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RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration

Abstract

In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity1. Reduction in synapse number is a consistent early feature of neurodegenerative diseases2,3, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders2,3, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity4,5. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice7, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders.

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Figure 1: The capacity for synaptic regeneration is lost early in neurodegenerative disease.
Figure 2: Failure to induce RBM3 parallels lost capacity for synaptic recovery in neurodegenerative disease models.
Figure 3: Early cooling induces RBM3 overexpression and is neuroprotective in prion-infected mice.
Figure 4: Lentivirally mediated overexpression of RBM3 restores structural synaptic plasticity and is neuroprotective in neurodegenerative disease.

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Acknowledgements

We thank J. Edwards, J. McWilliam and D. Read of the MRC Toxicology Unit; members of University of Leicester Department of Biological Services staff; and D. Morrison, University of Cambridge, for technical assistance. We thank J. Skepper, University of Cambridge, for advice about stereological procedures. This work was funded by the Medical Research Council, UK.

Author information

Affiliations

Authors

Contributions

D.P. performed most experimental procedures and analyses. A.B. analysed cold-shock proteins, N.V. and M.G.M. carried out prion inoculations and stereotaxic injections, C.M. performed behavioural tests, J.A.M. and H.R. carried out histological analyses, J.R.S. performed neurophysiological procedures, M.G.M. and T.S. performed ultramicrotomy and processed samples for electron microscopy, which was analysed by D.D. A.E.W. provided expertise on cold-shock and protein expression. G.R.M. conceived and directed the project. D.P. and G.R.M. wrote the paper. All authors contributed to discussion and analysis of data and to the final draft of the paper.

Corresponding author

Correspondence to Giovanna R. Mallucci.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Stereological assessment of volume and synapse size to validate 2D assumption-based approaches for counting synapse density.

a, CA1 volume and synapse mean length and area in the stratum radiatum remain essentially unchanged on cooling and rewarming in wild-type mice. Volume was measured using disector principle and synapse mean length and area determined in the same sections, as described1, n values as reported for Fig. 1a. b, c, Representative electron micrographs (pseudo-coloured for ease of synapse identification) for data not shown in Fig. 1b, c, from prion-infected mice at 4 and 6 w.p.i. (b) and for 5XFAD mice at 2 and 3 months (c) before cooling (black framed images) and cooled (blue framed images). d, Schematic showing lost capacity for structural plasticity precedes synapse loss and neuronal loss in both mouse models. Scale bar, 1 μm. All data in bar charts are mean ± s.e.m. Student’s t-test, two tailed. Non-significant P values.

Source data

Extended Data Figure 2 Synaptic protein levels during cooling–rewarming in prion and 5XFAD mice.

a, Levels of presynaptic (SNAP25, VAMP2) and postsynaptic (PSD95, NR1) proteins do not change before (black bars) and after cooling to 16–18 °C (blue bars) in prion-infected mice at 4 and 6 w.p.i., b, 5XFAD mice at 2 and 3 months. Representative western blots are shown for 3 mice per temperature and time point. Bar graphs show quantification of synaptic protein levels relative to GAPDH. All data represent means ± s.e.m. (n = 3–11 mice per time point). Student’s t-test, two tailed. n.s. = non-significant P values.

Source data

Extended Data Figure 3 Cooling does not induce changes in PrPSc or amyloid-β levels.

a, Levels of total PrP (upper blot) and PrPSc (lower blot) do not change notably before (white line), during (blue line) or after (red line) cooling to 16–18 °C in prion-infected mice. PrPSc is detected after digestion with proteinase K. Levels are undetectable by western bloting at 6 w.p.i., as expected. b, Cooling does not change levels of amyloid-β oligomers in 5XFAD mice, arrow indicates amyloid-β monomers (lane 1, synthetic amyloid-β oligomers; last lane, one-year-old 5XFAD control (C+)). Representative western blots are shown for 3 mice per temperature and time point. Non-significant P values.

