Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice
© Qiu et al.; licensee BioMed Central Ltd. 2013
Received: 21 August 2012
Accepted: 29 December 2012
Published: 3 January 2013
A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.
To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.
Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.
KeywordsVAPB ALS Motor neuron disease Neurodegeneration Transgenic mice
Motor neuron diseases (MND) are a group of diverse neurological disorders with motor neuron involvement that include amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, spastic paraplegias, progressive muscular atrophy, spinal muscular atrophy (SMA), and spinobulbar muscular atrophy . ALS, also referred to as Lou Gehrig’s disease, is the most common adult-onset motor neuron disease caused by degeneration of upper and lower motor neurons, accompanied by progressive weakness, muscle wasting and fasciculations, spasticity, dysarthria, dysphagia, and respiratory compromise. While 90% of ALS cases are sporadic without known genetic mutations, 10% of the cases are familial, which are caused by more than a dozen genetic mutations . Among them, a proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) in the highly conserved major sperm protein (MSP) domain causes some dominantly inherited forms of MND (ALS8), which show typical and atypical ALS symptoms or a mild late-onset spinal muscular atrophy (SMA) [3, 4]. A second missense mutation in VAPB causing an amino acid change from threonine to isoleucine at codon 46 (T46I), also in the MSP domain, has been proposed as a causative factor in a single case of familial ALS (fALS) .
VAPB belongs to a well conserved VAP family of proteins. VAPB is an integral membrane protein of the endoplasmic reticulum (ER) with an amino terminal MSP domain, a central coiled-coil motif and a carboxy-terminal transmembrane domain anchored in ER membrane [6–12]. VAPB has roles in neurotransmitter release , ER to Golgi transport [14, 15], bouton formation at the neuromuscular junction , Ca2+ homeostasis [6, 17] and signaling via Eph receptors  and Robo and Lar-like receptors . VAPB has also been implicated in other cellular processes, including endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), in which VAPBP56S is functionally abnormal in these processes [5, 10, 18, 20–22]. While some studies have shown that VAPBP56S can enhance ER stress and the UPR [18, 21], others have associated VAPBP56S with an inhibitory effect on the UPR [5, 10, 20, 22]. It is unclear what functional and structural changes induced by ALS/MND-related mutations in VAPB lead to neurodegeneration and muscle atrophy in patients.
Similar to many neurodegenerative disorders, the two known VAPB mutants can form aggregates [4, 5, 7, 12, 21, 23, 24], which also sequester wild type VAPB and VAPA [12, 25]. It is possible that VAPB mutations cause ALS/MND by a dominant-negative effect and/or a gain of toxicity from its protein aggregates and recruitment of wild type VAPB and VAPA proteins into these aggregates, especially in motor neurons. To test this hypothesis, we generated both wild type VAPB (wtVAPB) and VAPB P56S (muVAPB) transgenic mice with broad expression in the CNS and peripheral tissues and observed robust formation of VAPB positive aggregates in the spinal cord of the muVAPB, but not the wtVAPB transgenic mice. However, we detected neither motor dysfunction nor motor neuron degeneration. These results do not support the gain-of-toxicity mechanism for mutant VAPB-induced ALS.
It has been suggested that VAPB deficiency may play a broad role in the pathogenesis of familial ALS (fALS) and sporadic ALS (sALS) because decreased levels of VAPB have been reported in patients with sALS and the SOD1G93A mouse model [12, 26]. On the other hand, VAPBP56S may cause pathogenesis of the disease by a gain of toxic function . Therefore, we crossed both muVAPB and wtVAPB transgenic mice with SOD1G93A mice to determine whether the muVAPB and wtVAPB modulate the course of the disease caused by the SOD1 mutation. If there was a gain of toxic function or a dominant negative effect due to muVAPB overexpression, we expected to observe an adverse effect of muVAPB on SOD1G93A mice, such as accelerated disease onset and progression. Conversely, if the decreased level of VAPB played a significant role in the pathogenesis of ALS, we would observe a slowing in the disease progression in the SOD1G93A and wtVAPB double transgenic mice. However, neither muVAPB nor wtVAPB overexpression affected the disease onset or progression caused by mutant SOD1, suggesting that mutant VAPB does not have a gain of toxicity and VAPB levels do not modulate the disease course of ALS cases without the VAPB mutations.
