Author response: The critical role of membralin in postnatal motor neuron survival and disease (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Hitherto, membralin has been a protein of unknown function. Here, we show that membralin mutant mice manifest a severe and early-onset motor neuron disease in an autosomal recessive manner, dying by postnatal day 5–6. Selective death of lower motor neurons, including those innervating the limbs, intercostal muscles, and diaphragm, is predominantly responsible for this fatal phenotype. Neural expression of a membralin transgene completely rescues membralin mutant mice. Mechanistically, we show that membralin interacts with Erlin2, an endoplasmic reticulum (ER) membrane protein that is located in lipid rafts and known to be important in ER-associated protein degradation (ERAD). Accordingly, the degradation rate of ERAD substrates is attenuated in cells lacking membralin. Membralin mutations or deficiency in mouse models induces ER stress, rendering neurons more vulnerable to cell death. Our study reveals a critical role of membralin in motor neuron survival and suggests a novel mechanism for early-onset motor neuron disease. https://doi.org/10.7554/eLife.06500.001 eLife digest As new proteins are built inside a cell, many will pass into a structure called the endoplasmic reticulum for processing. There, the proteins are folded into the specific three-dimensional shapes that allow them to carry out their respective jobs. Sometimes the folding process goes awry, leading to a build-up of unfolded proteins that stress the endoplasmic reticulum and can kill the cell. Brain cells are particularly vulnerable to death from endoplasmic reticulum stress. To combat a deadly build-up of unfolded proteins, each cell has systems that respond when the endoplasmic reticulum is under stress. Unchecked stress on the endoplasmic reticulum has been linked to diseases like amyotrophic lateral sclerosis (called ALS for short). In diseases like ALS, the nerve cells that control muscle movements gradually die off, causing a loss of muscle control and eventually death. Scientists suspect that these nerve cells (called motor neurons) are particularly sensitive to endoplasmic reticulum stress because they are highly active. Drugs that help counteract stress on the endoplasmic reticulum extend the lives of mice with motor neuron disease, suggesting this may be a useful strategy for treating such diseases in humans. Now, Yang, Qu et al. identify a new protein that appears necessary for a healthy endoplasmic reticulum. Mice that lack the gene for a protein called membralin die within five or six days after birth because their motor neurons die off. Further experiments showed that re-introducing membralin in their nervous system can rescue these membralin-deficient mice. Yang, Qu et al. found that membralin interacts with another protein that helps eliminate poorly folded or unfolded proteins in the endoplasmic reticulum, and thus relieves stress on the cell. Mutations in this endoplasmic reticulum stress response protein have previously been linked to motor neuron diseases. The motor neurons in membralin-deficient mice show signs of endoplasmic reticulum stress and are extra vulnerable to chemicals that induce protein misfolding. Together, the experiments show membralin plays an important role in mitigating stress on the endoplasmic reticulum. More studies of mice lacking membralin may help explain why the endoplasmic reticulum stress increases in motor neuron diseases and may point to possible treatments. https://doi.org/10.7554/eLife.06500.002 Introduction The endoplasmic reticulum (ER) is a membrane-enclosed cellular organelle that plays an essential role in the folding of membrane-bound and secreted proteins, synthesis of lipids and sterols, and storage of free Ca2+. ER stress is often triggered by the accumulation of unfolded proteins due to pathological conditions, such as DNA sequence mutations, transcriptional and translational errors, or protein folding failure (Kim et al., 2008; Lin et al., 2008). Mammalian cells have evolved an intricate system with multiple signaling pathways to respond to ER stress, collectively termed the unfolded protein response (UPR) (Kim et al., 2008; Lin et al., 2008; Walter and Ron, 2011). If unchecked, ER stress eventually causes cell death, including neuronal death in neurodegenerative diseases (Lindholm et al., 2006; Scheper and Hoozemans, 2009; Hetz and Mollereau, 2014). Increased ER stress is thought to play an early role in motor neuron diseases, including amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth (CMT) (Atkin et al., 2006; Nagata et al., 2007; Nishitoh et al., 2008; Kanekura et al., 2009; Saxena et al., 2009). Manifestations and consequences of ER stress have been studied in the pathogenesis of SOD1 mutant mice, a commonly used model of ALS that expresses mutant human SOD1 protein as found in some inherited forms of ALS (Atkin et al., 2006; Kanekura et al., 