Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency
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© 2014 Nature America, Inc. All rights reserved. NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION ARTICLES Amyloid β-peptide (Aβ) is extensively deposited in the brains of patients with Alzheimer’s disease 1 and leads to impairment of central glutamatergic transmission and memory 2 . However, the underlying mechanism remains elusive and speculative. Neuroligin-1 (NLGN1) is a postsynaptic protein found in excitatory synapses that binds to the presynaptic neurexin to form a trans-synaptic complex with an intracellular domain containing PDZ-binding motifs 3 . NLGN1 dynamically shapes excitatory synaptic efficacy and plasticity 3–5 and participates in the formation of long-term memory 6 . Dysfunction of NLGNs and their associated proteins is linked to cognitive diseases such as autism 3 . However, the role of NLGN1 in amyloid-associated memory deficiency is unclear 7,8 . Epigenetic modifications of histone acetylation modulate hippocampal synaptic plasticity, learning and memory in rodent models of amyloid- induced memory deficiency 9,10 , although the precise mechanism remains elusive. Several subtypes of histone deacetylases (HDACs), which catalyze the hydrolysis of acetyl-l-lysine side chains in histone tails, leading to repressed gene transcription, exist in the mammal brain 11 . Upregulation of HDAC2 activity is associated with reduced expression of several genes important for learning and memory and is linked to memory deficiency in a rodent model of Alzheimer’s dis- ease 9,10 . Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional repressor that binds DNA sequences methylated at cytosine in the dinu- cleotide 5′-CpG (ref. 12), and it appears to be involved in the mainte- nance and development of synaptic plasticity and cognitive functions in the mammal brain 12,13 . Methylated DNA–binding proteins, including MeCP2, may repress gene transcription through their association with a nuclear receptor corepressor complex that possesses HDAC activity 14,15 . Although the loss of function or gain of function of MeCP2 mark- edly influences synaptic plasticity and memory in several diseases 13 , the functional adaptation and significance of the MeCP2-containing corepressor complex may be another contributor to amyloid-associated memory deficiency; however, this link remains unclear. Microglia are the resident immune cells in the brain and are acti- vated when tissue homeostasis is disturbed. Reactive microglia are found in the brains of patients with Alzheimer’s disease 16 . Although microglia appear to play an important role in promoting the clearance and phagocytosis of Aβ 17,18 , reactive microglia are predominantly detrimental because they induce neuroinflammation in the brain and impair central synapses and memory 19 . Currently, the molecular mechanism underlying neuroinflammation-induced synaptic dysfunction remains unclear. To make sense of the issues contributing to amyloid-associated memory deficiency, we explored the interaction between microglia- mediated neuroinflammation and epigenetic modulation of NLGN1 and its functional significance in the rodent model of amyloid- associated memory deficiency. The present study demonstrated that amyloid-induced neuroinflammation enhanced the activity of HDAC2-MeCP2 corepressor complex and suppressed NLGN1 expres- sion, thus leading to hippocampal glutamatergic dysfunction and memory deficiency in rodents. RESULTS Suppression of NLGN1 and amyloid-induced memory deficiency We bilaterally microinjected Aβ 1–40 fibrils (10 µg per side) or artificial CSF into the hippocampal CA1 area of rats 18,20 to determine the func- tional consequences. Consistent with previous reports 18,20 , treatment with amyloid fibrils led to memory deficiency as shown by extended escape latencies and less time (during the probe trial) spent in the tar- get quadrant in the Morris water maze test (Supplementary Fig. 1a,b). We also observed a substantial right shift in the input-output (stimula- tion intensities–current amplitudes) curve of glutamatergic excitatory postsynaptic currents (EPSCs) 18 (Supplementary Fig. 1c) and attenu- ated long-term potentiation (LTP) induced by high-frequency elec- trical stimuli 18 (HFS; Supplementary Fig. 1d) in the CA1 neurons. Microinjection of reverse sequence peptide, Aβ 40–1 fibrils (10 µg per 1 Department of General Anesthesiology, Anesthesiology Institute, Cleveland Clinic, Cleveland, Ohio, USA. 2 These authors contributed equally to this work. Correspondence should be addressed to M.N. ([email protected]). Received 17 October 2013; accepted 27 November 2013; published online 19 January 2014; doi:10.1038/nn.3618 Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency Bihua Bie 1,2 , Jiang Wu 1,2 , Hui Yang 1 , Jijun J Xu 1 , David L Brown 1 & Mohamed Naguib 1 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.
Transcript of Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency
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Amyloid β-peptide (Aβ) is extensively deposited in the brains of patients with Alzheimer’s disease1 and leads to impairment of central glutamatergic transmission and memory2. However, the underlying mechanism remains elusive and speculative. Neuroligin-1 (NLGN1) is a postsynaptic protein found in excitatory synapses that binds to the presynaptic neurexin to form a trans-synaptic complex with an intracellular domain containing PDZ-binding motifs3. NLGN1 dynamically shapes excitatory synaptic efficacy and plasticity3–5 and participates in the formation of long-term memory6. Dysfunction of NLGNs and their associated proteins is linked to cognitive diseases such as autism3. However, the role of NLGN1 in amyloid-associated memory deficiency is unclear7,8.
Epigenetic modifications of histone acetylation modulate hippocampal synaptic plasticity, learning and memory in rodent models of amyloid-induced memory deficiency9,10, although the precise mechanism remains elusive. Several subtypes of histone deacetylases (HDACs), which catalyze the hydrolysis of acetyl-l-lysine side chains in histone tails, leading to repressed gene transcription, exist in the mammal brain11. Upregulation of HDAC2 activity is associated with reduced expression of several genes important for learning and memory and is linked to memory deficiency in a rodent model of Alzheimer’s dis-ease9,10. Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional repressor that binds DNA sequences methylated at cytosine in the dinu-cleotide 5′-CpG (ref. 12), and it appears to be involved in the mainte-nance and development of synaptic plasticity and cognitive functions in the mammal brain12,13. Methylated DNA–binding proteins, including MeCP2, may repress gene transcription through their association with a nuclear receptor corepressor complex that possesses HDAC activity14,15. Although the loss of function or gain of function of MeCP2 mark-edly influences synaptic plasticity and memory in several diseases13, the functional adaptation and significance of the MeCP2-containing corepressor complex may be another contributor to amyloid-associated memory deficiency; however, this link remains unclear.
Microglia are the resident immune cells in the brain and are acti-vated when tissue homeostasis is disturbed. Reactive microglia are found in the brains of patients with Alzheimer’s disease16. Although microglia appear to play an important role in promoting the clearance and phagocytosis of Aβ17,18, reactive microglia are predominantly detrimental because they induce neuroinflammation in the brain and impair central synapses and memory19. Currently, the molecular mechanism underlying neuroinflammation-induced synaptic dysfunction remains unclear.
To make sense of the issues contributing to amyloid-associated memory deficiency, we explored the interaction between microglia- mediated neuroinflammation and epigenetic modulation of NLGN1 and its functional significance in the rodent model of amyloid-associated memory deficiency. The present study demonstrated that amyloid-induced neuroinflammation enhanced the activity of HDAC2-MeCP2 corepressor complex and suppressed NLGN1 expres-sion, thus leading to hippocampal glutamatergic dysfunction and memory deficiency in rodents.
RESULTSSuppression of NLGN1 and amyloid-induced memory deficiencyWe bilaterally microinjected Aβ1–40 fibrils (10 µg per side) or artificial CSF into the hippocampal CA1 area of rats18,20 to determine the func-tional consequences. Consistent with previous reports18,20, treatment with amyloid fibrils led to memory deficiency as shown by extended escape latencies and less time (during the probe trial) spent in the tar-get quadrant in the Morris water maze test (Supplementary Fig. 1a,b). We also observed a substantial right shift in the input-output (stimula-tion intensities–current amplitudes) curve of glutamatergic excitatory postsynaptic currents (EPSCs)18 (Supplementary Fig. 1c) and attenu-ated long-term potentiation (LTP) induced by high-frequency elec-trical stimuli18 (HFS; Supplementary Fig. 1d) in the CA1 neurons. Microinjection of reverse sequence peptide, Aβ40–1 fibrils (10 µg per
1Department of General Anesthesiology, Anesthesiology Institute, Cleveland Clinic, Cleveland, Ohio, USA. 2These authors contributed equally to this work. Correspondence should be addressed to M.N. ([email protected]).