Extended Data Figure 4 Cooling induces sustained increase in RBM3 levels but not in CIRP.

a, b, Levels of CIRP do not change after cooling in prion-infected mice at 4 and 6 w.p.i. (a) or in 5XFAD mice at 2 and 3 months (b). Representative western blots are shown for 3 mice per temperature and time point. Bar graphs show quantification of CIRP levels relative to GAPDH. All data represent means ± s.e.m. (n = 6–9 mice per time point). Student’s t-test, two tailed. n.s. = non-significant P values. c, Increased levels of RBM3 are sustained for at least 72 h after cooling in wild-type mice. Bar graph shows quantification of RMB3 against GAPDH in control (white bar), cooled (blue bar), and 12, 48 and 72 h recovery after cooling (red bars). All data represent means ± s.e.m., (n = 3–6 mice per time points, P < 0.05, Mann–Whitney U-test, two tailed).

Source data

Extended Data Figure 5 Early cooling induces sustained elevation of RBM3 levels.

RBM3 levels remain high after cooling to 16–18 °C in prion-infected mice (magenta boxes) compared to control prion-infected mice. These levels remained high up to 6 weeks later and declined at 12 w.p.i. Representative western blots are shown for 3 mice per time point.

Extended Data Figure 6 Exploration time in exposure phase of novel object testing is normal in all groups and RBM3 knockdown abolishes improved memory after cooling.

a, Exploratory behaviour measured in seconds is not different in mice with early cooling from prion-diseased mice and is not affected by the duration of disease (n as reported in Fig. 3d). b, c, Lentivirally mediated RNAi of RBM3 eliminates the protective effect of cooling on novel object memory impairment in prion disease (b) (dark green bar); but does not affect exploratory behaviour in training phase (c). All data represent means ± s.e.m. Data analysed using one way ANOVA, Brown–Forsythe test with Tukey’s post hoc analysis for multiple comparisons (n = 11–16 mice per time point, P < 0.01).

Source data

Extended Data Figure 7 Induction of hypothermia at time point when RBM3 induction fails is not neuroprotective.

Cooling at 5 and 6 w.p.i., when synaptic plasticity and RBM3 induction fails (see Fig. 1 and 2, main text), does not increase survival in prion-infected mice. Kaplan–Meier survival plots for prion-infected mice (black line, no cooling; n = 10; orange line, mice cooled at 5 and 6 w.p.i., n = 16). Student’s t-test, two tailed. Non-significant P values.

Source data

Extended Data Figure 8 PrPSc levels remain unchanged in prion with overexpression of RMB3.

a, b, In prion-infected mice total PrP and PrPSc levels do not alter after early cooling to 16–18 °C (a) (magenta boxes) or following treatment with LV-RBM3 (dark green) and LV-shRNA-RBM3 (pale green) (b). PrP and PrPSc levels tested in 9 w.p.i. and terminal mice. PrPSc is detected after digestion with proteinase K. Representative western blots are shown for 3 mice per temperature and time point, the lane marked C shows uninfected control mouse.

Extended Data Figure 9 Mild hypothermia also extends survival in prion-infected mice.

Kaplan–Meier plot showing that cooling to 26 °C at an early stage also significantly lengthens survival (n = 27 cooled vs n = 16 non-cooled mice); P < 0.01, Student’s t-test, two tailed.

Source data

Extended Data Figure 10 RNAi of RBM3 downregulation accelerates impaired structural synaptic plasticity in the 5XFAD mouse model, and also reduced synapse number and function in wild-type mice.

a, Impaired structural synaptic plasticity after cooling occurs in shRNA-RBM3 treated 5XFAD mice at 3 months. Representative electron micrographs are shown and are pseudo-coloured as in main text figures. Quantification shows significant reduction in synapse number by RNAi of RBM3 (n = 82–93 images from 3 mice per time point, Student’s t-test, two tailed). b, RBM3 knockdown reduces synapse number and novel object memory in wild-type mice (n = 93 images from 3 mice per time point, Student’s t-test, two tailed P < 0.0001; for novel object recognition task n = 11 mice, LV-shRNA-control and 10 mice, LV-shRNA-RBM3, Mann–Whitney U-test, P < 0.05). Scale bar, 1 μm.

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Peretti, D., Bastide, A., Radford, H. et al. RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration. Nature 518, 236–239 (2015). https://doi.org/10.1038/nature14142

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