2009; Saxena et al., 2009). Many ER stress-related molecules are upregulated in SOD1 mice at an early stage of the disease, and some are specific to motor neurons (Tobisawa et al., 2003; Atkin et al., 2006; Kikuchi et al., 2006; Nagata et al., 2007; Ito et al., 2009; Saxena et al., 2009). In addition, reduction in the ER co-chaperone SIL1 has been found to be associated specifically with ER stress-prone, fast-fatigable motor neurons (Filezac de L'Etang et al., 2015). ER upregulation of the UPR and aberrant modification of protein disulfide isomerase (PDI) also occur in human tissues from sporadic ALS (Atkin et al., 2008; Walker et al., 2010). Our studies on the activation of the type I interferon signaling in astrocytes at pre-symptomatic stages of SOD1 mice also suggest that ER stress in motor neurons may play a crucial role in disease onset (Wang et al., 2011). Moreover, genetic interruption of UPR signaling molecules, such as XBP-1 or apoptosis signal-regulating kinase1 (ASK1), protects motor neurons in SOD1 mutant mice (Nishitoh et al., 2008; Hetz and Mollereau, 2014). It has been suggested that the unusually heavy metabolic demand of motor neurons may make them particularly susceptible to ER stress (Vinay et al., 2000; Carrascal et al., 2005; Li et al., 2005). Importantly, ER stress inhibitors, such as salubrinal, guanabenz, and sphin1, delay disease onset and prolong the survival of these mutant mice (Saxena et al., 2009; Jiang et al., 2014; Wang et al., 2014; Das et al., 2015). This finding suggests that ER stress-related molecules may represent rational drug targets for motor neuron diseases. Previously, membralin had been predicted by genome sequencing to encompass an open reading frame (orf61 in mouse and c19orf6 in human), but the function of the encoded protein has, heretofore, remained unknown. Orf61 is highly conserved and found in the genome of most species except yeast and bacteria. cDNAs for membralin have been cloned from human and mouse tissues (Andersson and von Euler, 2002; Chen et al., 2005), and the protein encoded by orf61 was named membralin because it was predicted to be a membrane protein (Andersson and von Euler, 2002). Given the absence of known protein domains, membralin may represent a novel class of proteins. Here, we discovered during our cloning and characterization of the glutamate receptor subunit 3B gene (GluN3B, formerly designated NR3B, Nishi et al., 2001; Chatterton et al., 2002; Matsuda et al., 2002) that the 3′ end of the GluN3B gene overlaps with the 3′ end of the membralin gene on the opposite strand. Thus, our GluN3B knockout (KO) mice also carry a C-terminal truncation of membralin. We observed that GluN3B/membralin C-terminal double-knockout (DKO) mice die at postnatal day 5–6 of paresis due to severe motor neuron degeneration. Transgenic expression of membralin, but not GluN3B, rescued the phenotypes of DKO mice. Additionally, we generated membralin-specific KO mice and found that they phenocopied the DKO mice. These data suggest that membralin plays a critical role in motor neuron survival. We also demonstrate that membralin interacts with Erlin2, a protein that is enriched in ER lipid rafts and important for ER-associated protein degradation (ERAD) (Ikegawa et al., 1999; Browman et al., 2006). Additionally, neurons from membralin mutant mice display increased basal ER stress and increased vulnerability to ER stress-inducing agents. Our discovery that membralin mutations result in motor neuron disease provides mechanistic insight into pathophysiology and may offer a novel target for therapy. Results Membralin KO mice manifest severe motor neuron loss We generated GluN3B-deficienct mice by deletion of the 8-kb region encoding the entire GluN3B gene and found that it also resulted in partial deletion of membralin (Figure 1—figure supplement 1). Sequence analysis confirmed that the translation frame of the altered membralin transcript in null mice terminates 147 nucleotides (nts) beyond the end of partial exon XI, resulting in a slightly truncated membralin protein with 49 unrelated amino-acid (aa) residues replacing the wild-type (WT) 89 C-terminal aa residues. Thus, our targeting strategy generated a GluN3B/membralin C-terminal DKO mouse line. DKO mice appeared normal at birth but unexpectedly died at postnatal day 5–6 (P5-6). The motor function of these mice was indistinguishable from WT or heterozygous littermates during the first three postnatal days, but motor strength was severely impaired, thereafter, preceding death. DKO mice were also significantly smaller than WT and heterozygous littermates (Figure 1A) but displayed normal gross anatomy and histology in the brain (Figure 1—figure supplement 2), visceral organs, and skeletal muscle. Figure 1 with 4 supplements see all Download asset Open asset Motor neuron death in GluN3B/Membralin DKO mice. (A) DKO and WT littermate mice are shown at P5. The DKO mouse was unable to maintain correct posture. (B) Immunostaining with anti-Hb9 