1. Biochemistry and Chemical Biology
Download icon

Ataxin-1 oligomers induce local spread of pathology and decreasing them by passive immunization slows Spinocerebellar ataxia type 1 phenotypes

  1. Cristian A Lasagna-Reeves
  2. Maxime WC Rousseaux
  3. Marcos J Guerrero-Munoz
  4. Luis Vilanova-Velez
  5. Jeehye Park
  6. Lauren See
  7. Paymaan Jafar-Nejad
  8. Ronald Richman
  9. Harry T Orr
  10. Rakez Kayed
  11. Huda Y Zoghbi  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Texas Children's Hospital, United States
  3. University of Texas Medical Branch, United States
  4. Howard Hughes Medical Institute, Baylor College of Medicine, United States
  5. University of Minnesota, United States
  6. Baylor College of Medicine, United states
Research Advance
  • Cited 9
  • Views 1,209
  • Annotations
Cite this article as: eLife 2015;4:e10891 doi: 10.7554/eLife.10891

Abstract

Previously, we reported that ATXN1 oligomers are the primary drivers of toxicity in Spinocerebellar ataxia type 1 (SCA1; Lasagna-Reeves et al., 2015). Here we report that polyQ ATXN1 oligomers can propagate locally in vivo in mice predisposed to SCA1 following intracerebral oligomeric tissue inoculation. Our data also show that targeting these oligomers with passive immunotherapy leads to some improvement in motor coordination in SCA1 mice and to a modest increase in their life span. These findings provide evidence that oligomer propagation is regionally limited in SCA1 and that immunotherapy targeting extracellular oligomers can mildly modify disease phenotypes.

https://doi.org/10.7554/eLife.10891.001

Introduction

We recently demonstrated that soluble oligomers play an important role in Spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease caused by expansion of a CAG repeat that encodes for glutamine (Q) in ataxin-1 (ATXN1)(Lasagna-Reeves et al., 2015). In the same study, we showed that ATXN1 oligomers are internalized into the cell and seed the formation of new ATXN1 oligomers (Lasagna-Reeves et al., 2015). This 'seeding' concept has been explored in several other proteinopathies and is believed to be one of the drivers of neurodegeneration (Frost et al., 2009; Munch et al., 2011; Holmes and Diamond, 2012). What’s more, evidence has mounted that soluble aggregates of amyloidogenic proteins including Tau, Aβ and α–synuclein, can propagate pathology from cell-to-cell in a prion-like fashion, thereby causing pathology progression from one brain region to the another in a disease-specific pattern (Hardy and Revesz, 2012; Holmes and Diamond, 2012; Guo and Lee, 2014). Passive immunotherapy has been recently proposed as a feasible strategy to inhibit pathology propagation in mouse models for proteinopathy (Banks et al., 2007; Chai et al., 2011; Masliah et al., 2011; Yanamandra et al., 2013; Castillo-Carranza et al., 2014; Games et al., 2014; Tran et al., 2014). Despite these advances, it is not clear whether a mostly nuclear protein like ATXN1 will propagate from cell-to-cell in vivo and whether passive immunotherapy targeting oligomers will modify disease course.

In the present study, we report that polyQ ATXN1 oligomers act as a seed by inducing disease propagation to neighboring but not to distal cells and demonstrate that this cell-to-cell spread can be blocked using passive immunotherapy.

Results

ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo

We recently demonstrated that in Atxn1154Q/+mice ATXN1 oligomers are restricted to focal sub-populations of Purkinje cells (PCs) and are not evenly present throughout the cerebellum. Notably, this focal distribution coincided with cellular toxicity (Lasagna-Reeves et al., 2015). This observation, together with the finding that ATXN1 oligomers are able to penetrate cells in culture and seed the formation of new ATXN1 oligomers led us to hypothesize that if ATXN1 oligomers propagate in vivo, intracerebral injection of brain extract from a symptomatic SCA1 mouse into a disease-free mouse would predispose the latter to develop neuropathology. To test this hypothesis, we injected cerebellar extract (10 µg, 2.5 μL) either from Atxn1154Q/+or wild-type mice into the deep cerebellar nuclei of wild type, Atxn1-/- and Atxn178Q/+mice (Figure 1A). Atxn178Q/+mice express one allele of murine Atxn1 with a 78Q expansion. These mice do not display behavioral abnormalities nor any neuropathology, including ATXN1 inclusions, indicating that a single copy of 78Q-Atxn1 is insufficient to generate disease within the short lifespan of a mouse (Lorenzetti et al., 2000). These characteristics make this mouse an ideal model to determine the seeding abilities of ATXN1 soluble oligomers from Atxn1154Q/+mice lysates.

Figure 1 with 2 supplements see all
ATXN1 oligomers propagate further ATXN1 oligomerization in vivo. 

