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Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency

Nature Neuroscience volume 17, pages 223–231 (2014)Cite this article

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Abstract

Amyloid-induced microglial activation and neuroinflammation impair central synapses and memory function, although the mechanism remains unclear. Neuroligin 1 (NLGN1), a postsynaptic protein found in central excitatory synapses, governs excitatory synaptic efficacy and plasticity in the brain. Here we found, in rodents, that amyloid fibril–induced neuroinflammation enhanced the interaction between histone deacetylase 2 and methyl-CpG-binding protein 2, leading to suppressed histone H3 acetylation and enhanced cytosine methylation in the Nlgn1 promoter region and decreased NLGN1 expression, underlying amyloid-induced memory deficiency. Manipulation of microglia-associated neuroinflammation modulated the epigenetic modification of the Nlgn1 promoter, hippocampal glutamatergic transmission and memory function. These findings link neuroinflammation, synaptic efficacy and memory, thus providing insight into the pathogenesis of amyloid-associated diseases.

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Figure 1: Epigenetic suppression of NLGN1 underlies amyloid fibril–induced memory deficiency.
Figure 2: Enhanced HDAC2 activity was involved in amyloid fibril–suppressed NLGN1 expression.
Figure 3: Amyloid fibrils induced functional modification of MeCP2 activity in hippocampal CA1 tissue.
Figure 4: Suppression of MeCP2 activity ameliorated amyloid fibril–suppressed NLGN1 expression and memory.
Figure 5: LPS-induced suppression of NLGN1 expression.
Figure 6: LPS impaired hippocampal glutamatergic transmission and memory in naive rats.
Figure 7: Microinjection of minocycline (5 μg per side) ameliorated amyloid fibril–suppressed NLGN1 expression.
Figure 8: Microinjection of minocycline restored hippocampal glutamatergic transmission and memory impaired by amyloid fibrils.

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Acknowledgements

Support of the Lerner Research Institute at the Cleveland Clinic.

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Author notes

  1. Bihua Bie and Jiang Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of General Anesthesiology, Anesthesiology Institute, Cleveland Clinic, Cleveland, Ohio, USA

    Bihua Bie, Jiang Wu, Hui Yang, Jijun J Xu, David L Brown & Mohamed Naguib

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Contributions

B.B. and J.W. performed most of the experiments and contributed to a draft of the manuscript. J.W. performed all behavioral testing. H.Y. and J.J.X. conducted some of the molecular experiments. D.L.B. and M.N. designed and directed the project, reviewed the experimental data, performed data analyses and wrote the final manuscript. All authors commented on the final version of the manuscript.

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Correspondence to Mohamed Naguib.

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Integrated supplementary information

Supplementary Figure 1 Amyloid fibrils induced memory deficiency and impaired glutamatergic transmission in the hippocampal CA1 neurons.

Significantly extended escape latency (a, n = 10 rats in each group, F(2,27) = 17.0, P<0.0001) and less time spent in the target quadrant (b, n = 10 rats in each group, F(2,27) =8.6, P = 0.001) were observed in the rats microinjected with Aβ1-40 fibrils, but not Aβ40-1 fibrils nor artificial CSF (control). Significantly attenuated input (stimulus intensity) - output (EPSC amplitude) response of evoked EPSC (c, n = 11,11,10 neurons in each group, F(2,29) = 14.5, P<0.0001) and HFS-induced LTP (d, n = 11,9,11 neurons per group, F(2,28) =8.37, P = 0.001) were also observed in the hippocampal CA1 neurons of the rats microinjected with Aβ1-40 fibrils, but not Aβ40-1 fibrils nor artificial CSF. (e) Aβ1-40, but not Aβ40-1, fibrils significantly decreased hippocampal NLGN1 expression (n = 6 rats per group, F(2,15) = 9.3, P = 0.002). Data represent mean ± s.e.m. *, P<0.05, **, P<0.01. Full length blots are presented in supplementary figure 11.