Received 17 October 2013; accepted 27 November 2013; published online 19 January 2014; doi:10.1038/nn.3618
Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiencyBihua Bie1,2, Jiang Wu1,2, Hui Yang1, Jijun J Xu1, David L Brown1 & Mohamed Naguib1
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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side), did not induce appreciable impairment of behavioral perform-ance and glutamatergic transmission (Supplementary Fig. 1a–d). Aβ1–40 fibril–induced suppression of glutamatergic transmission may be attributable to decreased presynaptic glutamate release (indicated by increased paired-pulse ratio; Supplementary Fig. 2a) and the con-ductance of postsynaptic glutamate receptors (indicated by reduced exogenous 1 µM AMPA–evoked inward currents; Supplementary Fig. 2b). Although disturbance of GABAergic inhibitory synapse also existed in the amyloid-deposited brain21, microinjection of Aβ1–40 fibrils preferentially attenuated glutamatergic transmission in hip-pocampal CA1 neurons (Supplementary Fig. 2c–g).
Because NLGN1 plays a pivotal role in brain glutamatergic trans-mission and cognition3,4, we investigated the epigenetic modu-lation of NLGN1 expression in hippocampal CA1 tissues of the rats microinjected with amyloid fibrils. In these rats, the Nlgn1 mRNA level was significantly decreased (Fig. 1a), along with the NLGN1 protein level (Fig. 1b), whereas microinjection of reverse sequence peptide Aβ40–1 fibrils (10 µg per side) did not alter hip-pocampal NLGN1 expression (Supplementary Fig. 1e). In con-trast, we observed no appreciable changes in protein levels of neurexin-1α and NLGN2 in hippocampal CA1 after amyloid fibril injection (Supplementary Fig. 3a). Further chromatin immuno-precipitation (ChIP) assays22 revealed a significant decrease in histone H3 acetylation in the Nlgn1 promoter region (Fig. 1c) in hippocampal CA1 tissue of these rats. We observed no obvious changes in hippocampal ADAM10 and presenilin protein levels in the model rats (Supplementary Fig. 3b), although it has been suggested that cleavage of NLGN1 by ADAM10 and γ-secretase may negatively regulate the remodeling of excitatory synapse spines23. These find-ings indicate a potential epigenetic mechanism underlying decreased hippocampal NLGN1 expression induced by amyloid fibrils.
Next, to determine whether artificial suppression of NLGN1 impairs the glutamatergic synapses and memory function in a fashion similar to that noted with amyloid fibrils, we microinjected Nlgn1 short
interfering RNA (5 nmol per side) and observed appreciably decreased hippocampal Nlgn1 mRNA and NLGN1 protein levels in untreated rats (Supplementary Fig. 3c,d). In hippocampal slices from these rats, EPSCs in the input-output curve were attenuated (Fig. 1d) and HFS-induced LTP was significantly impaired (Fig. 1e). Furthermore, these rats showed significantly extended escape latencies, and they spent less time during the probe trial in the target quadrant in the Morris water maze test (Fig. 1f,g), indicating that suppression of NLGN1 expression induced memory deficiency. The same dose of scrambled RNA (scRNA) failed to alter Nlgn1 mRNA and NLGN1 protein expression (Supplementary Fig. 3c,d), basal glutamatergic strength (Fig. 1d), HFS-induced LTP (Fig. 1e) and performance in the Morris water maze (Fig. 1f,g). These findings provide a mechanistic link between suppression of NLGN1 and the synaptic dysfunction and memory deficiency induced by amyloid fibrils.
HDAC2 mediates epigenetic suppression of NLGN1Increased activity of HDAC2, a class I HDAC, is generally associ-ated with histone deacetylation and suppressed transcription of target genes24 and negative regulation of memory and synaptic plasticity in the rodent brain9,10,24. Here we explored the amyloid fibril–induced adaptation of HDAC2 activity and HDAC2 association with the epigenetic modification of NLGN1 expression in the hippocampal CA1 area. In accordance with a previous report10, our results showed increased hippocampal Hdac2 mRNA (Supplementary Fig. 4a) and HDAC2 protein (Fig. 2a), which largely colocalized with CA1 neurons (Supplementary Fig. 4c) in the rats injected with amyloid fibril, indi-cating amyloid-induced upregulation in HDAC2 activity. Intriguingly, further ChIP analysis revealed increased HDAC2 occupancy in the Nlgn1 promoter region (Fig. 2b), possibly explaining the amyloid-induced reduction of H3 acetylation in the Nlgn1 promoter region (Fig. 1c) in the hippocampal CA1 area.
We went on to assess whether suppressing HDAC2 activity, by local administration of the potent HDAC inhibitor suberoylanilide
Figure 1 Epigenetic suppression of NLGN1 underlies amyloid fibril–induced memory deficiency. (a,b) Significantly decreased Nlgn1 mRNA (n = 6 or 7 rats in two groups, t = 6.1, P < 0.0001) and NLGN1 protein (n = 6 rats in each group, t = 3.7, P = 0.004) in the hippocampal CA1 area in amyloid fibril–injected rats. (c) Significantly decreased histone H3 in the Nlgn1, but not Gapdh, promoter region in hippocampal CA1 tissue in amyloid fibril–injected rats (n = 6 rats in each group, t = 3.1, P = 0.01). (d–g) Microinjection of Nlgn1 siRNA (5 nmol per side) impaired hippocampal glutamatergic transmission and memory in naive rats. We observed significantly attenuated input-output (stimulus intensity–EPSC amplitude) response of evoked EPSCs (d, n = 10,11,9 neurons in each group, F2,27 = 14.0, P = 0.0001) and impaired HFS-induced LTP (e, n = 10,8,8 neurons in each group, F2,23 = 9.8, P = 0.001) in hippocampal CA1 neurons after microinjection of Nlgn1 siRNA, but not scRNA, in naive rats. Significantly extended escape latency (f, n = 10 rats in each group, F2,27 = 8.8, P = 0.001) and less time spent in the target quadrant (g, n = 10 rats in each group, F2,27 = 7.3, P = 0.003) in the rats microinjected with NLGN1 siRNA, but not scRNA. (g) Representative path tracings in each quadrant during the probe trial on day 6 (T, target quadrant; R, right quadrant; O, opposite quadrant; L, left quadrant). Full-length blots are presented in Supplementary Figure 11. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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hydroxamic acid (SAHA)25 or, more spe-cifically, Hdac2 siRNA, would confirm the involvement of HDAC2 in amyloid-induced reduction of NLGN1 by ameliorating the NLGN1 reduction, synaptic dysfunction and memory deficiency in the presence of amyloid fibrils. Local administration of Hdac2 siRNA (5 nmol per side), but not scRNA, effectively attenuated amyloid fibril–upregulated hippocampal Hdac2 mRNA and HDAC2 protein levels (Supplementary Fig. 4a–c). In addition, Hdac2 siRNA treatment significantly increased H3 acetyla-tion in the Nlgn1 promoter region (Fig. 2c) and, consequently, Nlgn1 mRNA and NLGN1 protein (Fig. 2d,e) in hippocampal CA1 tissue of amyloid-injected rats. Moreover, treatment with Hdac2 siRNA, but not scRNA, restored hippocampal dendritic spine numbers (Fig. 2f), synapse numbers (Supplementary Fig. 4e), evoked EPSC amplitudes in the input-output curve (Fig. 2g) and HFS-induced LTP (Fig. 2h) in hippocampal CA1 neurons of the amyloid-injected rats. In the Morris water maze test, the amyloid-injected rats exhibited significantly shortened escape latencies (Fig. 2i) and spent more time in the target quadrant (Fig. 2j) after treatment with Hdac2 siRNA. This was further confirmed by studying the effects of local administration of SAHA (0.3 nmol per side for 3 d) into hippocampal CA1 in the rats. Local admin-istration of SAHA restored the amyloid-induced reduction in hippo campal H3 acetylation in the Nlgn1 promoter region and consequently in Nlgn1 mRNA and NLGN1 protein levels as well (Supplementary Fig. 5a–c). In addition, SAHA substantially enhanced evoked EPSC amplitudes (Supplementary Fig. 5d) and HFS-induced LTP
(Supplementary Fig. 5e), indicating restoration of glutamatergic syn-aptic function in hippocampal CA1 neurons of the amyloid-injected rats. Moreover, SAHA treatment also shortened the escape laten-cies (Supplementary Fig. 5f) and increased the time the amyloid-injected rats spent in the target quadrant (Supplementary Fig. 5g) in the Morris water maze test. Microinjection of SAHA in the control animals induced an increase in NLGN1 expression (Supplementary Fig. 5a–c), with a trend showing enhanced synaptic plasticity (Supplementary Fig. 5d,e) and cognitive function (Supplementary Fig. 5f,g)9. These findings further confirmed that HDAC2 was involved in the mechanism underlying amyloid fibril–induced suppression in NLGN1 expression and cognitive capacities.