(A) Representative western blot brain lysates used for in vivo injections. F11G3 was used to detect oligomers (WT and Atxn1154Q/+, cerebellar samples). (B) Table of mouse genotypes and treatments used in the in vivo propagation assay. (C) ELISA for oligomers (F11G3, left panels) and ATXN1 (11750, right panels) was performed on WT, Atxn178Q/+ and Atxn1-/- mice injected with cerebellar lysate (WT or Atxn1154Q/+) in the indicated brain regions. x axis indicated groups from (B) * denotes p<0.05, ANOVA followed by Bonferroni’s post hoc test. (D) Representative histological staining for oligomers (F11G3) in groups indicated in (B) in the cerebellum. Arrowheads indicate the accumulation of oligomers in dendrites, arrows indicate their presence in the soma of PCs. Scale bar 15 μm. (E) Double staining using anti-ATXN1 antibody (green) and anti-oligomer antibody (red) confirmed the presence of ATXN1 oligomers in Purkinje cells of Atxn178Q/+ mice injected with Atxn1154Q/+ cerebellar soluble fraction. Scale bar 15 μm.

https://doi.org/10.7554/eLife.10891.003

Before performing injections, we confirmed by western blot analysis that the injected material from Atxn1154Q/+mouse cerebella was indeed enriched in oligomers (Figure 1A). Nine different groups of mice (each constituting 8 animals) were injected at 3 months of age (Figure 1B). Three months after injection, ELISA revealed that cerebellar extracts from the SCA1 Atxn1154Q/+ mice caused a two-to-three fold increase in oligomer formation in the cerebellum and brain stem of Atxn178Q/+mice compared to other groups (Figure 1C, left panels). Oligomer levels in the cortex, which were lower to begin with than in the cerebellum and brainstem, did not significantly change in any of the groups following injection of cerebellar lysate. Neuropathology confirmed that oligomers were located in cerebellum and brain stem, but not in cortex, hippocampus, striatum or olfactory bulb (data not shown). ATXN1 oligomers thus appear to propagate only to neighboring areas, and, at least under these conditions, trans-synaptic propagation was not detected. Nevertheless, we can’t exclude the possibility that a higher concentration of inoculum and/or a longer incubation time might lead to oligomer propagation to farther areas. In all of the groups of mice tested, we observed similar amounts of total ATXN1 (Figure 1C, right panels), which indicates that the increase of oligomers in the cerebellum and brain stem of Atxn178Q/+mice was produced by a seeding effect of ATXN1-154Q soluble oligomers rather than by inducing more expression of ATXN1-78Q. To evaluate whether or not pathology propagation was triggered by oligomers presented in the injected material from Atxn1154Q/+,we performed a cell-based seeding assay using the injected materials as previously described (Lasagna-Reeves et al., 2015). Specifically, we added the injected material from Atxn1154Q/+ alone or pre-incubated with the anti-oligomer antibody F11G3 to cells that express ATXN1(82Q) fused to mRFP. After a 10 hr incubation, we then quantified the amount of oligomeric inclusions. In comparison with cells incubated with the injected material from wild type or non-treated group, the cells exposed only to the injected material from Atxn1154Q/+ developed the largest proportion of oligomeric inclusions (37.25%). Moreover, when cells were exposed to the injected material from Atxn1154Q/+ pre-incubated with F11G3, the proportion of oligomeric inclusions (15.25%) did not increase in comparison with the control groups thus suggesting that the active material in the injected lysate are the oligomers (Figure 1—figure supplement 1). In addition, this oligomeric pathology did not result from the surgical procedure itself: only PCs of Atxn178Q/+knockin mice injected with cerebellar extract from Atxn1154Q/+ mice, not those injected with WT cerebellar extract, showed an increase in oligomers (Figure 1D). The observation that no oligomers were detected in PCs from either wild-type or Atxn1 null mice injected with Atxn1154Q/+ lysates suggests that host expression of polyQ-expanded ATXN1 is necessary for the de novo formation of ATXN1 oligomers in PCs. Immunofluorescence confirmed that the oligomers detected in Atxn178Q/+PCs were indeed ATXN1 oligomers (Figure 1E). Overall, these data suggest that the ATXN1 oligomers present in the injected material from Atxn1154Q/+ are responsible for inducing propagation of ATXN1 oligomers in Atxn178Q/+mice. Nevertheless, it is possible that other factors present in the inoculum play an important role in promoting propagation. Further, downstream exploration of the precise biochemical composition of these lysates will surely shed insight into this matter.

To determine if oligomer propagation was accompanied by motor deficit, we performed the rotarod assay in mice harboring brain lysate for 3 months. Even though the performance varied during training, there was no evidence of sustained deficit in the Atxn178Q/+knockin mice injected with cerebellar extract from Atxn1154Q/+ mice (Figure 1—figure supplement 2). Despite the fact that oligomers propagate through the cerebellum in Atxn178Q/+knockin mice, the newly formed 78Q ATXN1 oligomers appear insufficient to induce motor deficit or degeneration (data not shown). This lack of toxicity could be because in Atxn178Q/+knockin adult mice, the neurons are relatively healthy and thus can counteract the toxic effect of newly formed oligomers. In contrast, in Atxn1154Q/+mice, the neurons become dysfunctional by four weeks (Lasagna-Reeves et al., 2015) rendering them more susceptible to the toxicity of oligomers. Another possibility is that the newly formed 78Q ATXN1 oligomers adopt a different structure or conformation that is less toxic than the 154Q ATXN1 oligomers. Further studies will be necessary to determine if these newly formed ATXN1 oligomers are solely derived from 78Q ATXN1 molecules or whether they contain some persistent 154Q ATXN1 seeds.