antibody revealed a dramatic decrease in the number of motor neurons in the lumbar spinal cord of DKO mice relative to WT littermate mice at P5 but not at P0. (C) The number of motor neurons in the lumbar spinal cord of KO and WT mice was not significantly different at P0 and P3. However, severe motor neuron loss was observed by P5 in DKO mice (n = 3) compared with WT mice (n = 3, *p < 0.05 by Student's t-test). (D) Glial fibrillary-associated protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA-1) immunostaining in the spinal cord of WT and DKO mice. GFAP-positive astrocytes and IBA-1-positive microglial cells surrounding motor neurons (counterstained with Giemsa) of DKO mice showed swelling cell bodies and processes, a typical sign of astrogliosis. (E,F) Electron microscopic analysis of spinal roots showed that DKO mice at age P5 had severe damage of motor neuron axons in the ventral root (E) but not of sensory neuron axons in the dorsal root (F). Bar, 2 µm. (G,H) Damaged axons in the ventral root showed either vacuoles or loss of axoplasm (G), as an early sign of axonal damage. At a later stage of axonal damage, dark disintegrated myelin sheaths and amorphous lipid were present (H). Bar, 1 µm. (I) Percentage of injured axons in the ventral and dorsal roots of DKO and WT mice (*p < 0.05 by Student's t-test, n = 3). Data are mean ± s.e.m. https://doi.org/10.7554/eLife.06500.003 As DKO mice showed signs of paresis, we conducted histological examination of motor neurons in the lumbar spinal cord using the motor neuron-specific marker, Hb9. The number of motor neurons in DKO mice was unchanged at P0 and P3, but reduced by 40% at P5, compared to littermate WT controls (Figure 1B,C). The remaining motor neurons appeared smaller than those of WT mice. Motor neuron loss and surrounding astrogliosis were also apparent in the cervical spinal cords of P5 DKO mice (Figure 1D). We next examined Isg15 expression (interferon-induced 17 kDa protein), which is thought to play a role in innate immunity and is upregulated at both pre-symptomatic stages and post-symptomatic stages in a mouse model of ALS (Wang et al., 2011). WT mice manifested minimal Isg15 expression throughout the brain and spinal cord. In contrast, by P3, DKO mice had increased Isg15 expression in the ventral horn of the cervical and lumbar enlargement of the spinal cord (Figure 1—figure supplement 3A). By P5, DKO mice showed increased Isg15 expression in additional areas containing primary motor neurons, with highest expression in the ventral horn of all segments of the spinal cord and moderate expression in the facial nucleus of the brainstem (Figure 1—figure supplement 3A,B). No Isg15 expression was detectable in other brain areas, even in late-stage DKO mice. These results suggest that the motor neurons are progressively injured in DKO mice, starting with those in the spinal cord. Since the normal process of programmed cell death in embryonic motor neurons is complete at birth (Lance-Jones, 1982), the loss of motor neurons in DKO mice was most likely due to cellular events occurring perinatally. To confirm specific loss of motor neurons in DKO mice, we examined the spinal roots of the lumbar spinal cord by electron microscopy. Motor neuron axons in the ventral root of DKO mice showed advanced degeneration (Figure 1E), whereas sensory neuron axons in the dorsal root were intact (Figure 1F). Under high magnification of motor neuron axons, we saw intra-axonal vacuoles and the loss of axoplasm (Figure 1G), as well as disintegrated myelin and amorphous lipids (Figure 1H), as typically seen in Wallerian degeneration. The percentage of degenerated axons in the ventral root of DKO mice reached 48.0 ± 4.1% by P5, which was significantly greater (p < 0.01) than in WT mice (8.6 ± 1.5%) (Figure 1I). In contrast, the percentage of degenerated axons in the dorsal root of DKO mice (10.5 ± 1.9%) was similar to that of the WT ventral root, consistent with the normal background level of axonal degeneration that occurs during development. Moreover, the sensory afferent terminals in the spinal cord of DKO mice appeared normal (Figure 1—figure supplement 4A). In contrast, we found degeneration of phrenic motor nerve fibers and terminals in P5 DKO mice (Figure 1—figure supplement 4B). These results further confirm the selective death of motor neurons in DKO mice, potentially leading to respiratory failure due to loss of motor neuron innervation of the intercostal muscles and diaphragm. Taken together, our DKO mice present a phenotype of early-onset and apparently selective motor neuron degeneration. Causality of membralin deletion in motor neuron degeneration Since both membralin and GluN3B are mutated in DKO mice, we needed to determine which gene was responsible for the motor neuron degeneration. Therefore, we tested whether transgenically expressed intact membralin or GluN3B could rescue DKO mice. We generated a transgenic mouse line carrying full-length membralin under the control of the