Supplementary Figure 2 Amyloid fibrils preferentially attenuated hippocampal glutamatergic transmission.

(a) Amyloid fibrils (Aβ1-40) significantly increased the paired-pulse ratio of evoked EPSC, indicating decreased presynaptic glutamate release in hippocampal neurons (n = 11 and 9 neurons, F(1,18) = 14.1, P = 0.002), and decreased the exogenous AMPA (1 μM)-induced inward currents, indicating attenuated postsynaptic response in hippocampal CA1 neurons (b, n = 11 and 8 neurons, t = 2.5, P = 0.022). Representative traces of mEPSC and mIPSC of hippocampal CA1 neurons in control and amyloid-injected rats were shown (c). Amyloid fibrils significantly decreased the amplitude (d, n = 13 and 17 neurons, t = 2.8, P = 0.009) and frequency (e, n = 13 and 17 neurons, t = 2.4, P = 0.025) of mEPSC, but not mIPSC, in hippocampal CA1 neurons. (f) Amyloid fibrils decreased the mEPSC/mIPSC ratio of amplitude (n = 17 and 19 neurons, t = 2.3, P = 0.03) and frequency (n = 17 and 19 neurons, t = 4.8, P<0.001) in hippocampal CA1 neurons. (g) Immunofluorescence for inhibitory GABAergic presynaptic marker GAD67 (green) and excitatory glutamatergic presynaptic marker vGLUT1 (violet) in the hippocampal CA1 in control and amyloid-injected rats. Note a marked decrease in vGLUT1, but not GAD67, immunoreactivities was observed (n = 25 sections from 5 rats in each group, t = 2.456, P = 0.39, scale bar = 25 μm). Data represent mean ± s.e.m. *, P<0.05, **, P<0.01.

Supplementary Figure 3 Amyloid fibrils did not change the expression of NLGN2, neurexin-1α, ADAM10, or presenilin 1 in the hippocampal CA1 area, and microinjection of Nlgn1 siRNA significantly decreased the hippocampal expression of NLGN1.

(a) Amyloid fibrils did not significantly change the hippocampal protein levels of NLGN2 (n = 8 and 9 rats per group, t = 0.1, P = 0.907) and neurexin-1α (n = 8 and 9 rats per group, t = 0.3, P = 0.779). (b) Amyloid fibrils did not significantly affect the hippocampal protein levels of ADAM10 (n = 6 and 9 rats per group, t = 0.7, P = 0.497) and presenilin 1 (n = 9 rats per group, t = 0.3, P = 0.763). Significantly decreased mRNA (c, n = 6 rats in each group, F(2,15) = 5.9, P = 0.01) and protein (d, n = 6 rats in each group, F(2,15) = 4.0, P = 0.03) levels of NLGN1 were observed in the hippocampal CA1 area after Nlgn1 siRNA (5 nmol per side), but not scrambled RNA (scRNA) microinjection. Data represent mean ± s.e.m. Full length blots are presented in supplementary figure 11.

Supplementary Figure 4 Amyloid fibrils increased the HDAC2 immunosignal in the hippocampal CA1 neurons, which was attenuated by microinjection of Hdac2 siRNA.

Amyloid fibrils increased the HDAC2 immunosignal in the hippocampal CA1 neurons, which was attenuated by microinjection of Hdac2 siRNA. Microinjection of Hdac2 siRNA (5 nmol per side), but not scrambled RNA (scRNA), significantly attenuated amyloid-upregulated Hdac2 mRNA levels (a, n = 6 rats per group, F(3,20) = 8.8, P = 0.0006) and protein (b, n = 6 rats per group, F(3,20) = 8.1, P<0.001) levels in the hippocampal CA1 area. The expression of HDAC2 was largely colocalized with the immunofluorescence signal of NeuN (c), the marker of neuron, but not GFAP (d), the marker of astrocyte. Microinjection of Hdac2 siRNA, but not scrambled RNA (scRNA), significantly attenuated the amyloid fibrils-upregulated immunofluorescence signal of HDAC2 in the hippocampal CA1 neurons (n = 24 sections from 5 rats in each group, F(3,16) = 11.5, P = 0.0003, Scale bar = 25 μm). (e) Microinjection of Hdac2 siRNA also significantly attenuated amyloid-decreased synapse number in the hippocampal CA1 area. Total synapse density was calculated as the number of synapses per 100 μm2 of neuropil (n = 5 rats per group, F(3,16) = 12.77, P = 0.0002, scale bar = 0.5 μm). Data represent mean ± s.e.m. *, P<0.05, **, P<0.01. Full length blots are presented in supplementary figure 11.