Passive immunotherapy decreases ATXN1 oligomer pathology and improves motor coordination

Given the ability of the anti-oligomer antibody F11G3 to inhibit the internalization of ATXN1 oligomer complexes from Atxn1154Q/+ cerebellum in vitro (Lasagna-Reeves et al., 2015), we postulated that anti-oligomer (F11G3) immunotherapy could reduce the load of ATXN1 oligomers and mitigate the motor impairment observed in the Atxn1154Q/+ model. Because Atxn1154Q/+ mice develop motor incoordination as early as 5 weeks of age (Watase et al., 2002), we began treatment at 4 weeks of age, where Atxn1154Q/+ mice already have a modest accumulation of oligomers in the cerebellum (Figure 2—figure supplement 1). Wild-type and Atxn1154Q/+ mice were injected intraperitoneally with F11G3 or control IgM antibodies (5 mg/Kg) once a week for 6 weeks. One week after the last injection, we performed the rotarod assay, sacrificed the mice and performed pathological and biochemical analyses. Injected mice were separated into two cohorts, one for biochemical and pathological analysis and one for behavioral and survival analysis. We focused our pathological examination on the cerebellum. Brain sections were immunostained with the anti-oligomer antibody, A-11 (Kayed et al., 2003), to ensure depletion of oligomers at the site of interest. Atxn1154Q/+ mice treated with the anti-oligomer antibody showed fewer PCs with oligomers than the control group (Figure 2A and B). We further analyzed the cerebella by ELISA and noted a decrease in the amount of oligomers and ATXN1 (Figure 2C and D). Notably, the immunotherapy did not affect the formation and total number of nuclear inclusions in the cortex (Figure 2E): either these cortical inclusions are not preceded by oligomers or the antibody did not get internalized into these cells in vivo. Considering our previous study where we demonstrated that the anti-oligomer antibody blocks the seeding effect of ATXN1 oligomers in cell culture without getting internalized into the cell (Lasagna-Reeves et al., 2015), we suggest that the anti-oligomer antibody arrests the propagation of ATXN1 oligomer complexes to neighboring areas by targeting extracellular oligomeric entities rather than directly targeting intracellular oligomers. To confirm that the immunotherapy was indeed targeting ATXN1 oligomers, we analyzed the treated and control samples by western blot using F11G3 and anti-ATXN1 antibody (Figure 3A and B). The western blot analysis revealed a decrease in ATXN1 oligomers in the treated group, but no observable change at the level of monomeric ATXN1 (Figure 3A and B).

Figure 2 with 1 supplement see all
Anti-oligomer immunotherapy decreases pathology in vivo.

(A) Histological staining of PCs (Calbindin, top panels) and Oligomers (A11, bottom panel) of control (IgM) and treated (F11G3) Atxn1154Q/+ mice. Adjacent sections were used for comparison. (B) Quantification of (A), showing the percentage of PCs with oligomers. Data are represented as mean ± SEM., and ** denotes p<0.01, Student’s T-test. (C) ELISA for oligomer levels (A11) in the cerebellum of treated mice and the control group. Data are represented as mean ± SEM., and ** denotes p<0.01, Student’s T-test. (D) ELISA for ATXN1 (11750) levels in the cerebellum of treated mice and the control group. Data are represented as mean ± SEM., and ** denotes p<0.01, Student’s T-test. (E) Immunotherapy in the cortex of Atxn1154Q/+ mice produced no significant change in the number of cells with nuclear inclusions (NIs, stained with 11750 antibody).

https://doi.org/10.7554/eLife.10891.006
Anti-oligomer immunotherapy improves motor deficits and survival in vivo.

(A–B) Western blot detecting ATXN1 oligomers (F11G3, top panel) and ATXN1 monomer (11750, middle panel) in the cerebellum of Atxn1154Q/+following immunotherapy (anti-oligomer or control). * in (A) indicated change in exposure of the membrane. (B) Quantification of relative levels of oligomeric and monomeric ATXN1 from (A). ** denotes p<0.01, Student’s T-test. (C) Rotarod assay in all treatment groups over a four-day period (four trials per day, averaged) 6 weeks following onset of immunotherapy (mice 10 weeks of age). n = 12 per genotype; ** denotes p<0.01, ANOVA followed by Tukey’s post hoc test. (D) Kaplan-Meier survival curve shows that animals treated with anti-oligomer immunotherapy (blue line) lived, on average, 3.5 weeks longer than control animals (red line). No death was observed in WT mice receiving immunotherapy (black and grey lines). *** denotes p<0.001, Log-rank (Mantel-Cox) test. n = 12 per genotype.

https://doi.org/10.7554/eLife.10891.008

To determine the functional consequences of this immunotherapy, we tested motor performance in treated and control Atxn1154Q/+ mice. Rotarod analysis revealed a mildly significant improvement in coordination following six weeks of anti-oligomer immunotherapy (Figure 3C). Although treated mice still possessed a motor deficit in comparison with wild type mice, continued weekly immunotherapy treatments in Atxn1154Q/+ mice throughout their lifespan extended survival by approximately 3.5 weeks in comparison with the control-treated Atxn1154Q/+ mice (Figure 3D).