MeCP2 mediates epigenetic suppression of NLGN1We also studied the involvement of MeCP2, the corepressor that acts with HDACs14,15, in amyloid fibril–induced NLGN1 reduction, synaptic dysfunction and memory deficiency. First, we observed increased levels of Mecp2 mRNA (Supplementary Fig. 6c) and MeCP2 protein (Fig. 3a), which mostly colocalized with CA1 neurons (Supplementary Fig. 6a), in hippocampal CA1 tissue of the amyloid-injected rats. Phosphorylation of several serine residues (for example, pSer80, pSer421) modulates the association between MeCP2 and
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Figure 2 Enhanced HDAC2 activity was involved in amyloid fibril–suppressed NLGN1 expression. Amyloid fibrils induced significantly increased HDAC2 expression (a, n = 6 rats in each group, t = 9.3, P < 0.0001) and occupancy in the Nlgn1, but not Gapdh, promoter region (b, n = 6 rats in each group, t = 3.6, P < 0.01). Microinjection of Hdac2 siRNA (5 nmol per side), but not scRNA, significantly increased histone H3 acetylation in the Nlgn1 promoter region (c, n = 6 rats in each group, F3,20 = 6.3, P = 0.004), Nlgn1 mRNA (d, n = 6 rats in each group, F3,20 = 9.6, P = 0.0004) and NLGN1 protein (e, n = 8,6,6,6 rats in each group, F3,22 = 20.3, P < 0.0001) in hippocampal CA1 of the rats injected with amyloid fibrils. (f–j) Microinjection of Hdac2 siRNA (5 nmol per side) restored amyloid-impaired glutamatergic transmission and memory. Hippocampal dendritic spine numbers (f, n = 4,6,6,4 rats in each group, F3,16 = 7.1, P = 0.003; scale bars, 5 µm), input-output response of evoked EPSCs (g, n = 11,11,10,10 neurons in each group, F3,38 = 13.1, P < 0.0001) and HFS-induced LTP (h, n = 11,11,9,8 neurons in each group, F3,35 = 13.2, P < 0.0001) were significantly restored after siRNA, but not scRNA, microinjection. siRNA microinjection also shortened the escape latency (i, n = 10 rats in each group, F3,36 = 11.9, P < 0.0001) and increased the time in spent in the target quadrant (j, n = 10 rats in each group, F3,36 = 4.3, P = 0.011) in the rats microinjected with amyloid fibrils. (j) Representative path tracings in each quadrant during the probe trial on day 6. Full-length blots are presented in Supplementary Figure 11. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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target genes, thereby regulating gene expression in an activity-dependent manner26,27. In this regard, coimmunoprecipitation studies28 revealed increased phosphorylation of serine residues in MeCP2 (Fig. 3d) in hippocampal CA1 tissue of the amyloid-injected rats. MeCP2 pSer80 was elevated (Fig. 3d), while pSer421 remained unchanged (Fig. 3d). Moreover, the formation of a corepressor complex consisting of MeCP2 and HDAC2 (Fig. 3b) was also enhanced in hippocampal
CA1 tissue of these rats. These results indicate functional adaptation of MeCP2 in hippocampal CA1 tissue of the amyloid-injected rats. Meanwhile, our analysis revealed an increased MeCP2 occupancy (Fig. 3c) and enhanced cytosine methylation (Fig. 4c) in the Nlgn1 promoter region in the hippocampal CA1 tissue of these rats. These findings point toward MeCP2 involvement in amyloid-suppressed NLGN1 expression.
We went on to examine whether suppres-sion of MeCP2 activity ameliorates amyloid
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Figure 3 Amyloid fibrils induced functional modification of MeCP2 activity in hippocampal CA1 tissue. The expression of MeCP2 (a, n = 7 rats in each group, t = 12.7, P < 0.0001) and the HDAC2 in the immunocomplex pulled down by polyclonal MeCP2 antibody (b, n = 6 or 7 rats in each group, t = 6.6, P < 0.0001) were significantly increased in hippocampal CA1 of the rats injected with amyloid fibrils. Amyloid fibrils also increased MeCP2 occupancy in the Nlgn1, but not Gapdh, promoter region (c, n = 6 rats in each group, t = 3.0, P = 0.013). The phosphorylation of serine residues in MeCP2 (d, n = 6 rats in each group, t = 9.4, P < 0.0001) in hippocampal CA1 was induced by amyloid fibrils. The phosphorylation in Ser80 (d, n = 7 rats in each group, t = 3.4, P = 0.005), but not Ser421 (d, n = 6 rats in each group, t = 1.5, P = 0.177), in MeCP2 was significantly increased. Full-length blots are presented in Supplementary Figure 11. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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Figure 4 Suppression of MeCP2 activity ameliorated amyloid fibril–suppressed NLGN1 expression and memory. (a–e) Suppression of MeCP2 by siRNA (5 nmol per side) restored amyloid fibril–induced epigenetic suppression of NLGN1. (a) Mecp2 siRNA, but not scRNA, decreased amyloid-enhanced HDAC2 binding in the Nlgn1 promoter region (n = 6 rats in each group, F3,20 = 5.3, P = 0.007). It also increased H3 acetylation (b, n = 6 rats in each group, F3,20 = 5.4, P = 0.007) and decreased cytosine methylation (c, n = 6 rats in each group, F3,20 = 7.3, P = 0.002) in the Nlgn1 promoter region. Moreover, we also observed restored Nlgn1 mRNA (d, n = 6 rats in each group, F3,20 = 5.9, P = 0.005) and NLGN1 protein (e, n = 6 rats in each group, F3,20 = 15.4, P < 0.0001) levels after Mecp2 siRNA, but not scRNA, microinjection. (f–j) Microinjection of Mecp2 siRNA restored amyloid-impaired glutamatergic transmission and memory. Hippocampal dendritic spine numbers (f, n = 4,5,6,5 rats in each group, F3,16 = 6.9, P = 0.004; scale bars, 5 µm), input-output response of evoked EPSCs (g, n = 11,11,10,11 neurons in each group, F3,39 = 17.0, P < 0.0001) and HFS-induced LTP (h, n = 11,11,10,8 neurons in each group, F3,36 = 15.8, P < 0.0001) were significantly restored after Mecp2 siRNA, but not scRNA, microinjection. Microinjection of Mecp2 siRNA also shortened the escape latency (i, n = 10 rats in each group, F3,36 = 14.6, P < 0.0001) and increased the time spent in the target quadrant (j, n = 10 rats in each group, F3,36 = 5.4, P = 0.004) in the rats microinjected with amyloid fibrils. (j) Representative path tracings in each quadrant during the probe trial on day 6. Full-length blots are presented in Supplementary Figure 11. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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fibril–induced NLGN1 reduction, synaptic dysfunction and memory deficiency. Local administration of Mecp2 siRNA (5 nmol per side) decreased amyloid fibril–induced upregulation of Mecp2 mRNA and protein levels in hippocampal CA1 tissue (Supplementary Fig. 6a–d). Further ChIP analysis revealed that treatment with Mecp2 siRNA, but not scRNA, noticeably decreased HDAC2 occupancy (Fig. 4a), increased H3 acetylation (Fig. 4b) and decreased cytosine methyla-tion (Fig. 4c) in the Nlgn1 promoter region in hippocampal CA1 tissue of the amyloid-injected rats. Consequently, this treatment also significantly restored Nlgn1 mRNA (Fig. 4d) and protein (Fig. 4e) levels in hippocampal CA1 tissue of the amyloid-injected rats. In hippocampal slices from these rats, treatment with Mecp2 siRNA, but not scRNA, substantially restored hippocampal dendritic spine numbers (Fig. 4f) and synapse numbers (Supplementary Fig. 6e), and enhanced hippocampal EPSCs (Fig. 4g) and HFS-induced LTP (Fig. 4h). Furthermore, the water maze test also revealed that sup-pressing MeCP2 activity by siRNA significantly shortened the escape latencies (Fig. 4i) and increased the time spent in the target quadrant (Fig. 4j). These findings confirmed the involvement of MeCP2, via epi-genetic suppression of NLGN1 by partnering with HDAC2, in the amy-loid fibril–induced synaptic dysfunction and memory deficiency.
The MeCP2-containing corepressor complex may recruit other transcriptional factors, such as mSin3A and other HDACs29. We observed a substantially increased expression of mSin3A, but not HDAC1, in the hippocampal CA1 area of the amyloid-injected rats (Supplementary Fig. 7a). Binding between mSin3A, but not HDAC1, and MeCP2 was enhanced in the hippocampal CA1 area of these rats (Supplementary Fig. 7b). Moreover, the occupancy of mSin3A, but not HDAC1, in Nlgn1 promoter region was also increased in these rats (Supplementary Fig. 7c,d), suggesting that other transcrip-tional factors in MeCP2-containing corepressor complex may also be involved in amyloid-induced central synaptic dysfunction and memory deficiency.