Overexpression of human mutant or wild type VAPB in the transgenic mice
The construct was verified by expression of the transgene after transient transfection into HEK293 cells and subsequent Western blot for the FLAG-tagged VAPB protein (data not shown). After pronuclear injection, a total of 14 wtVAPB and 23 muVAPB transgenic founder lines were generated and then screened for the expression of the transgene in brains and spinal cords by Western blot with anti-FLAG or anti-VAPB antibody. One wtVAPB line (wtVAPB) and two muVAPB lines (muVAPB35 and muVAPB60) that showed the highest levels of expression in the spinal cords were further studied. In wtVAPB transgenic mice, the transgenic hVAPB protein level was 2–3 times higher than the mouse endogenous VAPB level in both the spinal cord and forebrain, while the protein levels of muVAPB in the lines muVAPB35 and muVAPB60 were slightly higher than the endogenous mouse VAPB level in the spinal cord and lower than the endogenous level in the forebrain (Figure 1B). To achieve a higher expression level of muVAPB, the two muVAPB lines were crossed to obtain double transgenic mice (muVAPB35/60), which expressed the muVAPB at a level approximately the same as the wtVAPB mice in the spinal cord and approximately equal to the endogenous VAPB level in the forebrain (Figure 1B). Notably, the expression of transgenic VAPB did not alter the expression level of the endogenous VAPB as compared with the level in the non-transgenic (NTG) control. In addition to the CNS, the transgene was also expressed in peripheral tissues, including the skeletal muscle, heart, spleen, liver and lung (data not shown).
Overexpression of muVAPB, but not wtVAPB, led to VAPB aggregation
muVAPB overexpression did not cause motor dysfunction or affect motor neuron survival
VAPB aggregation did not lead to typical ALS pathology
Protein aggregates in neurodegenerative diseases are marked by ubiquitin and p62, which implies an ongoing clearance process of these aggregates by the cellular protein quality control system . To examine whether there is a clearance process against the VAPB aggregates, we stained motor neurons for ubiquitin and p62. We detected no labeling of ubiquitin in the majority of VAPB aggregates and only occasional weak labeling in some aggregates (Figure 5B, arrows). We also detected no p62 labeling of the VAPB aggregates (data not shown). These results suggest that VAPB aggregates differ from the pathogenic aggregates such as those derived from mutant SOD1 (Figure 5B) that trigger the response from the cellular protein quality control system.
muVAPB did not cause structural change in ER or coaggregate with VAPA
muVAPB aggregation did not affect disease progression in SOD1G93A mice
Compared to the previous VAPB transgenic study , our results showed stronger and broader muVAPB aggregates, especially in motor neurons. Our muVAPB transgenic model with wide-spread aggregation formation could provide a useful tool to test the possible gain-of-toxic function due to protein misfolding and aggregation. Yet despite a systematic investigation with multiple analytic assays, we failed to find evidence for neurodegeneration or dysfunction in the transgenic mice. Although a two-fold overexpression of muVAPB may not have reached the toxic threshold, and therefore, may not be sufficient to trigger motor neuron degeneration, the failure of muVAPB to enhance mutant SOD1 toxicity diminishes the possibility of a harmful role of mutant VAPB in motor neuron degeneration. A further support for the innocuous nature of muVAPB is the absence of any pathological hallmarks, including structural changes of ER and mitochondria, evidence of UPR, reactive astrogliosis and microgliosis, redistribution of TDP-43, and activation of cellular protein quality control systems.