Discussion

Our previous study showed that ATXN1 oligomeric complexes were able to penetrate cells in culture and seed the formation of new ATXN1 oligomers (Lasagna-Reeves et al., 2015). It is widely accepted that amyloid formation is characterized by such seeding (Serio et al., 2000; Pedersen et al., 2004). This phenomenon has been explored in cell culture assays, several of which have demonstrated that extracellular aggregates that are internalized into the cell induce aggregation of intracellular proteins (Frost et al., 2009; Munch et al., 2011; Holmes and Diamond, 2012). Moreover, the concept of cell-to-cell spread of many amyloidogenic proteins in vivo has been demonstrated by the progression of protein aggregate pathology from one brain region to another in a disease-specific pattern (Hardy and Revesz, 2012; Holmes and Diamond, 2012). In our study, however, we found that injected oligomers induced formation of new ATXN1 oligomers only in areas proximal to the injection site. It thus seems unlikely that ATXN1 oligomers propagate transsynaptically in SCA1, as has been suggested with other neurodegenerative diseases (Harris et al., 2010; de Calignon et al., 2012; Liu et al., 2012; Pecho-Vrieseling et al., 2014). Rather, we suggest ATXN1 propagates through a secretion and reuptake mechanism between neighboring cells as has been proposed for other proteinopathies (Guo and Lee, 2014). Furthermore, no obvious signs of degeneration or motor incoordination were observed in the injected mice. This suggests that the cell must continuously express the toxic entity to trigger degeneration and behavioral deficits in mice. This notion is supported by the observation that immunotherapy targeting the ATXN1 oligomers slightly modified the phenotype in the Atxn1154Q/+ mice: the antibody hindered the local propagation of ATXN1 oligomers. This treatment is not curative, however, because neurons expressing polyQ ATXN1 will still form their own toxic oligomeric entities, and some non-oligomeric forms of PolyQ ATXN1 might contribute to toxicity.

Passive immunotherapy has been proposed as a feasible strategy to inhibit pathogenesis in mouse models for AD and PD (Banks et al., 2007; Chai et al., 2011; Masliah et al., 2011; Yanamandra et al., 2013; Castillo-Carranza et al., 2014; Games et al., 2014). Our findings suggest that such therapy might slightly impact disease progression by blocking the propagation of these oligomeric entities, but to halt or reverse symptoms it would likely be necessary to incorporate a joint therapy that targets the root cause of disease, the abnormal accumulation of polyQ ATXN1 (Park et al., 2013). Altogether, our findings revealed that ATXN1 oligomer propagation contributes to SCA1 pathogenesis, and that such propagation especially in the vicinity of affected cells, could be targeted through passive immunotherapy. That the benefit from such therapy was small highlights the need to develop therapies that target ATXN1, the protein driving the pathogenesis. The need for such combination therapy is likely to extend to other disease-driving proteins.

Materials and methods

Experimental procedures

Mouse models and preparation of brain extracts

Request a detailed protocol

All mouse procedures were approved by the Institutional Animal Care and Use Committee for Baylor College of Medicine and Affiliates. Atxn1154Q/+,Atxn178Q/+ and Atxn1-/- mice have been previously described (Lorenzetti et al., 2000; Watase et al., 2002) and were backcrossed to C57BL/6 for more than ten generations. Mouse cerebella were dissected and lysed in 0.5% Triton buffer (0.5% Triton X-100, 50 mM Tris pH 8, 75 mM NaCl) supplemented with protease and phosphatase inhibitors (Sigma, St-Louis, Mo). The protein lysate was then incubated on ice for 20 min and centrifuged at 13,200 r.p.m. for 10 min at 4°C, and the supernatants were portioned into aliquots, snap-frozen, and stored at -80°C until used.

Rotarod assay

View detailed protocol

Motor coordination was assessed on the Rotarod assay as previously described (Park et al., 2013), with four trials a day (separated by 1 hr each) for 4 days. The tester was blinded to animal genotype and treatment.

Immunotherapy

Request a detailed protocol

We used F11G3 and a control mouse IgM as antibodies for immunotherapy. Antibodies were administered at 5 mg/kg via intraperitoneal (i.p) injection once a week for six weeks. One week after completion of the treatment 12 mice per group were tested on the rotarod assay and sacrificed immediately afterward so that brains could be collected for biochemical and histopathological analysis. For survival studies, 12 mice per group were vaccinated once a week (5 mg/Kg) throughout their lifespan.

