ALS and FTLD are two distinctive diseases, but recent studies indicate that subsets of these diseases have common genetic causes (DeJesus-Hernandez et al., 2011; Neumann et al., 2006; Synofzik et al., 2012). Pathogenic mutations in ubiquilin 2 (UBQLN2) are causative of both ALS and FTLD (Deng et al., 2011; Synofzik et al., 2012; Williams et al., 2012). UBQLN2 belongs to ubiquilin family which consists of four members including UBQLN1, UBQLN2, UBQLN3, and UBQLN4 in mammals (Conklin et al., 2000; Wu et al., 2002). While ubiquilin proteins show a limited specialization in functions, they have the distinct patterns of expression (Conklin et al., 2000; Wu et al., 2002). UBQLN1 is ubiquitously expressed, UBQLN2 and UBQLN4 have more restricted but still widespread expression, and UBQLN3 is expressed exclusively in testes (Conklin et al., 2000; Wu et al., 2002). UBQLN2 consists of an ubiquitin-like domain (UBL) at the N-terminus and an ubiquitin-associated domain (UBA) at the C-terminus (Kleijnen et al., 2000). UBQLN2 shuttles between the nucleus and the cytoplasm to perform functions related to protein degradation via proteasomes and autophagy (Conklin et al., 2000; Kleijnen et al., 2000; N’Diaye et al., 2009; Wu et al., 2002). Proteasome and autophagy are impaired in transgenic rodents overexpressing mutant UBQLN2 though further studies are required to unravel the mechanisms underlying the pathologies (Gorrie et al., 2014; Wu et al., 2015).

One prominent feature of UBQLN2-linked diseases is protein aggregation (Deng et al., 2011; Synofzik et al., 2012; Williams et al., 2012), which is well reproduced in transgenic rats and mice overexpressing mutant human UBQLN2 (Gorrie et al., 2014; Wu et al., 2015). While overexpression of mutant UBQLN2 in rodents causes progressive neurodegeneration (Gorrie et al., 2014; Wu et al., 2015), deletion of Ubqln2 in knockout rats does not cause noticeable neuropathology (Wu et al., 2015), indicating that Ubqln2 is dispensable in neurons. Genetic studies in rodents strongly suggest that mutation of UBQLN2 causes neurodegeneration mainly through a gain rather than a loss of function. What toxic gain in mutant UBQLN2 is not known and thus requires follow-up studies. Pathogenic mutation of TDP-43 or FUS is causative of ALS and FTLD (Kwiatkowski et al., 2009; Sreedharan et al., 2008; Vance et al., 2009). When overexpressed in rodents, human TDP-43 and FUS, with or without pathogenic mutations, elicits indistinguishable neurodegeneration (Huang et al., 2011; Mitchell et al., 2013; Wegorzewska et al.; Wils et al., 2010; Zhou et al., 2010), suggesting that a toxic gain of function in the mutant genes is likely related to the enhancement of undefined physiological functions. To determine whether elevated wildtype UBQLN2 is detrimental or beneficial to neurons, we created transgenic rats overexpressing human or rat wildtype Ubqln2. Overexpression of wildtype human or rat Ubqln2 all caused prominent proteinopathy and progressive neuron death in transgenic rats. These findings suggest that excess UBQLN2 causes neurotoxicity rather than provides neuroprotection.

Materials and methods

Approval of animal study

Animal use was in accord with NIH guidelines and the animal use protocol was approved by the Institutional Animal Care and Use Committees at Thomas Jefferson University.