Our results contrast with some previous literature reports. Overexpression of muVAPB has been reported to alter ER morphology and modulate UPR in vitro[5, 7, 12, 18]. Our data do not support these roles in vivo. In an analysis of a VAPBP56S transgenic mouse line, Tudor and colleagues reported a redistribution of TDP-43 from nucleus to the cytoplasm and the presence of ubiquitin- and p62-labeled structures in motor neurons . We have not confirmed their observation in our muVAPB mice. The reason of this discrepancy is not clear. A simple explanation might be that the mutant VAPB was expressed at a higher level in their mice than in the mice used in this study. Consistent in both studies, however, is the observation that overexpression of muVAPB does not cause motor neuron degeneration phenotypes. Therefore, the results from both of these studies do not support the possibility that a gain of toxicity in mutant VAPB causes motor neuron degeneration.
Common in neurodegenerative diseases are cytoplasmic protein aggregates, including mutant or the wild type proteins of SOD1 [37, 38], TDP-43 [39–41], and FUS/TLS . However, it is not clear what roles these aggregates play in the pathogenesis of the disease. Indeed, it is controversial whether the aggregation is detrimental to cell survival. For example, neurodegeneration is still induced by ALS-related TDP-43 mutations in the absence of cytoplasmic TDP-43 aggregates, suggesting TDP-43 aggregation may not be required for pathogenesis [43, 44]. Furthermore, inclusion body formation might protect neurons by reducing the levels of toxic soluble forms of mutants, as has been shown in mutant huntingtin . It is possible that the neurodegeneration is not due to the presence of cytoplasmic aggregates, but by altered function of the mutant protein [46, 47]. In human MND caused by VAPB mutations, the cellular mechanism leading to motor neuron degeneration and muscle atrophy is not known. Although robust VAPB aggregates were formed in the muVAPB transgenic mice, especially in the motor neurons, our results have revealed that motor neurons could tolerate the VAPB aggregates without degeneration, suggesting that VAPB protein aggregation may not play a significant role in the pathogenesis.
Whether VAPB aggregation has a role in human patients with VAPB mutations is not clear. In fact, it remains to be established that VAPB cytoplasmic aggregates exist in ALS8 patients. Motor neurons derived from iPS cells from ALS8 patients with the known VAPB-P56S mutation have decreased levels of VAPB without VAPB aggregation , which is consistent with the loss-of-function hypothesis. In this study, we detected no decrease in the endogenous VAPB level, nor did we attain evidence for involvement of VAPA or endogenous VAPB in the VAPB aggregates. This result does not support the possibility that the mutant VAPB exerts a dominant negative effect on the normal allele by coaggregation. Furthermore, in the presence of widespread VAPB aggregates, especially in the CNS, no neurodegeneration or motor dysfunction was observed, which does not support the toxic gain-of-function hypothesis. Therefore, future studies to determine whether a loss of function is the underlying pathogenic mechanism for the mutant VAPB is of critical importance.
Previous studies have shown that VAPB levels were reduced in sporadic ALS patients and SOD1G93A mice [12, 26], suggesting that VAPB dysfunction may contribute to ALS in general. We tested this possibility by overexpression of either the mutant or the wild type VAPB in SOD1G93A mice. Our results demonstrate that neither the mutant nor the wild type VAPB had any impact on the course of the disease in the SOD1G93A mice. These results do not support a general role of VAPB in ALS cases that do not have VAPB mutations.
Since protein aggregation is common in many neurodegenerative diseases, it has been proposed that the protein aggregation weakens the capacity of cellular proteostasis, thus enhancing the cell vulnerability to secondary assault(s) from another misfolding-prone protein . Our muVAPB/SOD1G93A double transgenic mice enabled us to test whether mutant SOD1 aggregation enhances mutant VAPB aggregation and vice versa in a mammalian system. Our data showed that VAPBP56S and SOD1G93A aggregate independently and do not enhance each other’s aggregation. Therefore, the synergistic protein misfolding and aggregation may not occur in all experimental systems. In addition, since VAPB aggregation did not cause motor neuron degeneration, and in particular, did not enhance mutant SOD1 toxicity, some protein aggregation may be innocuous and all protein aggregation does not inevitably lead to cellular toxicity.