murine prion promoter, followed by internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) cDNA sequences (Figure 2A). The membralin transgenic mice [Tg (membralin)] were fertile, normal in size, and did not display any gross physical or behavioral abnormalities compared with their littermate controls. When Tg (membralin) mice were crossbred with DKO mice, the DKO/Tg (membralin) mice were apparently normal, manifesting neither paresis nor premature death (Figure 2B). Thus, the membralin transgene rescued DKO mice, even though membralin mRNA levels remained somewhat lower than in WT mice (Figure 2C). GFP immunostaining in the ventral horn of the spinal cord showed that membralin/GFP transgene expression was predominantly neuronal, although we could not rule out relatively weak expression in glial cells (Figure 2—figure supplement 1). The DKO/Tg (membralin) mice showed no sign of motor defects or motor neuron loss, despite the absence of GluN3B expression (Figure 2D). In contrast, the GluN3B transgene did not rescue DKO mice (Figure 2—figure supplement 2), and GluN3B KO mice generated by deleting the first exon did not show any defect in motor neurons (Niemann et al., 2007). Taken together, these findings are consistent with the hypothesis that the loss of intact membralin in DKO mice causes motor neuron degeneration. Figure 2 with 2 supplements see all Download asset Open asset Membralin transgene [Tg (membralin)] rescues GluN3B/membralin (DKO) mice. (A) Generation of the transgenic constructs expressing membralin. The murine prion promoter was cloned with full-length mouse membralin cDNA followed by the IRES and the EGFP sequence. The entire transgenic sequence was isolated by enzymatic digestion and used to generate Tg (membralin) mice. (B) DKO/Tg (membralin) mice were viable and fertile. Left: genotyping by PCR showed that 3 littermates (#3, 4 and 5) from a breeding of DKO and Tg (membralin) mice were positive to DKO primers and negative to WT primers (same primers used as in Figure 1). Of the three DKO mice, the two receiving membralin transgene (#3 and 5, as positively identified by GFP primers) were rescued. Right: Weight of the 7 littermates was monitored after birth. Only DKO mouse #4, which was negative for the membralin transgene, showed weight loss after P3 and died at P5.5. Data shown here are from one representative litter (5 independent litters were analyzed with similar results). (C) Gene expression analysis by RT-PCR for mice #1, 3, 4 of the same litter as in B. Membralin transgene expression is seen in the brains of littermates #1 and 3, but not 4, whereas GluN3B is only expressed in #1. Membralin and GluN3B are not expressed in muscle samples. GAPDH is expressed in both brain and muscles of all mice and served as a positive control. (D) Rescued DKO/Tg (membralin) mice lived to adulthood without any sign of paresis. https://doi.org/10.7554/eLife.06500.008 Since heterozygous DKO mice did not manifest motor defects, and expression of the truncated form of membralin appears to lack any deleterious effect. This conclusion is also supported by results from a second membralin KO mouse line we generated using a gene trapping strategy (Figure 3A). RT-PCR experiments using primers from exon 1 and 2 further confirmed that homozygous mice carrying membralin/trapping vector alleles did not express membralin mRNAs with sequences beyond exon 2. Membralin protein was also reduced in heterozygotes and absent in homozygotes. These membralin KO mice phenocopied the DKO mice in motor defects, with ∼50% motor neuron loss preceding death around P5 (Figure 3B,C). Figure 3 Download asset Open asset Membralin KO mice die of motor neuron degeneration and consequent paresis. (A) Gene trapping was used to generate membralin KO mice by inserting a trapping vector that contained a splicing acceptor sequence between exon 1 and 2 to disrupt normal RNA splicing. Primers (P1, P2, P3, red arrows) were designed to detect normal and trapped membralin transcripts. RT-PCR experiments showed that PCR products using the P1 and P3 primer pair were only detected in WT mouse brain (lane 1), liver (lane 2), and kidney (lane 3), whereas PCR products using the P1 and P2 primers were only detected in these tissues of KO mice. (B) Membralin KO mice phenocopied GluN3B/membralin DKO mice and died of paresis around P5. (C) Lumbar motor neurons, identified by anti-Hb9 staining (top panels), were significantly reduced (lower panel) in membralin KO mice compared to WT mice (n = 3 for each group of mice, *p < 0.05, Student's t-test). Data are mean +s.e.m. https://doi.org/10.7554/eLife.06500.011 Membralin resides in the ER membrane and interacts with Erlin2 Membralin is a novel protein with no conserved domains. RT-PCR analysis showed that membralin mRNA was expressed in multiple tissues, including brain and spinal cord, but not heart, skeletal muscle, or spleen (Figure 4A). Western blot analysis of spinal cord samples from WT and KO mice showed specific expression