Animal studies

Transgenic rats were created by pronuclear injection and were maintained on Sprague Dawley genomic background as described (Huang et al., 2012a; Huang et al., 2012b; Tong et al., 2013; Wu et al., 2015). Both male and female rats of similar numbers were used in the study. Wildtype Sprague Dawley rats were purchased from Charles River (Wilmington, Massachusetts) and were maintained in the lab for multiple years. Mutant human UBQLN2 (TRE-hUBQLN2P497H) and CaMKα2-tTA transgenic rats have been characterized (Huang et al., 2012a; Tong et al., 2012; Wu et al., 2015). Wildtype human UBQLN2 transgene construct was built as described previously (Wu et al., 2015). The open reading frames (ORF) of rat Ubqln2 were amplified by PCR from the cDNA pools that were generated with mRNA isolated from Sprague Dawley rat brains. The ORF of wildtype human and rat Ubqln2 was inserted between the tetracycline-responsive element (TRE promoter) and SV40 late poly (A) sequence as previously described (Wu et al., 2015). Transgenic rats were identified by PCR amplification of a promoter region with the following primers: 5′-TTGTTTGTGGATCGCTGTGA-3′ (forward) and 5′-GACAAACTTCACGTCAGGGT-3′ (reverse). Copy number of the transgenes was determined by quantitative PCR with the same set of primers and the copy standard was established by mixing transgenic DNA with rat genomic DNA as described (Zhou et al., 2009). TRE-Ubqln2 transgenic lines were crossed with CaMKα2-tTA transgenic line to produce double-transgenic offspring, in which the Ubqln2 transgene was expressed in the forebrain neurons (Huang et al., 2012a; Tong et al., 2012). Breeding rats were given Doxycycline (Dox) in drinking water (50 μg/ml) to suppress transgene expression during embryonic development. Ubqln2 transgenic rats and their controls were deprived of Dox at birth to allow for transgene expression and disease induction.

Rats were examined with a Barnes Maze (Med Associates) to reveal any deficits in spatial learning and memory as described (Huang et al., 2011). Rats were given one training session and four test sessions for 5 consecutive days. During training or testing sessions, rats were placed in the same initial orientation inside a transparent cylinder (start box) that was located at the center of the maze disk and the rats remained in the start box for 1 minute such that a standard starting context was ensured. Latency to locate the fixed escaping box was calculated from the time testing started to the time when the animal entered, or its four paws touched, the box.

Antibodies and immunoblotting

The following primary antibodies were used in this study: mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA), rabbit anti-GFAP (DAKO North America, Carpinteria, USA), and rabbit anti-Iba-1 (Wako Chemicals, USA), mouse monoclonal antibody recognizing human and rat Ubqln2 (Novus, Littleton, USA), rabbit polyclonal antibody against p62 (Novus, Littleton, USA), chicken anti-ubiquitin (Sigma-Aldrich, Saint Louis, USA), and rabbit anti-Rpt1 antibodies (Novus, Littleton, USA). Primary antibodies were used at the lowest dilutions recommended by manufacturers. For immunoblotting, rat’s forebrains were homogenized in RIPA buffer and total proteins in cleared lysates were separated on SDS-PAGE as described (Zhou et al., 2010). Proteins were resolved on SDS-PAGE and were transferred onto nitrocellulose membrane. Immunoreactivity for a target protein was detected with specific primary antibodies as described (Wu et al., 2015).

Histology and immunostaining

Numbers of neurons in the frontal cortex and dentate gyrus were estimated with unbiased stereological cell counting as described (Huang et al., 2012a; Tong et al., 2012). Rat’s forebrains were cut into coronal sections (20 μm) with a Cryostat. Tissue sections containing the whole frontal cortex (from the apical forebrain to the first occurrence of corpus callosum) or dentate gyrus of one cerebral hemisphere were collected and every 10th section was counted for neurons in defined brain regions. Tissue sections were stained with Cresyl violet and mounted in sequential order (rostral-caudal). The number of targeted neurons was estimated using a fractionator-based unbiased stereology software program (Stereologer), which was run on a PC computer that was attached to a Nikon 80i microscope with a motorized XYZ stage (Prior). The detail of stereological cell counting was previously described (Huang et al., 2012a; Tong et al., 2012). For immunostaining, rats were perfused with 4% paraformaldehyde under deep anesthesia and rat’s brains were dissected thereafter. The brains were further fixed in the same fixative at 4°C overnight and then dehydrated in 30% sucrose as described (Huang et al., 2012b; Huang et al., 2011). Coronal sections (15 μm) of rat forebrain were immunostained first with primary antibodies and then with dye-labeled secondary antibodies. Immunoreactivity for specified proteins was observed under Nikon fluorescence microscope and documented with a digital camera. For determining protein colocalization, tissue sections were examined with a confocal microscope (Imaging Facility of Kimmel Cancer Center at Jefferson). Single-layer images were scanned with a Zeiss LSM510 META confocal system.