Brain sections immunofluorescence

Request a detailed protocol

Paraffin sections were deparaffinized, rehydrated, and washed in 0.01 M PBS 3 times for 5 min each time. After blocking in normal goat serum for 1 hr, sections were incubated overnight with rabbit anti-ATXN1 antibody 11750 (1:700). The next day, the sections were washed in PBS 3 times for 10 min each and then incubated with goat anti-rabbit IgG Alexa Fluor 568 (1:700; Invitrogen) for 1 hr. The sections were then washed 3 times for 10 min each time in PBS before incubation overnight with mouse anti-oligomers F11G3 (1:300). The next day, the sections were washed in PBS 3 times for 10 min each before incubation with goat anti-IgM Alexa Fluor 488 (1:700; Invitrogen) for 1 hr. Sections were washed and mounted in Vectashield mounting medium with DAPI (Vector Laboratories). The sections were examined using a Zeiss LSM 710 confocal microscope.

Immunohistochemistry

Request a detailed protocol

IHC was performed on paraffin-embedded sections. In brief, sections (5 μm) were deparaffinized and rehydrated. Primary antibodies were detected with biotinylated goat anti-mouse IgG (1:2000; Jackson ImmunoResearch Laboratories), biotinylated goat anti-mouse IgM (1:1500), or biotinylated goat anti-rabbit IgG (1:1800) (all from Jackson ImmunoResearch Laboratories) and visualized using an ABC reagent kit (Vector Laboratories, Burlingame, CA), according to the manufacturer’s recommendations. Bright-field images were acquired using a Carl Zeiss Axio Imager M2 microscope, equipped with an Axio Cam MRc5 color camera (Carl Zeiss, Oberkochen, Germany). Sections were counterstained with hematoxylin (Vector Laboratories) for nuclear staining. The following antibodies were used for immunostaining: rabbit anti-oligomer antibody A-11 (1:600), mouse anti-oligomer antibody F11G3 (1:100), and mouse anti-calbindin antibody (1:450).

Stereotaxic surgery

Request a detailed protocol

Atxn178Q/+, wild type or null Atxn1 mice were anaesthetized with a mixture of ketamine (110 mg/kg body weight) and xylazine (20 mg/kg body weight) in saline. Bilateral stereotaxic injections of 2.5 μl brain extract (3.9 μg/μL) from Atxn1154Q/+or Wild Type mice were placed with a Hamilton syringe into the cerebellum (From Bregma; Post -6.0 mm, lat +/- 2.0 mm, dv -2.2 mm). Injection speed was 1.25 μl/minute and the needle was kept in place for an additional 2 min before it was slowly withdrawn. The surgical area was cleaned with sterile saline, the incision was sutured, and the mice were monitored until recovery from anesthesia. If not otherwise stated, 8 mice/group were used. Injected material was obtained from mouse brain extracts prepared from the cerebellum of aged 30-week-old Atxn1154Q/+ and age-matched, non-transgenic wild-type control mice. All animal experiments were in compliance with protocols approved by the local Animal Care and Use Committee.

Cell-based seeding assay

Request a detailed protocol

A stable Daoy mRFP-ATXN1(82Q) cell line was generated as previously described (Park et al., 2013). Cells were plated in 24-well plates (2*104 cells/ml). 24 hr later, cells were treated with 2.5 μl brain extract (3.9 μg/μL) from Atxn1154Q/+or Wild Type mice. After 10 hr of treatment, cells were fixed with methanol at −80˚C for 45 min. RFP-positive inclusions ranging from 350 to 900 nm were considered oligomeric; inclusions larger than 900 nm and not detected by F11G3 were considered fibrillar. One hundred cells were quantified per group in triplicates. Analyses were manually performed with Image J. For blocking assays, each sample was mixed with F11G3 antibody (2.5 mg/ml) in a ratio 1:1 (vol/vol) for 1 hr and then added to the cells.

ELISA

Request a detailed protocol

For ELISA, plates were coated with 10 μl of the soluble fraction of brains using 0.1 M sodium bicarbonate (pH 9.6) as a coating buffer, followed by incubation for 1 hr at 37°C, washing three times with Tris-buffered saline with very low (0.01%) Tween 20 (TBS-T), and then blocking for 1 hr at 37°C with 10% BSA. The plates were then washed three times with TBS-T; F11G3 (1:500), A-11 (1:1000), 11750 (1:2000) or Tubulin (1:2000) antibodies (diluted in 5% nonfat milk in TBS-T) were added and allowed to react for 1 hr at 37°C. The plates were then washed three times with TBS-T, and 100 μl of horseradish peroxidase-conjugated anti-mouse IgM, anti-mouse IgG or anti-rabbit IgG (diluted 1:10,000 in 5% nonfat milk in TBS-T; Promega, Madison, WI) were added, followed by incubation for 1 hr at 37°C. Finally, plates were washed 3 times with TBS-T and developed with 3,3,5,5-tetramethylbenzidine (TMB-1 component substrate) from KPL (Gaithersburg, MD). The reaction was stopped with 100 μl of 1 M HCl, and samples were read at 450 nm.

Analysis of ATXN1 pathology

Request a detailed protocol

To determined the percent of PCs with ATXN1 oligomers, 5 μm brain sections were stained with anti-Calbindin antibody as described above to quantify the total number of PCs in the cerebellum. The number of PCs positive for Calbindin was consider as the 100%. The adjacent sections were immunostained for oligomers using F11G3 or A-11. For the quantification of nuclear inclusions, we stained ATXN1 using 11750 antibody and performed nuclear staining with hematoxylin. We considered the total amount of nuclei in the cortex as the 100% of nucleus. Data were analyzed using post-hoc test.