HDAC2-MeCP2 complex suppresses NLGN1 in transgenic miceAdult Tg-APPsw/PSEN1DE9 transgenic mice display extensive accu-mulation amyloid fibrils and neuroinflammation in the brain and exhibit amyloid-associated memory deficiency30. In the hippocampal CA1 area of these mice, we observed noticeably increased expres-sion of HDAC2 and MeCP2, along with the increased formation of HDAC2-MeCP2 complexes (Supplementary Fig. 8a,b). Moreover, the occupancy of HDAC2 and MeCP2 in the Nlgn1 promoter region
was also enhanced in the hippocampal CA1 area of these mice (Supplementary Fig. 8c,d). Accordingly, we observed decreased histone H3 acetylation, as well as increased cytosine methylation, in the Nlgn1 promoter region of these rats (Supplementary Fig. 8e,f). In addition, we observed significantly decreased hippocampal expres-sion of NLGN1 in these mice (Supplementary Fig. 8g). Together, our findings support the presence of epigenetic modulation of NLGN1 expression in this transgenic rodent model of Alzheimer’s disease with extensive deposition of amyloid fibrils.
Microglial activation induces epigenetic NLGN1 modificationBecause microglia play a pivotal role in central synaptic dysfunction and memory deficiency in several neuroinflammatory diseases31,32, we investigated whether amyloid-induced neuroinflammation leads to the epigenetic suppression of NLGN1 in hippocampal CA1 neurons of the amyloid-injected rats. Consistent with the earlier reports18, we observed increased CD11b signal (a microglia marker), cell complexity (rounded cell bodies and thicker processes) (Supplementary Fig. 9a) and IL-1β (Supplementary Fig. 10b), all indicating amyloid fibril–induced microglial activation and neuroinflammation in hippocam-pal CA1 tissue of the amyloid-injected rats. To determine whether artificial activation of microglia mimics amyloid fibril–induced gluta-matergic synaptic dysfunction and memory deficiency, we delivered lipopolysaccharide (LPS, 5 µg per side) into the hippocampal CA1 area in naive rats, which resulted in increased hippocampal CD11b signal (Supplementary Fig. 9b) and IL-1β (Supplementary Fig. 10b), indicating microglia activation. In these rats, LPS increased HDAC2 and MeCP2 expression (Fig. 5a) in the hippocampal CA1 area. Substantially increased HDAC2 and phosphorylation of Ser80 in MeCP2 were detected in the immunocomplex pulled down by poly-clonal anti-MeCP2 antibody (Fig. 5b), indicating an LPS-induced functional modification of the MeCP2 corepressor complex, as noted with amyloid fibrils. In addition, increased HDAC2 (Fig. 5c) and MeCP2 (Fig. 5d) occupancy and, consequently, decreased H3 acetyla-tion (Fig. 5e) and increased cytosine methylation (Fig. 5f) in the Nlgn1 promoter region were observed after LPS injection. We also observed significantly decreased hippocampal Nlgn1 mRNA (Fig. 5g) and protein (Fig. 5h) in these rats.
In hippocampal CA1 from these rats, we also noted decreased den-dritic spine numbers (Fig. 6a) and synapse numbers (Supplementary Fig. 9c), weakened evoked EPSCs in the input-output curve (Fig. 6b) and weakened HFS-induced LTP (Fig. 6c), indicating LPS-impaired
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glutamatergic transmission. The LPS- microinjected rats also exhibited extended escape latencies (Fig. 6d) and spent less time in the target quadrant (Fig. 6e) in the Morris water maze test, indi-cating LPS-induced memory deficiency. Collectively, these findings suggest that the artificial activation of microglia may mimic, via a similar epigenetic mechanism, amyloid fibril–induced NLGN1 reduc-tion, synaptic dysfunction and memory deficiency.
We went on to test the hypothesis that suppression of microglial activation would ameliorate amyloid fibril–induced suppression of NLGN1 expression and restore synaptic and memory function. Microinjection of minocycline (5 µg per side) attenuated hippocampal amyloid-induced upregulation of CD11b (Supplementary Fig. 10a)
and IL-1β (Supplementary Fig. 10b), indicating suppression of microglial activation in the Alzheimer’s disease model rats. In these rats, minocycline significantly decreased amyloid-upregulated HDAC2 and MeCP2 expression (Fig. 7a) in the hippocampal CA1 area. In addition, we detected significantly decreased phosphorylation of serine residues, pSer80 MeCP2 and HDAC2 in the immunocom-plex pulled down by anti-MeCP2 antibody in the amyloid-injected rats after treatment with minocycline (Fig. 7b), indicating that sup-pression of neuroinflammation by minocycline ameliorated amyloid- induced functional adaptation of MeCP2 in the hippocampal
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Figure 6 LPS impaired hippocampal glutamatergic transmission and memory in naive rats. We observed significantly decreased hippocampal dendritic spine numbers (a, n = 5 rats in each group, t = 4.0, P = 0.004; scale bars, 5 µm), attenuated input-output response of evoked EPSCs (b, n = 9 neurons in each group, F1,16 = 17.9, P < 0.0001) and impaired HFS-induced LTP (c, n = 11 and 9 neurons in each group, F1,18 = 14.4, P = 0.002) in hippocampal CA1 neurons after LPS microinjection. We also observed significantly extended escape latency (d, n = 10 rats in each group, F1,18 = 4.9, P = 0.04) and less time spent in the target quadrant (e, n = 10 rats in each group, F1,18 = 5.17, P = 0.036) in the rats microinjected with LPS. (e) Representative path tracings in each quadrant during the probe trial on day 6. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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Figure 7 Microinjection of minocycline (5 µg per side) ameliorated amyloid fibril–suppressed NLGN1 expression. (a) Minocycline (mino) decreased amyloid- induced HDAC2 (n = 7,7,6,6 rats in each group, F3,22 = 14.2, P < 0.0001) and MeCP2 expression (n = 7,7,6,6 rats in each group, F3,22 = 22.7, P < 0.0001). (b) Minocycline decreased the amount of amyloid-induced phosphorylated serine residues (n = 7,7,5,5 rats in each group, F3,20 = 85.2, P < 0.0001), pSer80 MeCP2 (n = 6 rats in each group, F3,20 = 5.9, P = 0.0048), and HDAC2 (n = 7,7,6,6 rats in each group, F3,22 = 76.4, P < 0.0001) in the immunocomplex precipitated by MeCP2 antibody from hippocampal CA1 area lysates (n = 6 or 7 rats in each group). Minocycline decreased amyloid-induced HDAC2 (c, n = 6 rats in each group, F3,20 = 4.5, P = 0.015) and MECP2 binding (d, n = 6 rats in each group, F3,20 = 6.2, P = 0.004), increased H3 acetylation (e, n = 6 rats in each group, F3,20 = 4.4, P = 0.016) and decreased cytosine methylation (f, n = 6 rats in each group, F3,20 = 5.1, P = 0.009) in the Nlgn1, but not Gapdh, promoter region and, consequently, restored the hippocampal Nlgn1 mRNA (g, n = 6 rats in each group, F3,20 = 4.4, P = 0.015) and NLGN1 protein (h, n = 8,8,6,6 rats in each group, F3,24 = 5.6, P = 0.005) that had been reduced by amyloid fibrils. Full-length blots are presented in Supplementary Figure 11. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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CA1 area. Further, ChIP assays revealed that minocycline induced a significant decrease in HDAC2 and MeCP2 occupancy (Fig. 7c,d) and, consequently, increased the H3 acetyla-tion (Fig. 7e) and decreased cytosine meth-ylation (Fig. 7f) in the Nlgn1 promoter region in the hippocampal CA1 area of amyloid-injected rats. Minocycline also significantly restored amyloid-suppressed hippocampal Nlgn1 mRNA (Fig. 7g) and NLGN1 pro-tein levels (Fig. 7h) in the amyloid-injected rats. Minocycline had no obvious effect on epigenetic modulation of NLGN1 expres-sion in the control rats microinjected with artificial cerebrospinal fluid (Fig. 7a–h).