of membralin in the ER but not in the cytosol or mitochondria (Figure 4B). Since our anti-membralin antibody proved ineffective for immunostaining, we made a fusion protein by adding a myc tag to the of membralin that with an ER but not with for or mitochondria (Figure was not expressed on the cell it was not detected in cells without first the membrane not for from the for that the membralin is most likely a protein with containing an between 1 and 2. Additionally, the protein has multiple and at both the and C-terminal (Figure supplement These data suggest that membralin is a membrane protein most likely to the ER Figure 4 with 1 supplement see all Download asset Open asset expression and of membralin. (A) RT-PCR analysis showed the expression of membralin mRNA in brain spinal cord liver and kidney but not in spleen and muscle tissues of WT mice. (B) Western blot analysis showed the expression of membralin protein in the ER but not in the or the of the WT mouse (C) of membralin in were with membralin. At after of membralin was detected by were with specific for for and for of shown at magnification in show membralin with the ER marker, study human ERAD an strategy identified a of membralin with Erlin2 et al., Erlin2 is a protein that is enriched in ER lipid rafts (Ikegawa et al., 1999; Browman et al., and mutations in Erlin2 have been linked to human diseases with motor et al., 2006; et al., et al., et al., et al., We designed experiments to the We found that membralin not only with but also manifested a with Erlin2 in the ER (Figure The was significantly for membralin and Erlin2 ± than the negative control and ± consistent with a of the two molecules (Figure Additionally, full-length membralin with Erlin2 (Figure This of membralin with Erlin2 further the that membralin is an ER membrane protein and also suggests that loss of membralin could potentially ER stress by we found that the ER membrane was more in mouse embryonic from membralin KO mice than from WT mice (Figure whereas was no in the of the ER (Figure supplement 1). These results suggest that membralin deficiency degradation of ER membrane proteins, which could potentially ER stress. Thus, we next analyzed ER stress in membralin KO mice. Figure with 1 supplement see all Download asset Open asset Membralin interacts with Erlin2 and protein (A) cells were with control and or target and or were shown in and (B) was for control and target data shown are mean ± s.e.m. n = < by Student's (C) The of membralin with Erlin2 was confirmed by experiments using Erlin2 and membralin fusion proteins into were with or and using (D) from both WT and membralin KO mouse were with and to analysis of degradation after with at the were to with to shown the of and the of three (n = 3, *p < 0.05 by Student's t-test). data shown are mean ± s.e.m. Increased ER stress in membralin KO mice Increased ER stress is thought to be in some forms of motor neuron disease, including ALS (Atkin et al., 2006; Nagata et al., 2007; Nishitoh et al., 2008; Kanekura et al., 2009; Saxena et al., 2009). tested whether the absence of membralin ER stress and Ron, 2011). Western blot analysis showed that the levels of and two molecules during ER stress activation of the signaling were in the spinal cord of P3 membralin KO mice than WT mice ± and ± of n = n = Figure In contrast, levels of activation of the signaling during ER stress, were not altered (Figure supplement an of ER stress, a greater of cell death in from KO mice than from WT mice (Figure also increased and levels and to a greater in from KO levels were also at (Figure levels of but not were in membralin KO mice than WT mice (Figure but XBP-1 splicing was not altered (Figure supplement these data suggest that loss of membralin increases basal ER stress and cells more vulnerable to additional ER Figure with 1 supplement see all Download asset Open asset ER stress in membralin KO mice. (A) of and in spinal cord of membralin KO mice. Left: analysis levels of and in the spinal cord of P3 membralin KO mice compared to that of littermate WT mice. Right: increased expression level of and to in membralin KO mice WT mice. (B) of after to or by a (C) The expression level of and in from KO and WT mice is shown after to at different of and are and in from KO than that from WT mice. representative is data from three each data are mean ± n = 3 for each *p < 0.05 by Student's Discussion motor is into two spinal and which without sensory In to a group of muscles with both autosomal and recessive et al., 2006; 2008). of were on age of of and of a number of forms of were adding more genetic of gene and other associated have been identified for of type these for a group of proteins, as in et al., 2006; 2008). The of the proteins stress proteins protein in the ER RNA protein 2 and and axonal It is not these mutated proteins of cellular function to motor neuron survival In their expression is not even to motor similar is found for membralin in this Therefore, further studies on