Statistical analysis

Request a detailed protocol

Experimental analysis and data collection were performed in a blinded fashion. p-values were determined using the appropriate statistical method via GraphPad Prism, as described throughout the manuscript. For simple comparisons, Student’s t-test was used. For multiple comparisons, ANOVA followed by the appropriate post hoc analysis were utilized. All data is presented as mean ± SEM. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
    A native interactor scaffolds and stabilizes toxic ATAXIN-1 oligomers in SCA1
    1. CA Lasagna-Reeves
    2. MWC Rousseaux
    3. MJ Guerrero-Muñoz
    4. J Park
    5. P Jafar-Nejad
    6. R Richman
    7. N Lu
    8. U Sengupta
    9. A Litvinchuk
    10. HT Orr
    11. R Kayed
    12. HY Zoghbi
    (2015)
    eLife, 4, 10.7554/eLife.07558.
  13. 13
  14. 14
  15. 15
  16. 16
    Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells
    1. C Munch
    2. J O'Brien
    3. A Bertolotti
    (2011)
    Proceedings of the National Academy of Sciences of the United States of America 108:3548–3553.
    https://doi.org/10.1073/pnas.1017275108
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23

Decision letter

  1. Bart De Strooper
    Reviewing Editor; VIB Center for the Biology of Disease, KU Leuven, Belgium

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Ataxin-1 oligomers induce local spread of pathology and decreasing them by passive immunization slows SCA1 phenotypes" for peer review at eLife. Your submission has been favorably evaluated by a Senior editor, a Reviewing editor, and three reviewers.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

This is a timely and intriguing follow up to the author's recent paper showing that oligomers are primary drivers of toxicity. Here the authors test whether injected oligomers can drive regional propagation of SCA1 pathology and whether passive immunotherapy can reverse disease phenotypes. The authors show data that oligomers can propagate within a localized region and that this propagation is limited only to neighboring areas. Immunotherapy results provide some indication that this approach could have limited efficacy. The major points are that for ataxin-1, propagation does not appear to be the major driver of toxicity, therefore immunotherapy against extracellular oligomers would require additional therapeutic strategies in combination. This is a very important point relating to treatment that passive immunotherapy may not be curative because neurons will form toxic oligomeric entities internally and that one would need to target abnormal accumulation of polyQ ATXN1.

Overall some type of more direct test of either the injected oligomers being the driver of seeding or whether immunotherapy impacts oligomers directly versus reducing mutant ATXN1 levels would have been helpful. We would welcome any additional experimental back up to address the major criticisms mentioned below but consider it crucial that the authors indicate very clearly the limitations of the work in their final manuscript by addressing in their revised text the questions raised in the essential revisions section.

Essential revisions:

1) Cerebellar extracts from control or Atxn1 154Q/+ mice were injected into Atxn1 78Q/+ or wild type mice and limited spreading to neighboring areas within the cerebellum and brain stem was detected. The authors state that the fact ATXN1 levels were the same indicates that oligomers were induced by the seeding effect of ATXN1-154Q soluble oligomers. It would have been helpful to use similar approaches as used in the initial published studies to deplete lysates of ataxin-1 (using oligomer antibody) to show that the effect was directly a consequence of oligomers, but minimally the previous study and discussion of this depletion experiment should be discussed. There are other factors that could provide this effect including inflammatory cytokines or other external (and limiting) factors. Further, it is not mentioned until the Discussion whether this localized spreading had an effect on behavior or neurodegeneration in the Atxn1 78Q/+ mice and should be discussed in the Results.

2) The authors perform passive immunotherapy with F11G3 using IP injection in 154Q/+ mice. Decreased oligomers and ATXN1 were observed in cerebella. What is status at 4 weeks when injected? The authors postulate that the antibody targets extracellular oligomeric entities. This experiment could also be done in the 78Q/+ mice using the conditions with injected lysates. This would provide a direct test of whether immunotherapy targeted the oligomers.

3) Could the phenotypic effect of the passive immunity experiment be due to the decrease of ATXN1 (panel 2D), not the oligomers since increasing the oligomers in the 78Q background does not result in a phenotype?

4) It is briefly mentioned that the increase in oligomers in the 78Q background post-injection is not associated with neurodegeneration or motor incoordination. The authors interpret this to mean that the "toxic entity" (presumably the 154Q) needs to be continuously expressed to observe a phenotype. As the 78Q expansion is abnormal yet insufficient to cause disease in the mouse, presumably due to their short lifespan (subsection “ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo”, first paragraph), seeding the formation of oligomers was insufficient to increase the pathogenicity of this abnormal repeat as hypothesized by the authors. This would call into question the true pathogenicity of the oligomers if their presence does not alter phenotype. Can the authors comment on whether pure 78Q oligomers are likely formed following seeding from the 154Q lysate or whether all the oligomers likely contain one or more 154Q molecules? If pure 78Q oligomers are formed post-seeding, then why is there no phenotype? If the 78Q is simply insufficient to cause disease, does injection of the oligomers back into 154Q itself worsen that phenotype?