Overexpression of mutant but not wild type VAPB leads to robust VAPB-positive aggregates. However, motor neurons are capable of tolerating these aggregates without degeneration, thus suggesting that mutant VAPB does not cause ALS through toxicity associated with its aggregation. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate protein aggregation and the course of the ALS in SOD1G93A mice, suggesting that VAPB does not play a significant role in ALS patients that do not have VAPB mutations. Importantly, mutant VAPB and mutant SOD1 aggregate independently and do not augment each other’s aggregation, thus suggesting that aggregation-prone proteins do not inevitably lead to deterioration of cellular proteostasis. Overall, our results do not support a gain of toxic function or a dominant negative effect as the underlying mechanism for muVAPB-induced neurodegeneration. Therefore, future experiments need to test the loss-of-function hypothesis.
Human VAPB (hVAPB) cDNA clone in the vector pCMV6-XL5 was purchased from OriGene. Proline at position-56 was changed to Serine by converting C to T at the 166th position using a site-directed mutagenesis kit (Qbiogene) with the PCR primers 5′-CCACGTAGGTACTGTGTGAGGTCCAACAGCGGAATCATCGAT-3′ and 5′-ATCGATGATTCCGCTGTTGGACCTCACACAGTACCTACGTGG-3′. The mutation was confirmed by sequencing both strands. Wild type hVAPB (hVAPBWT) and hVAPBP56S fragments were amplified by PCR with the primers flanked by EcoRI and KpnI sites and cloned in the plasmid vector p3XFLAG-CMV-7 (Sigma) with the same restriction enzyme sites to obtain two intermediate vectors (all enzymes were obtained from New England Biolabs unless otherwise indicated). The 3XFLAG tag was added to differentiate the transgene from the mouse endogenous VAPB gene. We used a previously described pCAG-EGFP/RFP-miRNAint plasmid  to construct the transgenic constructs (Figure 1A). The EGFP encoding fragment in the plasmid was cut out with Cla1 digestion, blunted by Klenow, and then cut by Sph1. The large fragment of the vector was gel-purified for subsequent cloning. The 3xFLAG-hVAPBWT and 3xFLAG-hVAPBP56S fragments were amplified by PCR from the intermediate vectors, blunted with T4 polymerase, and digested with SphI. The transgenic vectors pCAG-wtVAPB/RFP and pCAG-muVAPB/RFP were constructed by ligation of the large vector fragment and the 3xFLAG hVAPB WT or 3xFLAG hVAPB P56S fragment, respectively. All cloning procedures were verified by DNA sequencing.
Generation and propagation of transgenic mice
The expression vectors, pCAG-wtVAPB/RFP and pCAG-muVAPB/RFP, were linearized by digestion with the restriction enzymes Ssp1 and Pci1 and subsequently purified as previously described . The linearized DNA constructs were injected into C57BL/6 × SJL F2 hybrid mouse embryos by the UMassTransgenic Animal Modeling Core. Mouse tails of 2 mm in length were cut for PCR-based genotyping. Tail DNA samples were prepared by DirectPCR Lysis Reagent (Viagen Biotech) according to the provided instructions. Positive transgenic mice were identified by a 185 bp fragment within the open reading frame of the human VAPB gene produced by PCR using the primer pair 5′- AGTCTCTGAGTTCTTCTTTGG -3′ and 5′-CTTCCCAGTTGGGGCTA-3′. The PCR program was set as 4 minutes at 95°C, followed by 40 cycles of 95°C for 30 seconds, 51°C for 20 seconds, and 72°C for 30 seconds. The transgenic mice were maintained as heterozygotes by crossing with FVB/NJ mice. SOD1G93A (B6.Cg-Tg(SOD1*G93A)1Gur/J) transgenic mice  were obtained from Jackson Laboratory and crossed with FVB/NJ for ten or more generations. All mice were maintained at the University of Massachusetts Medical School animal facility according to the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) and all animal procedures were approved by the IACUC.