Further studies revealed substantially restored dendritic spine numbers (Fig. 8a), synapse numbers (Supplementary Fig. 10c), amyloid-impaired evoked EPSCs (Fig. 8b) and HFS-induced LTP (Fig. 8c) in hippocampal neurons after treatment with minocycline. Functionally, these amyloid-injected rats, after treatment with mino-cycline, exhibited significantly shorter escape latencies (Fig. 8d) and spent more time in the target quadrant (Fig. 8e) in the Morris water maze test, indicating improved memory. Notably, in the amyloid-injected rats that were treated with minocycline, coadministration of Nlgn1 siRNA (5 nmol per side) to decrease NLGN1 expression, as noted above, significantly attenuated evoked EPSCs and HFS-induced LTP in hippocampal CA1 neurons and impaired performance in the Morris water maze test (Fig. 8). Together, these findings show that suppression of microglia activation attenuated amyloid fibril–induced epigenetic suppression of NLGN1, glutamatergic synapse dysfunction and memory deficiency.
DISCUSSIONNLGN1 is primarily localized postsynaptically at central glutamater-gic synapses3,33. Promoting the interaction between NLGN1 and its presynaptic receptor neurexin may positively impact synaptogenesis
and the stability and efficiency of central glutamatergic synapses5,33,34. Perturbing endogenous NLGN-neurexin complex formation leads to decreased synaptic function35,36 and impairs synaptic plasticity4, while overexpression of NLGNs enhances the number and efficacy of functional central synapses33,36. Genetic mutations of NLGNs and/or neurexins have been detected in patients with autism3, and artificial deletion of NLGNs markedly impairs spatial memory in rodents6 and conditional learning behavior in Aplysia4. In our study, we found amyloid fibril–induced reduction in hippocampal NLGN1 expres-sion. Artificial suppression of NLGN1 mimicked amyloid-induced hippocampal glutamatergic dysfunction and memory deficiency, and recovery of NLGN1 expression by epigenetic modification improved the function of central glutamatergic synapses and the memory impaired by amyloid. Thus, we provide evidence that establishes the critical involvement of NLGN1 in amyloid-induced glutamatergic synaptic dysfunction and memory deficiency, which had been pre-viously hinted at by a few emerging reports7,8.
In the rodent, amyloid alters the expression of several key proteins pertinent to synaptic plasticity and memory37,38 through unknown mechanisms. Epigenetic modification, including histone acetylation and cytosine methylation, serves as one of the main mechanisms inducing long-term adaptation of gene expression and, conse-quently, alteration of synaptic plasticity in several pathological con-ditions9,10,25. In the present study, we observed extensive modification of histone acetylation and cytosine methylation, which underscores
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Figure 8 Microinjection of minocycline restored hippocampal glutamatergic transmission and memory impaired by amyloid fibrils. We observed significantly restored hippocampal dendritic spine numbers (a, n = 4,6,6,4 rats in each group, F3,16 = 3.8, P = 0.031, scale bars, 5 µm), input-output response of evoked EPSCs (b, n = 11,10,10,9,9 neurons in each group, F4,44 = 13.1, P < 0.0001) and HFS-induced LTP (c, n = 11,11,8,8,8 neurons in each group, F4,41 = 9.9, P < 0.0001) in hippocampal CA1 neurons after minocycline (mino) microinjection. It also shortened the escape latency (d, n = 10,10,10,10,10 rats in each group, F4,45 = 16.7, P < 0.0001) and increased the time spent in the target quadrant (e, n = 10,10,10,10,10 rats in each group, F4,45 = 6.2, P = 0.0005) in the rats microinjected with amyloid fibrils. Notably, minocycline failed to restore amyloid fibril–impaired EPSC input-output response (b, n = 9 neurons in each point, P = 0.1), HFS-induced LTP (c, n = 8 neurons, P = 0.1) and performance in the water maze test (d,e, n = 10 rats, P = 0.1) when Nlgn1 siRNA was simultaneously delivered into the hippocampal CA1 area. (e) Representative path tracings in each quadrant during the probe trial on day 6. Data represent mean ± s.e.m. *P < 0.05; **P < 0.01.
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the significance of amyloid-induced alteration of NLGN1 expression and the synaptic plasticity that in turn goes on to regulate amyloid-associated memory deficiency.
Abundant evidence suggests that HDAC2 is critical in maladapta-tion of synaptic plasticity in a variety of pathological conditions9,39, including Alzheimer’s disease10. Inhibition of HDAC2 activity sig-nificantly enhances brain-derived neurotrophic factor expression and facilitates LTP induction in aged rats39. In an animal model of Alzheimer’s disease, suppression of HDAC2 activity reinstates central structural and synaptic plasticity and abolishes neurodegeneration-associated memory impairments10. Our study reveals upregulated HDAC2 activity induced by amyloid fibrils and confirms the benefi-cial effect of HDAC2 inhibition in reversing amyloid-induced synap-tic dysfunction and memory deficiency. More importantly, our study suggests that HDAC2-induced epigenetic blockade of NLGN1 func-tion serves as one of the main mechanisms associated with amyloid fibril–induced memory deficiency.
MeCP2 is an abundant nuclear protein that binds methylated DNA in the brain12,40 and serves as a transcriptional repressor that is critical for neural development40,41. Functional modification of MeCP2 activity is tightly correlated with central glutamatergic syn-aptic plasticity42. Either down- or upregulation of the function of MeCP2 markedly influences synaptic plasticity and memory func-tion in several diseases13. Genetic analysis has identified sporadic mutations of MECP2 linked to autism spectrum disorder and Rett syndrome43. Phosphorylation of several distinct serine residues confers contradictory effects on specific genes44–46, which provides a potential mechanism underlying the MeCP2-mediated dynamic epigenetic modification of target genes47. Coupling with other transcriptional repressors, such as HDACs and mSin3A29, appears essential for MeCP2-containing repressor complex-mediated histone deacetylation and suppression of the expression of target genes14,15. Meanwhile, MeCP2 may also form a complex with DNA methyl-transferase 1 (DNMT1), the enzyme that catalyzes DNA (cytosine-5) methylation at CpG sites in promoter regions, to maintain DNA methylation in the genome48. It was also reported that elevation of histone acetylation by disturbing the HDAC-containing repressor complex may facilitate DNA demethylation in the promoter regions of some target genes49. In our study, amyloid-induced hyperactiv-ity of MeCP2 was demonstrated by increased expression and phos phorylation of residue Ser80 and formation of repressor complex with HDAC2 and mSin3A in the hippocampal CA1 area, and inhibition of MeCP2 activity alleviated histone H3 deacetylation and cytosine methylation in the Nlgn1 promoter region, although the mechanisms remain speculative. Our electrophysiological and behavioral results further suggested that the increased MeCP2 activity was closely associated with the amyloid-induced glutamatergic synaptic dysfunc-tion and memory deficiency.
In the brain, activated microglia synthesize and release neurotoxic cytokines, including IL-1β, which underlies amyloid-induced impair-ment of synaptic plasticity and cognitive function31,32. Activation of microglia also induces HDAC activity and suppresses histone acetyla-tion and specific gene transcription in culture cells50. Here we have shown that artificial activation of microglia mimics amyloid-induced epigenetic modification of NLGN1, synaptic dysfunction and memory deficiency, while suppression of microglial activity largely prevents amyloid-impaired synaptic plasticity and memory by attenuating activated microglia-induced epigenetic suppression of NLGN1 expres-sion. Together these findings reveal a new epigenetic mechanism that links amyloid-induced neuroinflammation, synaptic dysfunction and memory deficiency.
In conclusion, we show that amyloid-induced neuroinflammation leads to epigenetic suppression of NLGN1 expression, which under-lies amyloid fibril–induced glutamatergic synapse dysfunction and memory deficiency in rodents. Microglia-mediated inflammation may increase the activity of several transcriptional repressors, such as HDAC2 and MeCP2, and epigenetically suppress NLGN1 expres-sion in hippocampal neurons, which consequently impairs central glutamatergic synapses and memory in the brain. Understanding the epigenetic mechanism leading to the interaction between neuro-inflammation and glutamatergic transmission may provide insight into the pathogenesis of amyloid-associated diseases.
METHODSMethods and any associated references are available in the online version of the paper.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.
AcknowledgmentSSupport of the Lerner Research Institute at the Cleveland Clinic.
AUtHoR contRIBUtIonSB.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.
comPetIng FInAncIAl InteReStSThe authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
1. Hardy, J. & Selkoe, D.J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353–356 (2002).
2. Middei, S., Geracitano, R., Caprioli, A., Mercuri, N. & Ammassari-Teule, M. Preserved fronto-striatal plasticity and enhanced procedural learning in a transgenic mouse model of Alzheimer’s disease overexpressing mutant hAPPswe. Learn. Mem. 11, 447–452 (2004).
3. Südhof, T.C. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903–911 (2008).
4. Choi, Y.B. et al. Neurexin-neuroligin transsynaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in Aplysia. Neuron 70, 468–481 (2011).