Supplementary Figure 5 Microinjection of suberoylanilide hydroxamic acid (SAHA) significantly restored amyloid-impaired hippocampal NLGN1 expression, glutamatergic transmission and memory.

SAHA (0.3 nmol per side for 3 days) significantly increased histone H3 acetylation across the Nlgn1 promoter region (a, n = 6 rats per group, F(3,20) = 5.3, P = 0.008), mRNA (b, n = 6 rats per group, F(3,20) = 6.7, P = 0.003) and protein (c, n = 8,8,6,6 rats per group, F(3,20) = 9.4, P = 0.0003) levels of the hippocampal NLGN1 in the rats injected with amyloid fibrils. SAHA significantly enhanced evoked EPSC amplitudes (d, n = 11,11,10,9 neurons in each group, F(3,37) = 10.8, P<0.0001) and HFS-induced LTP (e, n = 10,11,9,9 neurons in each group, F(3,35) = 7.96, P = 0.0004) in the hippocampal CA1 neurons of the amyloid-injected rats. It also significantly shortened the escape latencies (f, n = 10 rats per group, F(3,36) = 13.7, P<0.0001) and increased the time spent in the target quadrant (g, n = 10 rats per group, F(3,36) = 8.9, P = 0.0002) for amyloid-injected rats in the Morris water maze test. Data represent mean ± s.e.m. *, P<0.05, **, P<0.01. Full length blots are presented in supplementary figure 11.

Supplementary Figure 6 Amyloid fibrils increased the MeCP2 immunosignal in the hippocampal CA1 neurons, which was attenuated by microinjection of Mecp2 siRNA.

Amyloid-induced increased in the MeCP2 immunosignal was significantly attenuated by microinjection of Mecp2 siRNA (5 nmol per side), but not scrambled RNA (scRNA) (a, n = 24 sections from 5 rats in each group, F(3,16) = 5.8, P = 0.0071, scale bar = 25 μm). The expression of MeCP2 was largely colocalized with the immunofluorescence signal of NeuN (a), the marker of neuron, but not GFAP (b), the marker of astrocyte. Microinjection of Mecp2 siRNA significantly decreased the mRNA (c, n = 6 rats per group, F(3,20) = 10.1, P<0.01) and protein (d, n = 6 rats per group, F(3,20) = 34.4, P<0.0001) levels of hippocampal MeCP2 in the amyloid-injected rats. Microinjection of Mecp2 siRNA also significantly attenuated amyloid-decreased synapse number in the hippocampal CA1 area (e, n = 5 rats per group, F(3,16) = 25.6, P<0.0001, scale bar = 0.5 μm). Total synapse density was calculated as the number of synapses per 100 μm2 of neuropil. *, P<0.05, **, P<0.01. Data represent mean ± s.e.m. Full length blots are presented in supplementary figure 11.

Supplementary Figure 7 Amyloid fibrils induced significant change in mSin3A but not HDAC1 expression in the hippocampal CA1 area.