Statistical analysis

Numbers of neurons in frontal cortex or dentate gyrus were compared between Ubqln2 transgenic rats and control rats using paired t-tests. A P-value of less than 0.05 was considered statistical significance.

Results

Transgenic rats are created to express wildtype human UBQLN2

To examine the consequence of enhanced UBQLN2 expression in an intact model system, we first created transgenic rats that overexpressed wildtype human UBQLN2 (Fig 1). A tetracycline-regulatory gene expression system has proved to be effective for the temporal and spatial expression of transgenes in rats (Huang et al., 2014; Huang et al., 2012a; Huang et al., 2012b; Tong et al., 2013; Zhou et al., 2010; Zhou et al., 2009), and thus, it was used to drive wildtype human UBQLN2 transgene (Fig 1: A). As the promoter of mouse Camk2a drives transgene expression specifically in the forebrain neurons (Huang et al., 2012a; Tong et al., 2012), this promoter was chosen to achieve neuronal expression of wildtype human UBQLN2 in transgenic rats (Fig 1). We have previously characterized a transgenic rat line that overexpresses human UBQLN2 with a pathogenic mutation (P497H substitution) and develops progressive neuron death and learning deficit (Wu et al., 2015). A transgenic line that expressed wildtype human UBQLN2 at a level comparable to the mutant UBQLN2 transgenic line was chosen for characterization and comparison (Fig 1: B). Immunostaining revealed that human UBQLN2 was substantially expressed in the hippocampus and in the cortex of transgenic rats carrying wildtype human UBQLN2 transgene (Fig 1: C–V).

Figure 1.

Figure 1

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Overexpressed wildtype human UBQLN2 accumulated in transgenic rats. (A) A diagram showing a strategy by which wildtype human UBQLN2 was overexpressed in neurons of transgenic rats. (B) Immunoblotting revealed that human UBQLN2 was expressed at comparable levels between mutant (line 20 carrying P497H substitution) and wildtype UBQLN2 transgenic lines. Proteins in rat’s forebrain lysates were loaded at equal amount which was confirmed by probing the same membrane with a GAPDH antibody. (C–V) Fluorescence staining revealed that human UBQLN2 was substantially expressed in the hippocampus (C–R) and cortex (S–V) of rat forebrain. Note that UBQLN2-expressing cells were progressively reduced in rats (P–Q). Scale bars: 100 μm (C–J) and 30 μm (K–V).

Overexpression of wildtype human UBQLN2 causes neuronal death and cognitive deficits

Our previous studies have showed that expression of mutant human UBQLN2 in postnatal rats causes a progressive loss of neurons (Wu et al., 2015). For comparison, a similar strategy was used to activate wildtype human UBQLN2 transgene in postnatal rats (Figs 1–2). Doxycycline was withdrawn from rats at birth such that UBQLN2 transgene was activated thereafter in the forebrain (Fig 1). At the ages of 40 and 80 days, transgenic rats displayed in the dentate gyrus a progressive loss of neurons which was revealed by Cresyl violet staining (Fig 2: A–F). Neuronal loss also was evident in the frontal cortex (Fig 2: G and H). In response to neuron death, astrocytes and microglia changed morphologies indicative of reactive status (Fig 2: I–L). Quantification with unbiased stereological cell counting verified that neurons were significantly lost in the whole frontal cortex and the dentate gyrus of one cerebral hemisphere in transgenic rats (Fig 2: M and N). We examined rat’s cognitive function using Barnes maze assay and found that transgenic rats showed a deficit in spatial learning and memory when they overexpressed wildtype human UBQLN2 (Fig 2: O).