5. Ye, X. & Carew, T.J. Transsynaptic coordination of presynaptic and postsynaptic modifications underlying enduring synaptic plasticity. Neuron 70, 379–381 (2011).
6. Blundell, J. et al. Neuroligin-1 deletion results in impaired spatial memory and increased repetitive behavior. J. Neurosci. 30, 2115–2129 (2010).
7. Dinamarca, M.C., Weinstein, D., Monasterio, O. & Inestrosa, N.C. The synaptic protein neuroligin-1 interacts with the amyloid beta-peptide. Is there a role in Alzheimer’s disease? Biochemistry 50, 8127–8137 (2011).
8. Saura, C.A., Servian-Morilla, E. & Scholl, F.G. Presenilin/gamma-secretase regulates neurexin processing at synapses. PLoS ONE 6, e19430 (2011).
9. Guan, J.S. et al. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459, 55–60 (2009).
10. Gräff, J. et al. An epigenetic blockade of cognitive functions in the neuro-degenerating brain. Nature 483, 222–226 (2012).
11. Lombardi, P.M., Cole, K.E., Dowling, D.P. & Christianson, D.W. Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes. Curr. Opin. Struct. Biol. 21, 735–743 (2011).
12. Guy, J., Cheval, H., Selfridge, J. & Bird, A. The role of MeCP2 in the brain. Annu. Rev. Cell Dev. Biol. 27, 631–652 (2011).
13. Na, E.S., Nelson, E.D., Kavalali, E.T. & Monteggia, L.M. The impact of MeCP2 loss- or gain-of-function on synaptic plasticity. Neuropsychopharmacology 38, 212–219 (2013).
14. Yoon, H.G., Chan, D.W., Reynolds, A.B., Qin, J. & Wong, J. N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso. Mol. Cell 12, 723–734 (2003).
15. Jones, P.L. et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19, 187–191 (1998).
16. Wyss-Coray, T. Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Nat. Med. 12, 1005–1015 (2006).
©20
14 N
atu
re A
mer
ica,
Inc.
All
rig
hts
res
erve
d.
nature neurOSCIenCe advance online publication
a r t I C l e S
17. Grathwohl, S.A. et al. Formation and maintenance of Alzheimer’s disease beta-amyloid plaques in the absence of microglia. Nat. Neurosci. 12, 1361–1363 (2009).
18. Wu, J. et al. Activation of the CB2 receptor system reverses amyloid-induced memory deficiency. Neurobiol. Aging 34, 791–804 (2013).
19. Solito, E. & Sastre, M. Microglia function in Alzheimer’s disease. Front. Pharmacol. 3, 14 (2012).
20. Chacón, M.A., Barria, M.I., Soto, C. & Inestrosa, N.C. Beta-sheet breaker peptide prevents Aβ-induced spatial memory impairments with partial reduction of amyloid deposits. Mol. Psychiatry 9, 953–961 (2004).
21. Sun, B. et al. Imbalance between GABAergic and glutamatergic transmission impairs adult neurogenesis in an animal model of Alzheimer’s disease. Cell Stem Cell 5, 624–633 (2009).
22. Bie, B. et al. Upregulation of nerve growth factor in central amygdala increases sensitivity to opioid reward. Neuropsychopharmacology 37, 2780–2788 (2012).
23. Suzuki, K. et al. Activity-dependent proteolytic cleavage of neuroligin-1. Neuron 76, 410–422 (2012).
24. Fischer, A., Sananbenesi, F., Mungenast, A. & Tsai, L.H. Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol. Sci. 31, 605–617 (2010).
25. Zhang, Z., Cai, Y.Q., Zou, F., Bie, B. & Pan, Z.Z. Epigenetic suppression of GAD65 expression mediates persistent pain. Nat. Med. 17, 1448–1455 (2011).
26. Tao, J. et al. Phosphorylation of MeCP2 at serine 80 regulates its chromatin association and neurological function. Proc. Natl. Acad. Sci. USA 106, 4882–4887 (2009).
27. Zhou, Z. et al. Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron 52, 255–269 (2006).
28. Bie, B. et al. Nerve growth factor-regulated emergence of functional delta-opioid receptors. J. Neurosci. 30, 5617–5628 (2010).
29. Nan, X. et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386–389 (1998).
30. Jankowsky, J.L. et al. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum. Mol. Genet. 13, 159–170 (2004).
31. Ransohoff, R.M. & Cardona, A.E. The myeloid cells of the central nervous system parenchyma. Nature 468, 253–262 (2010).
32. Prinz, M., Priller, J., Sisodia, S.S. & Ransohoff, R.M. Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat. Neurosci. 14, 1227–1235 (2011).
33. Dean, C. & Dresbach, T. Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function. Trends Neurosci. 29, 21–29 (2006).
34. Ting, J.T., Peca, J. & Feng, G. Functional consequences of mutations in postsynaptic scaffolding proteins and relevance to psychiatric disorders. Annu. Rev. Neurosci. 35, 49–71 (2012).
35. Chih, B., Engelman, H. & Scheiffele, P. Control of excitatory and inhibitory synapse formation by neuroligins. Science 307, 1324–1328 (2005).
36. Levinson, J.N. et al. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificity. J. Biol. Chem. 280, 17312–17319 (2005).
37. Lee, S.T. et al. miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model. Ann. Neurol. 72, 269–277 (2012).
38. Sultana, R., Banks, W.A. & Butterfield, D.A. Decreased levels of PSD95 and two associated proteins and increased levels of BCl2 and caspase 3 in hippocampus from subjects with amnestic mild cognitive impairment: insights into their potential roles for loss of synapses and memory, accumulation of Aβ, and neurodegeneration in a prodromal stage of Alzheimer’s disease. J. Neurosci. Res. 88, 469–477 (2010).
39. Zeng, Y. et al. Epigenetic enhancement of BDNF signaling rescues synaptic plasticity in aging. J. Neurosci. 31, 17800–17810 (2011).
40. Skene, P.J. et al. Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol. Cell 37, 457–468 (2010).
41. Kishi, N. & Macklis, J.D. MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Mol. Cell. Neurosci. 27, 306–321 (2004).
42. Adkins, N.L. & Georgel, P.T. MeCP2: structure and function. Biochem. Cell Biol. 89, 1–11 (2011).
43. Samaco, R.C. & Neul, J.L. Complexities of Rett syndrome and MeCP2. J. Neurosci. 31, 7951–7959 (2011).
44. Cohen, S. et al. Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function. Neuron 72, 72–85 (2011).
45. Li, H., Zhong, X., Chau, K.F., Williams, E.C. & Chang, Q. Loss of activity-induced phosphorylation of MeCP2 enhances synaptogenesis, LTP and spatial memory. Nat. Neurosci. 14, 1001–1008 (2011).
46. Gonzales, M.L., Adams, S., Dunaway, K.W. & LaSalle, J.M. Phosphorylation of distinct sites in MeCP2 modifies cofactor associations and the dynamics of transcriptional regulation. Mol. Cell. Biol. 32, 2894–2903 (2012).
47. Rutlin, M. & Nelson, S.B. MeCP2: phosphorylated locally, acting globally. Neuron 72, 3–5 (2011).
48. Kimura, H. & Shiota, K. Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1. J. Biol. Chem. 278, 4806–4812 (2003).
49. Dong, E., Guidotti, A., Grayson, D.R. & Costa, E. Histone hyperacetylation induces demethylation of reelin and 67-kDa glutamic acid decarboxylase promoters. Proc. Natl. Acad. Sci. USA 104, 4676–4681 (2007).
50. Correa, F., Mallard, C., Nilsson, M. & Sandberg, M. Activated microglia decrease histone acetylation and Nrf2-inducible anti-oxidant defence in astrocytes: restoring effects of inhibitors of HDACs, p38 MAPK and GSK3beta. Neurobiol. Dis. 44, 142–151 (2011).
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14 N
atu
re A
mer
ica,
Inc.
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rig
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res
erve
d.
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ONLINE METHODSAnimals. All animal procedures were approved by the Animal Care and Use Committee of Cleveland Clinic. Adult male Sprague–Dawley rats (200–250 g, Charles River) were used, and all experiments were performed during the light cycle. Tg-APPsw/PSEN1DE9 (APP/PS1) and C57BL/6 mice were purchased from Jackson Laboratory and were maintained following standard protocols; they were used at 6 months old. The animals were randomly assigned to different groups with specific treatment, and another party blinded the experimenter to the indi-vidual groups. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications9,18.
microinjection into the hippocampal cA1 area. The rats were anesthetized with sodium pentobarbital (45 mg/kg i.p.) and restrained in a stereotaxic apparatus28. Aβ1–40 fibrils were formed as described previously20. Aβ1–40 fibrils (10 µg per 3 µl), Aβ40–1 fibrils (10 µg per 3 µl) or 3 µl of artificial cer-ebrospinal fluid were injected stereotaxically and bilaterally into each hippo-campus (anteroposterior, −3.5 mm; mediolateral, ± 2.0 mm; dorsoventral, −3.0 mm)51 using a 10-µl Hamilton syringe with a 27-G stainless steel needle at a rate of 0.5 µl/min. This experimental model has been used for studying Alzheimer’s disease20,52,53.