(a) Significant increase in protein levels of mSin3A (n = 6 and 9 rats per group, t = 5.6, P<0.0001), but not HDAC1 (n = 9 rats in each group, t = 0.9, P = 0.360), in the hippocampal CA1 of amyloid-injected rats. (b) Significant increase in binding of mSin3A (n = 9 rats per group, t = 4.1, P = 0.0008), but not HDAC1 (n = 6 rats per group, t = 0.4, P = 0.718), with MeCP2 in the hippocampal CA1 of amyloid-injected rats. Significant increase in binding of mSin3A (d, n = 6 rats per group, t = 2.7, P = 0.024), but not HDAC1 (c, n = 6 rats per group, t = 0.3, P = 0.772), in Nlgn1 promoter region in the hippocampal CA1 of amyloid-injected rats. Data represent mean ± s.e.m. *, P<0.05, **, P<0.01. Full length blots are presented in supplementary figure 11.

Supplementary Figure 8 Epigenetic modification of NLGN1 expression in APP/PSEN1 mice.

Significantly increased expression of HDAC2 (a, n = 9 mice per group, t = 3.8, P = 0.002) and MeCP2 (n = 5 and 6 mice per group, t = 12.0, P<0.0001) as well as increased formation of complex containing HDAC2 and MeCP2 (b, n = 5 mice per group, t = 4.9, P = 0.001) were observed in the hippocampal CA1 of APP/PSEN1 mice. The occupancy of HDAC2 (c, n = 6 mice per group, t = 2.7, P = 0.023) and MeCP2 (d, n = 6 mice per group, t = 4.1, P = 0.002) were increased in Nlgn1, but not Gpdh, promoter region in these transgenic mice. Significantly decreased histone H3 acetylation (e, n = 6 mice per group, t = 3.1, P = 0.01) and increased cytosine methylation (f, n = 6 mice per group, t = 3.7, P = 0.004) were also observed in Nlgn1, but not Gpdh, promoter region in these transgenic mice. The expression of NLGN1 was decreased in hippocampal CA1 of these transgenic mice (g, n = 8 and 10 mice in each group, t = 7.2, P<0.0001). Data represent mean ± s.e.m. *, P<0.05, **, P<0.01. Full length blots are presented in supplementary figure 11.

Supplementary Figure 9 Microinjection of lipopolysaccharide (LPS, 5 μg per side), like that of amyloid fibrils, induced significant activation of microglia in the hippocampal CA1 area.

Significantly increased CD11b expression was observed in the hippocampal CA1 area of the rats injected with amyloid fibrils (a, n = 3-4 sections from 5 rats, t = 13.5, P<0.0001, scale bar = 40 μm.) or LPS (b, n = 3-4 sections from 5 rats in each group, t = 15.1, P<0.0001). Microinjection of LPS also significantly decreased synapse number in the hippocampal CA1 area (c, n = 5 rats per group, t = 6.862, P = 0.0001, scale bar = 0.5 μm). Total synapse density was calculated as the number of synapses per 100 μm2 of neuropil. Data represent mean ± s.e.m.**, P<0.01.

Supplementary Figure 10 Microinjection of minocycline (5 μg per side) suppressed amyloid fibril—induced microglial activity in the hippocampal CA1 area.

Immunostaining revealed significantly decreased CD11b expression in the hippocampal CA1 area of the modeled rats injected with minocycline (mino) (a, n = 3-4 sections from 5 rats in each group, F(3,72) = 98.9, P<0.0001, **, P<0.01, scale bar = 40 μm). Microinjection of minocycline also prevented amyloid fibril-induced upsurge in IL-1β synthesis (b, n = 6 rats in each group, F(4,25) = 9.4, P=0.001, *, P<0.05). Microinjection of minocycline (5 μg per side) significantly attenuated amyloid-decreased synapse number in the hippocampal CA1 area (c, n = 5 rats per group, F(3,16) = 21.4, P<0.0001, scale bar = 0.5 μm). Total synapse density was calculated as the number of synapses per 100 μm2 of neuropil. Data represent mean ± s.e.m. *, P<0.01, **, P<0.01.

Supplementary Figure 11 Full-length blots.

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Bie, B., Wu, J., Yang, H. et al. Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency. Nat Neurosci 17, 223–231 (2014). https://doi.org/10.1038/nn.3618

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