Figure 2.

Figure 2

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Overexpression of wildtype human UBQLN2 caused neuron death and learning deficit in transgenic rats. (A–H) Cresyl violet staining revealed neuronal loss in the dentate gyrus (A–F) and frontal cortex (G–H) of transgenic rats. (I–L) Immunofluorescence staining revealed activation of astrocytes (I and J) and microglia (K and L) in rats overexpressing wildtype human UBQLN2. (M, N) Stereological cell counting confirmed the loss of neurons in the frontal cortex and dentate gyrus of wildtype human UBQLN2 rats. Neurons were estimated for the whole frontal cortex and dentate gyrus of one cerebral hemisphere. Data are means ± SD (n = 6). * p < 0.01. (O) Barnes maze assay revealed spatial learning deficits in CaMKα2-tTA/TRE-hUBQLN2 double-transgenic rats (hUB2) as compared to CaMKα2-tTA single-transgenic rats (tTA) at the age of 80 days. Rats were trained to locate an escape hole in a Barnes maze in the first day and then were daily examined for improvement in locating fixed escape hole. Data are means ± SD (n = 6). * p < 0.05. Scale bars: 100 μm (A–C) and 30 μm (D–L).

Overexpression of wildtype human UBQLN2 leads to p62 and PSMC2 accumulation

Pathogenic mutation of UBQLN2 predisposes it to aggregation and this property of mutant UBQLN2 is well reproduced in rodent models (Gorrie et al., 2014; Wu et al., 2015). Overexpression of mutant human UBQLN2 in rodents causes the accumulation of autophagy substrates and proteasomal components (Gorrie et al., 2014; Wu et al., 2015). We examined wildtype human UBQLN2 transgenic rats by immunofluorescence staining and we observed that p62 and Rpt1 (also called PSMC2) accumulated in the neurons overexpressing wildtype human UBQLN2 (Fig 3). While Rpt1 is a key component of proteasome, p62 is related to autophagy function. P62 accumulation is indicative of autophagy deficits. Similar accumulation of p62 and Rpt1 was detected in transgenic rats expressing mutant human UBQLN2 (Fig 3: G and I). In mutant human UBQLN2 transgenic rats, UBQLN2 accumulates initially in the neurites and cytoplasm and further in the nuclei (Wu et al., 2015). Following mutant UBQLN2 translocation, p62 accumulates first in the neurites and then in the nuclei (Wu et al., 2015). In contrast to mutant human UBQLN2, wildtype human UBQLN2 was constantly accumulated in the cytoplasm and p62 was accordingly accumulated in the cytoplasm (Fig 3: B–F). Ubiquitin inclusions were detected in the rats overexpressing wildtype human UBQLN2 (Fig 4). Confocal microscopy revealed that ubiquitin was partially colocalized with Ubqln2 in protein inclusions (Fig 4: I–K).

Figure 3.

Figure 3

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Overexpression of wildtype human UBQLN2 caused p62 and Rpt1 accumulation in transgenic rats. (A–E) Immunofluorescence staining revealed p62 accumulation in wildtype human UBQLN2 transgenic rats. Dentate gyrus was examined for CaMKα2-tTA transgenic rats (tTA) serving as controls and for CaMKα2-tTA/TRE-hUBQLN2 transgenic rats overexpressing wildtype human UBQLN2 (hUB2wt) in the forebrain. Protein inclusion was examined for the rats at indicated ages. (F) Confocal microscopy revealed localization of UBQLN2 and p62. Transgenic rats overexpressing hUB2wt were examined at the age of 60 days. (G) Immunofluorescence staining revealed the accumulation and translocation of p62 in transgenic rats overexpressing mutant human UBQLN2 in the forebrain (P497H). (H–J) Immunostaining revealed Rpt1 accumulation in mutant (I: P497H) and wildtype human UBQLN2 (J: hUB2wt) transgenic rats. Scale bars: 100 μm (A–B), 30 μm (C–E and G–J) and 8 μm (F1–3).