For in vivo treatment by microinjection, a 26-gauge double-guide cannula was inserted into the brain, aimed at the hippocampal CA1 area (same coordi-nates as above)28. The guide cannula was then cemented in place to the skull and securely capped. The rat was allowed to recover for at least 5 d before subsequent treatment. siRNA for Nlgn1, Hdac2 or Mecp2 were commercially designed and synthesized by Invitrogen. siRNA of all types (5 nmol in 3 µl per side), lipo-polysaccharide (LPS, 5 µg per side), SAHA (0.3 nmol per side), minocycline (5 µg per side) or saline was delivered into the hippocampal CA1 area through a 33-G double injector with a rate of 0.5 µl/min. The injection sites for hippocampal CA1 were histologically verified afterward by injecting same volume of ink18,54.
morris water maze test. The Morris water maze test (n = 10 in each group) was employed to determine memory function in rats18,20. The water maze test was performed in a circular tank (diameter 1.8 m) filled with water. A platform (15 cm in diameter) was submerged below the water’s surface in the center of the target quadrant. The platform was camouflaged by placing opacifying material (tempera paint) in the water. The swimming path of the rat was recorded by a video cam-era and analyzed by EthoVision XT software (Noldus Information Technology). For each training session, the rats were placed into the maze consecutively from four random points of the tank and were allowed to search for the platform for 120 s. If the rat did not find the platform within 120 s, it was gently guided to it. Rats were allowed to remain on the platform for 20 s. The latency for each trial was recorded for analysis. During the probe trial, the platform was removed from the tank, and the rats were allowed to swim in the maze for 60 s.
chromatin immunoprecipitation (chIP). The ChIP assay was performed as previously described22 with minor modifications. Hippocampal CA1 tissues were collected and cross-linked with 2% formaldehyde for 5 min, and chromatin was solubilized and sonicated with a sonic Dismembrator 250 (Fisher Scientific) on ice six times for 30 s each, followed by 1 min cooling on ice, to produce frag-ments of approximately 200–500 bp. The polyclonal antibody against histone H3 acetylated at the lysine residues of N terminus, anti-HDAC2 antibody and anti-MeCP2 antibody (1:100, Millipore) were added to each sample and incub-ated overnight at 4 °C with gentle mixing. Mouse or rabbit IgG was used as the negative control, and monoclonal anti–RNA polymerase II antibody (1:100, Millipore) was used as a positive control. Immunocomplexes were recovered by salmon sperm DNA–protein A agarose beads and sequentially extensively washed. The cross-linking between histones and DNA was reversed, and DNA fragments were purified with phenol-chloroform extraction followed by acid ethanol precipitation.
Real-time PCR was performed to amplify approximately 200-bp fragments within the Nlgn1 transcriptional control region. Primers sets were as follows: Nlgn1, 5′-TGCTCTATCACGGCACATTC-3′, 5′-AAAGGAAGCATCCAATC GAA-3′; Gapdh, 5′-AGACAGCCGCATCTTCTTGT-3′, 5′-CGTCC TCTACCATCCTCTGC-3′. Primer sets for mice were as follows: Nlgn1, 5′-CCTTTGATCTCCCACAGACAG-3′, 5′-GGAAAGTGCCATGAAA CACC-3′; Gapdh (5′-CTCCTGTGTTCTCCCCTCAC-3′, 5′-GTT
GAATTGGAGGAGGCTCA-3′. Amplifications were run in triplicate. ∆Ctnormalized ChIP = CtChIP − [CtInput − log2(input dilution factor)]; and ChIP/input ratio was calculated as 2(–∆Ct[normalized ChIP]).
methylated dnA immunoprecipitation. Methylated DNA immunopre-cipitation was performed as described previously with minor modification55. Hippocampal CA1 tissue was homogenized, and genomic DNA was sonicated on ice six times for 30 s each. Polyclonal antibody against 5-methylcytosine (Millipore) was added to each sample and incubated overnight at 4 °C with gentle mixing. Immunocomplexes were recovered by salmon sperm DNA–protein A agarose beads and sequentially extensively washed, and DNA frag-ments were purified with phenol-chloroform extraction followed by acid ethanol precipitation. Immunoprecipitated DNA was subjected to quantita-tive real-time PCR using primers specific for about 250-bp segments cor-responding to CpG sites within the Nlgn1 promoter region. Primers sets for rats were as follows: Nlgn1, 5′-TGCTCTATCACGGCACATTC-3′, 5′-CCCTGAATCAAGACCCCATA-3′; Gapdh, 5′-AGACAGCCGCATCT TCTTGT-3′, 5′-CTGCGGGAGAAGAAAGTCAG-3′. Primer sets for mice were as follows: Nlgn1, 5′-CCTTTGATCTCCCACAGACAG-3′, 5′-GCCTCCATGTGAGTTCAGGT-3′; Gapdh, 5′-CTCCTGTGTTCTCC CCTCAC-3′, 5′-GTTGAATTGGAGGAGGCTCA-3′. Amplifications were run in triplicate, and the PCR data were analyzed as above.
Quantitative Rt-PcR for mRnA analysis. RNA was isolated from hippocampal CA1 tissue using a single-step RNA isolation protocol and quantified as previ-ously described56. Reverse transcription and real-time RT-PCR were performed in triplicate with Nlgn1 exon primers (5′-CTTTCCAGCTGGGCTGTTAG-3′, 5′-ATCGATCACAGGTCCAAAGG-3′), Gapdh primers (5′-AGACAGCC GCATCTTCTTGT-3′, 5′-CTTGCCGTGGGTAGAGTCAT-3′), Mecp2 primers (5′-CCGGGGACCTATGTATGATG-3′, 5′-GGTGTCTCCCACCTTTTC AA-3′) and Hdac2 primers (5′-TGGCCTTTCTGAGCTGATTT-3′, 5′-AG AGGGTCTCTGCCACTGAA-3′). Fold change was calculated using the ∆∆Ct method. The entire protocol was repeated in triplicate, and the mean and s.e.m. were calculated.
elISA. Hippocampal CA1 IL-1β concentrations were measured using a commercially available ELISA kit (R&D Systems) following the manufacturer’s protocol.
Immunostaining. Immunostaining on the serial sections containing hippo-campal CA1 area in all groups (30 µm, 15–20 per rat, n = 5 rats per group) was performed as previously described18,28. Mouse monoclonal antibody against the microglial marker CD11b (1:200, Abcam, Cambridge, MA), monoclonal anti-HDAC2 (Millipore, 1:1,000), monoclonal anti-MeCP2 (1:1,000, Cell Signaling), polyclonal anti-GFAP (Abcam, 1:500), polyclonal anti-NeuN antibody (Millipore, 1:500), guinea pig polyclonal anti-vGLUT1 (Millipore, 1:250), mouse monoclonal anti-GAD67 (Millipore, 1:1,000) and rabbit monoclonal anti-synapsin (Millipore, 1:200) were used. Then the sections were incubated with FITC-Cy3–conjugated secondary antibody (1:500, Jackson ImmunoResearch) or Alexa Fluor 633 (1:500, Invitrogen) for 1 h. Omission of primary or secondary antibodies resulted in no immunostaining. All stained sections were blindly examined and analyzed.