Mice under deep anesthesia were transcardially perfused with cold PBS followed by cold fixation buffer containing 4% paraformaldehyde in PBS. Tissues were dissected out and immersed in the same fixative overnight at 4°C. Tissues were treated in PBS containing 15% sucrose for 2–3 days at 4°C, and then frozen in OCT media (Sakura, Torrance, CA). Frozen sections at 10 μm thickness were cut by a cryostat and air-dried on slides at room temperature for 30 minutes. For immunofluorescence staining, slides were incubated in the blocking solution (5% normal goat serum and 0.2% Triton X-100 in PBS, pH7.4) for 30 min at RT, rinsed three times in PBS, and incubated with primary antibodies in the blocking solution overnight at 4°C. The slides were subsequently washed with PBS three times and incubated with secondary antibodies in the blocking solution for 2 hr at RT in dark. After washing, the slides were mounted with Vectashield mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI, Vector Laboratories). Images were taken with a Nikon Eclipse Ti microscope equipped with NIS elements software and further processed by the three-dimensional deconvolution program AutoQuant X2 (Media Cybernetics). The following antibodies were used: VAPB (ProteinTech Group), Iba1 (BioCare Medical), GFAP (Cell Signaling), TDP-43 (ProteinTech Group and Encor Biotechnology), NF-H (Covance), Ubiquitin (Millipore), PDI (Stressgen), and SOD1 (Biodesign). The secondary goat anti-rabbit, goat anti-mouse or donkey anti-sheep IgG (Invitrogen) conjugated by Alexa Fluor-488 or Alexa Fluor-555 were used.
For muscle histology, samples were obtained from fixed mice, embedded in paraffin blocks, sectioned and stained with Hematoxylin & Eosin (H&E).
For nerve and spinal cord plastic sections, the cervical and lumbar spinal cords and the L3 and L4 nerve roots were dissected from mice fixed with 4% paraformaldehyde and further fixed in 4% paraformaldehyde plus 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH7.6 for 3 to 5 days. Plastic sections at 1 μm were processed and stained with toluidine blue. For electron microscopy, thin sections of ventral horn were cut from the Epon tissue blocks, stained with uranyl acetate and lead citrate, and proceeded for examination under a Philips CM-10 transmission electron microscope.
In all experiments, NTG mice were processed in parallel as negative controls. For ubiquitin and p62 staining, the end stage mutant SOD1 mice were used as positive controls.
Mouse tissues were homogenized at a ratio of 100 mg/ml in homogenization solution (50 mM Tris HCl pH7.4, 1% SDS, 0.05% Triton X-100, 1 mM EGTA and protease inhibitor mixture) for 1 minute by using the Fast Prep-24 (MP Biomedicals). The resultant tissue lysates were heated at 95°C for 5 min followed by vortexing at low speed. After centrifugation at 13000 rpm for 5 min, the supernatants were stored at −80°C.
Protein samples (50 μg each) were resolved on a 10% SDS-polyacrylamide gel (Bio-Rad) for subsequent Western blotting. The membrane was probed with anti-FLAG (Stratagene,1:2000), anti-VAPB (ProteinTech Group, 1:1000), or anti-SOD1 antibody (Biodesign,1:10,000). The protein bands were visualized using the SuperSignal West Pico kit (Pierce). After detecting VAPB, the blot was stripped and reprobed with anti-tubulin primary antibody (Sigma, 1:10,000).
Before the actual test, mice were trained on an automated 4-lane rotarod unit (Columbus Instruments) for three days. The rotarod was set to an accelerating mode from 13 to 72 rpm over a period of 5 minutes. The animals were tested for three consecutive days and three times each day with a rest of 15 minutes between each trial. The length of time that each animal was able to stay on the rod was recorded. The best performance of each animal on each day was used to calculate the average performance for the three days.