https://doi.org/10.7554/eLife.10891.009

Author response

Essential revisions:

1) Cerebellar extracts from control or Atxn1 154Q/+ mice were injected into Atxn1 78Q/+ or wild type mice and limited spreading to neighboring areas within the cerebellum and brain stem was detected. The authors state that the fact ATXN1 levels were the same indicates that oligomers were induced by the seeding effect of ATXN1-154Q soluble oligomers. It would have been helpful to use similar approaches as used in the initial published studies to deplete lysates of ataxin-1 (using oligomer antibody) to show that the effect was directly a consequence of oligomers, but minimally the previous study and discussion of this depletion experiment should be discussed. There are other factors that could provide this effect including inflammatory cytokines or other external (and limiting) factors. Further, it is not mentioned until the Discussion whether this localized spreading had an effect on behavior or neurodegeneration in the Atxn1 78Q/+ mice and should be discussed in the Results.

In our previous study, we pre-incubated F11G3 with Atxn1154Q/+ brain homogenate to demonstrate that the seeding effect was a direct consequence of ATXN1 oligomers in the brain (please refer to Figure 3A in (Lasagna-Reeves et al., 2015)). In the current study, using this same strategy, we performed a cell-based seeding assay using the Atxn1154Q/+ brain homogenate (injected material) alone or pre-incubated with F11G3 antibody. Pre-incubating the Atxn1154Q/+ brain homogenate with F11G3 completely abolished the seeding nature of the homogenate (Figure 1—figure supplement 1; subsection “ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo”, second paragraph). This suggests that the seeding/propagation observed in vivo is due to the oligomers present in the injected material. Nevertheless, in the manuscript we acknowledge that other factors in the injected material such as inflammatory responses could also play a role in propagation. We also discussed the necessity to perform future studies where lysate depleted of ATXN1 oligomers are injected in Atxn178Q/+ mice.

With regards to whether the localized spreading had an effect on behavior in Atxn78Q/+ mice, we performed Rotarod analysis to test for the presence of motor coordination defects in these mice (Figure 1—figure supplement 2). We found that – consistent with our pathological findings – only mild and unsustained defects were observed in the Atxn78Q/+ mice injected with Atxn1154Q/+ brain homogenate (in the last paragraph of the aforementioned subsection).

2) The authors perform passive immunotherapy with F11G3 using IP injection in 154Q/+ mice. Decreased oligomers and ATXN1 were observed in cerebella. What is status at 4 weeks when injected? The authors postulate that the antibody targets extracellular oligomeric entities. This experiment could also be done in the 78Q/+ mice using the conditions with injected lysates. This would provide a direct test of whether immunotherapy targeted the oligomers.

Our new data demonstrate that ATXN1 oligomers are already present in Atxn1154Q/+ mice cerebellum at 4 weeks of age. Nevertheless the amount of oligomers at this age is modest in comparison with Atxn1154Q/+ mice at 11 weeks of age (Figure 2—figure supplement 1; subsection “Passive immunotherapy decreases ATXN1 oligomer pathology and improves motor coordination”, first paragraph).

We agree with the reviewers’ observation regarding the administration of F11G3 in Atxn78Q/+ mice after lysate injection in order to provide direct test of whether immunotherapy targeted oligomers. However, this experiment would take several months as we would have to restart essentially all experiments (to have the proper littermate and age matched controls). Thus, to answer this question as a proxy, we showed that immunotherapy using F11G3 in Atxn1154Q/+ mice specifically targets ATXN1 oligomers but not monomeric ATXN1 (Figure 3A and B; in the aforementioned paragraph). These data lend further support that the effects observed throughout these experiments are from the immunotherapy specifically targeting ATXN1 oligomers.

3) Could the phenotypic effect of the passive immunity experiment be due to the decrease of ATXN1 (panel 2D), not the oligomers since increasing the oligomers in the 78Q background does not result in a phenotype?

Our new data demonstrate how the immunotherapy in Atxn1154Q/+ mice specifically targets ATXN1 oligomers but not monomeric ATXN1 (Figure 3A and B; subsection “Passive immunotherapy decreases ATXN1 oligomer pathology and improves motor coordination”, first paragraph). Therefore, the decrease of total ATXN1 levels measured by ELISA (Figure 2D) is mainly due to a decrease in the levels of ATXN1 oligomers.

Regarding the fact that no phenotype is observed in the Atxn178Q/+ mice injected with Atxn1154Q/+ brain homogenate; we added new comments in the Results section (subsection “ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo”, last paragraph). Furthermore, we address this point in more detail in question 4.

4) It is briefly mentioned that the increase in oligomers in the 78Q background post-injection is not associated with neurodegeneration or motor incoordination. The authors interpret this to mean that the "toxic entity" (presumably the 154Q) needs to be continuously expressed to observe a phenotype. As the 78Q expansion is abnormal yet insufficient to cause disease in the mouse, presumably due to their short lifespan (subsection “ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo”, first paragraph), seeding the formation of oligomers was insufficient to increase the pathogenicity of this abnormal repeat as hypothesized by the authors. This would call into question the true pathogenicity of the oligomers if their presence does not alter phenotype. Can the authors comment on whether pure 78Q oligomers are likely formed following seeding from the 154Q lysate or whether all the oligomers likely contain one or more 154Q molecules? If pure 78Q oligomers are formed post-seeding, then why is there no phenotype? If the 78Q is simply insufficient to cause disease, does injection of the oligomers back into 154Q itself worsen that phenotype?