Figure 4.

Figure 4

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Overexpression of wildtype human UBQLN2 causes ubiquitin aggregation in transgenic rats. (A–H) Double-labeling fluorescence staining revealed that ubiquitin accumulated in wildtype human UBQLN2 transgenic rats. Dentate gyrus was examined of protein inclusions for CaMKα2-tTA single-transgenic rats (tTA) serving as a control and for CaMKα2-tTA/TRE-UBQLN2 double-transgenic rats overexpressing wildtype human UBQLN2 (hUB2wt) in the forebrain at indicated ages. (I–K) Confocal microscopy revealed the localization of UBQLN2 and ubiquitin aggregates. Scale bars: 30 μm (A–H) and 8 μm (I–K).

Overexpression of rat Ubqln2 causes neuron death and ubiquitin aggregation in rats

Ubqln2 is highly conserved across species including human and rats; however, difference in amino acid composition is noticed for human and rat Ubqln2 proteins (Ko et al., 2004; Wu et al., 2002). Human UBQLN2 may not behave properly in the rat genome and thus may cause unexpected toxicity. To examine this possibility, we created new transgenic rats overexpressing rat own Ubqln2 (Fig 5). Similar to wildtype human UBQLN2, wildtype rat Ubqln2 of elevated levels caused progressive neuron death which was revealed by Cresyl violet staining (Fig 5: E–J). Immunostaining revealed upregulation of the astrocyte marker GFAP and the microglia marker Iba1 (Fig 5: K–N), indicating that glial reaction occurred. Stereological cell counting confirmed neuronal loss in transgenic rats overexpressing rat Ubqln2 (Fig 5: O). Similar to wildtype human UBQLN2, excess wildtype rat Ubqln2 induced p62 and ubiquitin aggregation in transgenic rats (Fig 6).

Figure 5.

Figure 5

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Overexpression of rat Ubqln2 caused neuron death in transgenic rats. (A–D) Immunofluorescence staining revealed that rat Ubqln2 was overexpressed in the CaMKα2-tTA/TRE-rUbqln2 double-transgenic rats (rUB2) as compared to CaMKα2-tTA single-transgenic rats (tTA). Frontal cortex (A, B) and dentate gyrus (C, D) were examined of Ubqln2 expression for tTA and rUB2 rats at the age of 40 days. (E–J) Cresyl violet staining revealed a progressive loss of neurons in rUB2 transgenic rats as compared to the control tTA rats. (K–N) Immunofluorescence staining revealed activation of astrocytes (K and L) and microglia (M and N) in rats overexpressing its Ubqln2. (O) Stereological cell counting confirmed neuron loss in transgenic rats overexpressing rat Ubqln2 (rUB2). Data are means ± SD (n = 5). * p < 0.01. Scale bars: 30 μm (A–D and H–N) and 100 μm (E–G).

Figure 6.

Figure 6

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Overexpression of rat Ubqln2 caused accumulation of p62 and ubiquitin in transgenic rats. (A–F) Immunofluorescence staining revealed p62 accumulation in Ubqln2 transgenic rats. Dentate gyrus (A–B and D–E) and frontal cortex (C, F) were examined CaMKα2-tTA transgenic rats (tTA) serving as controls and for CaMKα2-tTA/TRE-rUbqln2 transgenic rats overexpressing rat Ubqln2 (rUB2) in the forebrain. (G–L) Fluorescence staining revealed ubiquitin accumulation in transgenic rats overexpressing rat own Ubqln2. Protein inclusions were examined for tTA and rUB2 rats at the age of 40 days. Scale bars: 100 μm (A, D) and 30 μm (B–C and E–L).