Grip strength test
A device with a force gauge (Chatillon, DFM2) connected with a stainless steel grid was used for the test. For forelimb grip strength measurement, the mouse was gently lowered over the top of the grid so that only its front paws can grip the grid. The mouse was pulled away from the grid until it released the grip. For combined forelimb and hind limb measurement, the mouse was lowered over the top of the grid so that both its front and hind paws can grip the grid. The mouse was pulled away horizontally and steadily until it released its grip. Each animal was measured three times and the peak grip strength value of the animal was recorded as its performance.
The RT-PCR was done as previously described . Briefly, total RNA was extracted from mouse tissues by Trizol reagent (Invitrogen) followed by treatment with DNase I (Promega) and cDNA synthesis by using SuperScript III kit (Invitrogen). Quantitative PCR was performed by using Sybr Green mix (Qiagen). The primer pair 5′-TCCTGTCCTCAGATGAAATTGG-3′ and 5′-CCACTCTGTTTCCGTTTCCTA-3′ were designed for the mouse Chop gene (Ddit3) with a product size of 241 bp. The primer pair 5′-TTCAATGGCAAGGAGCCATC-3′ and 5′-TGATTATCGGAAGCCGTGGA-3′ were designed for the mouse BiP gene (Hspa5) with a product size of 239 bp. Mouse reference gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was amplified with the primer pair 5′-CTGGAGAAACCTGCCAAGTA-3′ and 5′-TGTTGCTGTAGCCGTATTCA-3′, which produce a product of 223 bp. The PCR program was set as follows: 3 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, 30 seconds at 58°C, and 20 seconds at 72°C. ΔCt values were calculated by subtracting the Ct values of Gapdh from those of Chop or BiP. ΔΔCt values were derived by subtracting the ΔCt values of the wild type from those of the transgenic mice, which were then used to calculate the levels of the mRNA in the transgenic mice relative to those in the controls. All the RT-PCR reactions were done by using the CFX96 Real-Time System (Bio-Rad).
Mice of different genotypes were generated from crossing wtVAPB transgenic or muVAPB35/60 double transgenic mice with SOD1G93A mice and were analyzed at the age of 95 to 100 days. Mouse tissues were mixed at a ratio of 1:10 with pre-chilled lysis buffer containing 50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 1 mM EDTA, 150 mM KCl, and 1% multiple protease inhibitors (Sigma). Homogenization was done using the handheld polytron for 20 sec on ice. The lysates were centrifuged at 12,000 g for 5 min at 4°C. The supernatants were collected as the soluble fraction. The pellets were homogenized in the same buffer and centrifuged. The supernatants were discarded. The pellets were homogenized for 10 sec in the buffer containing 50 mM Tris HCl pH 7.4, 1% SDS, 0.05% Triton X-100, 1 mM EGTA and protease inhibitor mixture. One fifth of the original lysis buffer volume was used. Following the homogenization, the homogenate was vortexed vigorously for 10 sec. The resulting lysates were incubated at 95°C for 10 min and vortexed vigorously again for 10 sec. The samples were left at room temp to cool and then centrifuged at 13000 rpm for 5 min at room temperature. These supernatants were collected as the insoluble fraction. All the soluble and insoluble parts were used for Western blotting as stated above.
Student’s t-test was used for statistical analysis.
Vesicle-associated membrane protein-associated protein B
Motor neuron disease
Amyotrophic lateral sclerosis
Spinal muscular atrophy
Unfolded protein response
Vesicle-associated membrane protein-associated protein A
Proline-to-serine substitution at position-56
Major sperm protein
Threonine to isoleucine at codon 46
Central nervous system
TAR-DNA binding protein
Frontotemporal lobar degeneration
Cu/Zn superoxide dismutase-1
Hematoxylin & Eosin
3X poly A sequences.
The authors thank Ms. Sili Zhou and Dr. Thaya Venugopal for technical assistance, the UMass Transgenic Animal Modeling Core for pronuclear injection and the technical support from the Core Electron Microscopy Facility, which receives support from the National Center for Research Resources (S10RR027897). This work was supported by a grant (5R01NS059708) from National Institute of Neurological Disorders and Stroke to ZX.
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