At this time, we cannot ascertain the exact nature of the oligomeric entities in the brains of injected animals without proper genetic tools (such as having differentially tagged knockin mice for Atxn1154Q/+ and Atxn178Q/+ and probing for their respective tags biochemically). Nevertheless, we comment in the results section whether pure 78Q ATXN1 oligomers are likely formed following seeding from the Atxn1154Q/+ brain lysate or whether all the oligomers likely contain one or more 154Q ATXN1 molecules (subsection “ATXN1 oligomers seed the formation of new endogenous ATXN1 oligomers in vivo”, last paragraph). In the same section, we also discussed that the newly formed oligomers are insufficient to cause disease, possibly either because the healthy neuronal environment in Atxn178Q/+ mice can counteract the toxic effect of these oligomers or because they are structurally different from 154Q toxic oligomers.

We have not had to opportunity to inject oligomers into Atxn1154Q/+ mice yet given their sensitivity to stereotaxic surgery. However, considering the abundance of published studies from several proteinopathies indicating that seeding inoculate into presymptomatic mice accelerates neurodegeneration (Guo and Lee, 2014), we believe that the injected material would accelerate and perhaps worsen phenotypes in Atxn1154Q/+ mice.

https://doi.org/10.7554/eLife.10891.010

Article and author information

Author details

  1. Cristian A Lasagna-Reeves

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    CAL-R, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  2. Maxime WC Rousseaux

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    MWCR, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  3. Marcos J Guerrero-Munoz

    Department of Neurology, University of Texas Medical Branch, Galveston, United States
    Contribution
    MJG-M, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  4. Luis Vilanova-Velez

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    LV-V, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  5. Jeehye Park

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    JP, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  6. Lauren See

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    LS, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  7. Paymaan Jafar-Nejad

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    PJ-N, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  8. Ronald Richman

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    3. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States
    Contribution
    RR, Performed experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  9. Harry T Orr

    Institute for Translational Neuroscience, University of Minnesota, Minnesota, United States
    Contribution
    HTO, Assisted in design and interpretation of experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  10. Rakez Kayed

    Department of Neurology, University of Texas Medical Branch, Galveston, United States
    Contribution
    RK, Assisted in design and interpretation of experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  11. Huda Y Zoghbi

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    3. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States
    4. Department of Neuroscience, Baylor College of Medicine, Houston, United states
    Contribution
    HYZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    hzoghbi@bcm.edu
    Competing interests
    HYZ: Senior editor, eLife.

Funding

Howard Hughes Medical Institute

  • Huda Y Zoghbi

Robert and Renee E Belfer Family Foundation

  • Cristian A Lasagna-Reeves
  • Maxime WC Rousseaux
  • Paymaan Jafar-Nejad

National Institute of Neurological Disorders and Stroke (R01 NS027699-17)

  • Jeehye Park
  • Huda Y Zoghbi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the members of the Zoghbi, Orr and Kayed laboratories for suggestions and discussions, and V Brandt for critical reading of the manuscript. This work was supported by a Howard Hughes Medical Institute Collaborative Innovation Awards grant, the Robert A and Renee E Belfer Family Foundation and grant NIH/NINDS R01 NS027699-17. The NIH/NINDS 3R01 NS027699-25S1 and 1K22NS092688-01 to CALR. MWCR gratefully acknowledges The Canadian Institutes of Health Research Fellowship (201210MFE-290072-173743). We also appreciate the assistance of the confocal microscopy and mouse behavioral cores of the Baylor College of Medicine (BCM) Intellectual and Developmental Disabilities Research Center (1U54 HD083092).

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocols (#AN-1013) of Baylor College of Medicine

Reviewing Editor

  1. Bart De Strooper, VIB Center for the Biology of Disease, KU Leuven, Belgium

Publication history

  1. Received: August 17, 2015
  2. Accepted: December 15, 2015
  3. Accepted Manuscript published: December 17, 2015 (version 1)
  4. Version of Record published: March 23, 2016 (version 2)

Copyright

© 2015, Lasagna-Reeves et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,209
    Page views
  • 391
    Downloads
  • 9
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Qi Yang et al.
    Research Article

    The Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    İbrahim Avşar Ilik et al.
    Research Article

    The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (Banani et al., 2017; Shin and Brangwynne, 2017). First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (Spector and Lamond, 2011). Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified (Chen and Belmont, 2019; Galganski et al., 2017). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (Fu and Maniatis, 1990). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large (~300 kDa), spliceosome-associated (Jia and Sun, 2018) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (Blencowe et al., 1994). Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS as reported previously (Ahn et al., 2011; Fei et al., 2017; Sharma et al., 2010), in contrast, combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.