Protein extraction and immunoblotting. The protocol for protein extraction and immunoblotting was generally based on previous reports28,57. Hippocampal CA1 tissues were collected and lysed in ice-cold lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.02 mM NaN3, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1% Triton X-100 and proteinase inhibitor cocktail. Cytoplasmic and nuclear protein were extracted12 and subjected to 7.5% SDS-PAGE followed by immunoblotting. The blots were incubated overnight at 4 °C with the pri-mary antibodies as follows: monoclonal anti-NLGN1 primary antibody (1:1,000; Synaptic System, or 1:200; Santa Cruz Biotechnology), monoclonal anti-HDAC2 antibody (1:1,000; Cell Signaling), monoclonal anti-HDAC1 antibody (1:1,000; Cell Signaling), rabbit polyclonal anti-MeCP2 antibody (1:1,000; Millipore), rabbit polyclonal anti-ADAM10 antibody (1:500, Millipore), rabbit monoclonal anti-presenilin 1 antibody (1:1,000, Cell Signaling), monoclonal anti-HDAC1 (1:1,000, Millipore), monoclonal anti-mSin3A (1:1,000, Cell Signaling) or polyclonal anti-β-actin antibody (1:2,000; Cell Signaling). The membranes
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were washed extensively and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibody (1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit; Amersham Biosciences). The intensity of the bands was captured digitally and analyzed quantitatively with ImageJ software. The immunoreactivity of all proteins was normalized to that of β-actin. Full length pictures of the blots are presented in Supplementary Figure 11.
coimmunoprecipitation and immunoblotting. Coimmunoprecipitation and immunoblotting were performed as previously described28. Hippocampal CA1 tissues were homogenized in lysis buffer (0.1% Triton X-100, 150 mM NaCl, 2.5 mM KCl and 10 mM Tris-HCl, pH 7.4) with protease inhibitors and phos-phatase inhibitors). Cleared lysate was incubated overnight at 4 °C with rabbit polyclonal anti-MeCP2 antibody (1:100, Cell Signaling) and precipitated for 5 h by protein A–agarose beads (Sigma). Precipitates were washed extensively, and the proteins were separated on a 7.5% SDS-PAGE gel and blotted to nitro-cellulose membrane. The blots were incubated overnight at 4 °C with monoclonal anti-HDAC2 (1:1,000, Cell Signaling), anti-HDAC1 (1:1,000, Cell Signaling) or anti-mSin3A (1:1,000, Cell Signaling). The membrane was washed with Tris-buffered saline and incubated with HRP-conjugated anti-mouse IgG antibody (1:10,000, GE Healthcare) with ECL detection thereafter. For detecting serine phosphorylation of MeCP2, the membrane was incubated with monoclonal anti-phosphoserine (1:1,000, Cell Signaling), anti-pSer80 MeCP2 (1:1,000, ECM Biosciences) or anti-pSer421 MeCP2 (1:1,000, ECM Biosciences), extensively washed and then incubated with HRP-conjugated anti-mouse IgG antibody (1:10,000) for ECL detection. The total amount of immunoprecipitated MeCP2 was detected by incubating the stripped membranes with monoclonal anti-MeCP2 antibody (1:1,000, Millipore). Full length pictures of the blots are presented in Supplementary Figure 11.
golgi impregnation. Golgi impregnation was performed with the FD rapid GolgiStain kit (FD NeuroTechnologies Inc.)9. Golgi–Cox-stained brains were cut to cross-sections 100 µm thick with a Vibratome (Leica), and digitized and analyzed using Image-Pro Plus software (Media Cybernetics, Warrendale, PA). For each experimental group, a minimum of ten cells per slice (five animals) were analyzed. The number of apical and basal spines on hippocampal CA1 pyramidal neurons was counted blindly.
transmission electron microscopy. Rat brains (4 or 5 per group) were per-fused in 0.2 M sodium cacodylate buffer, pH 7.4, containing 2.5% glutaraldehyde and 4% formaldehyde. After washing 3 times in the same buffer, hippocampal CA1 tissues were postfixed with 1% aqueous osmium tetroxide for 1 h at room temperature (22–24 °C). The tissues were washed in sodium cacodylate buffer, followed by maleate buffer, pH 5.1, and then dehydrated with ascending grades of ethanol and propylene oxide. Samples were embedded in LX-112 medium, polymerized at 70 °C for 48 h, trimmed, sectioned at 70–90 nm, poststained in 50% saturated uranyl acetate and 0.2% lead citrate, and examined with a transmis-sion electron microscope (Philips CM120)58. Sections were blinded and analyzed independently by two different researchers.
electron microscopy quantification. At least 21 single-section transmission electron micrographs were randomly taken from dense neuropil of the hippo-campal CA1 area59. The area of each image taken at 23,000 × was approximately 27.7 µm2. Synapses were identified on the basis of generally accepted criteria59.
Hippocampal slice preparation and whole-cell recordings. Brain slices contain-ing hippocampal CA1 area were prepared as previously described28,54,60. The brain was quickly removed and cut on a Vibratome in cold physiological saline
to obtain coronal slices (300 µm thick) containing the hippocampus. Whole-cell voltage-clamp recordings from the CA1 area were taken using an Axopatch 200B amplifier (Molecular Devices) with 2–4 MΩ glass electrodes containing an internal solution consisting of (in mM) potassium gluconate or cesium meth-anesulfonate, 125; NaCl, 5; MgCl2 1; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; pH 7.3; 290–300 mOsm. A seal resistance of ≥2 GΩ and an access resistance of 15–20 MΩ were considered acceptable. The series resistance was optimally compensated by ≥70% and constantly monitored throughout the experiments. Schaffer collateral–commissural fibers were stimulated by ultrathin concentric bipolar electrodes (FHC Inc.), and the excitatory postsynaptic currents (EPSCs) were recorded in the CA1 area in the presence of bicuculline (30 µM). The evoked EPSCs were filtered at 2 kHz, digitized at 10 kHz, and acquired and analyzed using AxoGraph X software. The amplitude of the EPSCs was monitored for a baseline period of at least 15 min. If synaptic transmission was stable (<15% change in EPSC amplitude over 15 min), long-term potentiation (LTP) was induced by a single high-frequency electric stimuli train (100 Hz for 1 s)61. mEPSCs were recorded in the presence of tetrodotoxin (TTX, 1 µM) and bicuculline (10 µM) at holding potential of −70 mV, and mIPSCs were recorded in the presence of TTX (1 µM), APV (10 µM) and CNQX (10 µM) at holding potential of −50 mV. All electrophysiological experiments were performed at room tempera-ture (22–24 °C).
compounds and data analysis. Aβ peptide consisting of residues 1–40 of the human wild-type sequence (Aβ1–40); Aβ40–1 was purchased from Bachem (Torrance, CA). AP-5 (d-2-amino-5-phosphonopentanoate), CNQX (6-cyano-2,3-dihydroxy-7-nitroquinoxaline), bicuculline and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Tocris (Ellisville, MO).
For electrophysiological and behavioral analysis, the data were compared with repeated measures ANOVA followed by post hoc analysis. The data from histo-logical studies, western blots, real-time PCR and other studies were analyzed using Student’s t-test or one-way ANOVA test followed by post hoc analysis. All statistical analyses were performed with BMDP statistical software (Statistical Solutions, Saugus, MA). Normality was tested using Shapiro and Wilk’s test for normality. All data were expressed as means ± s.e.m. and were considered sig-nificant when P < 0.05.
51. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates (Academic, New York, 1998).
52. Shin, R.W. et al. Amyloid beta-protein (Aβ) 1–40 but not Aβ1–42 contributes to the experimental formation of Alzheimer disease amyloid fibrils in rat brain. J. Neurosci. 17, 8187–8193 (1997).
53. Ahmed, T., Enam, S.A. & Gilani, A.H. Curcuminoids enhance memory in an amyloid-infused rat model of Alzheimer’s disease. Neuroscience 169, 1296–1306 (2010).
54. Bie, B., Zhu, W. & Pan, Z.Z. Ethanol-induced delta-opioid receptor modulation of glutamate synaptic transmission and conditioned place preference in central amygdala. Neuroscience 160, 348–358 (2009).
55. Lubin, F.D., Roth, T.L. & Sweatt, J.D. Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J. Neurosci. 28, 10576–10586 (2008).
56. Bie, B., Peng, Y., Zhang, Y. & Pan, Z.Z. cAMP-mediated mechanisms for pain sensitization during opioid withdrawal. J. Neurosci. 25, 3824–3832 (2005).
57. Bie, B., Brown, D.L. & Naguib, M. Increased synaptic GluR1 subunits in the anterior cingulate cortex of rats with peripheral inflammation. Eur. J. Pharmacol. 653, 26–31 (2011).
58. Gallicano, G.I. et al. Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage. J. Cell Biol. 143, 2009–2022 (1998).
59. DeFelipe, J., Marco, P., Busturia, I. & Merchán-Pérez, A. Estimation of the number of synapses in the cerebral cortex: methodological considerations. Cereb. Cortex 9, 722–732 (1999).
60. Bie, B., Zhu, W. & Pan, Z.Z. Rewarding morphine-induced synaptic function of delta-opioid receptors on central glutamate synapses. J. Pharmacol. Exp. Ther. 329, 290–296 (2009).
61. Shipton, O.A. et al. Tau protein is required for amyloid β-induced impairment of hippocampal long-term potentiation. J. Neurosci. 31, 1688–1692 (2011).
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