Discussion

Our findings in wildtype UBQLN2 transgenic rats show that excess UBQLN2 is detrimental to neurons. This conclusion is supported by substantial evidence. Rats overexpressing human wildtype UBQLN2 displayed a progressive loss of neurons which were targeted for transgene expression. Neuron loss was detected by Cresyl violet staining and was verified by unbiased stereological cell counting. In response to neuronal death, astrocytes and microglia became reactive. Considering that human UBQLN2 may not function properly in the rats, we further overexpressed rat Ubqln2 in transgenic rats. Similar to wildtype human UBQLN2, rat Ubqln2 caused neuronal death and glial reaction when overexpressed in transgenic rats. In fact, a detrimental effect is observed for UBQLN1 (Ganguly et al., 2008). Overexpression of wildtype human UBQLN1 in drosophila causes progressive retinal degeneration and this toxicity is likely related to the antagonizing effect of UBQLN1 against presenilin (Ganguly et al., 2008). As Ubiquilin proteins have a limited specialization in functionality (Conklin et al., 2000; Wu et al., 2002), observation on Ubqln1 and Ubqln2 could be generalized to the other members of Ubiquilin family. Studies on in vivo models suggest that an increase in Ubqln2 expression may produce toxicity rather than provide protection to neurons.

Similar to mutant Ubqln2 (Wu et al., 2015), wildtype Ubqln2 of increased expression also caused protein aggregation in transgenic rats. When overexpressed, both human and rat wildtype Ubqln2 aggressively formed inclusions in the cytoplasm. Aggregating Ubqln2 induced p62 and Rpt1 to accumulate in protein inclusions. Overexpression of Ubqln2 with or without a pathogenic mutation reproduced a key feature of ALS and FTLD associated with UBQLN2 mutations (Deng et al., 2011; Synofzik et al., 2012; Williams et al., 2012). As disease progresses in rat models, mutant UBQLN2 accumulates initially in the cytoplasm and further in the nuclei (Wu et al., 2015). By contrast, wildtype human and rat Ubqln2 accumulated in the cytoplasm. A similar localization of p62 is observed for wildtype and mutant UBQLN2 transgenic rats, suggesting that p62 accumulation is associated with UBQLN2. The colocalization of UBQLN2 with p62 in aggregates was indeed observed with confocal microscopy. Overexpression of mutant human UBQLN2 in transgenic rats causes prominent dendritic pathology which is visualized with Golgi staining and immunofluorescence staining (Wu et al., 2015). By contrast, dendritic pathology was less evident in new transgenic rats overexpressing wildtype human or rat Ubqln2. A possibility is that a fast progression of neuropathology in wildtype Ubqln2 transgenic rats prevents a full development of dendritic pathology. Along with our previous findings (Wu et al., 2015), our studies in rat models show that wildtype and mutant Ubqln2 cause some indistinguishable neuropathology. In contrast to transgenic overexpression of Ubqln2, deletion of Ubqln2 causes no abnormality in knockout rats (Wu et al., 2015). Existing evidence from in vivo studies collectively suggests that the enhancement of undefined UBQLN2 functions is implicated in the toxic gain of function in UBQLN2 mutations.

Acknowledgments

This work is supported by the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NS095972 to C. H., NS073829 and NS089701 to H. Z. and NS095962 and NS084089 to X.G.X). The content is the author’s responsibility and does not necessarily represent the official view of the NIH institutes.

Abbreviation used

UBQLN2

ubiquilin 2

ALS

amyotrophic lateral sclerosis

FTLD

frontotemporal lobar degeneration

p62

sequestosome-1

Rpt1

26S proteasome regulatory subunit 7

Footnotes

Conflict of interest

The authors declare that no conflict of interest exists.

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