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The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid (ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing. Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome editing.

Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica–metal–organic framework hybrid nanoparticle (SMOF NP) consisting of both silica and zeolitic imidazole framework (ZIF). This unique SMOF NP offers a high loading content and efficiency, excellent stability, and robust intracellular delivery of a variety of payloads, including hydrophilic small molecule drugs (e.g., doxorubicin hydrochloride), nucleic acids (e.g., DNA and mRNA), and genome-editing machineries (e.g., Cas9-sgRNA ribonucleoprotein (RNP), and RNP together with donor DNA (e.g., RNP + ssODN)). The superior drug delivery/gene transfection/genome-editing efficiencies of the SMOF NP are attributed to its pH-controlled release and endosomal escape capabilities due to the proton sponge effect enabled by the imidazole moieties in the SMOF NPs. Moreover, the surface of the SMOF NP can be easily customized (e.g., PEGylation and ligand conjugation) via various functional groups incorporated into the silica component. RNP-loaded SMOF NPs induced efficient genome editing in vivo in murine retinal pigment epithelium (RPE) tissue via subretinal injection, providing a highly promising nanoplatform for the delivery of a wide range of hydrophilic payloads.

Aging is a significant factor in the development of age-related diseases but how aging disrupts cellular homeostasis to cause age-related retinal disease is unknown. Here, we further our studies on transmembrane protein 135 (Tmem135), a gene involved in retinal aging, by examining the transcriptomic profiles of wild-type, heterozygous and homozygous Tmem135 mutant posterior eyecup samples through RNA sequencing (RNA-Seq). We found significant gene expression changes in both heterozygous and homozygous Tmem135 mutant mouse eyecups that correlate with visual function deficits. Further analysis revealed that expression of many genes involved in lipid metabolism are changed due to the Tmem135 mutation. Consistent with these changes, we found increased lipid accumulation in mutant Tmem135 eyecup samples. Since mutant Tmem135 mice have similar ocular pathologies as human age-related macular degeneration (AMD) eyes, we compared our homozygous Tmem135 mutant eyecup RNA-Seq dataset with transcriptomic datasets of human AMD donor eyes. We found similar changes in genes involved in lipid metabolism between the homozygous Tmem135 mutant eyecups and AMD donor eyes. Our study suggests that the Tmem135 mutation affects lipid metabolism as similarly observed in human AMD eyes, thus Tmem135 mutant mice can serve as a good model for the role of dysregulated lipid metabolism in AMD.

Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing. Polymer nanocapsules, assembled around a ribonucleoprotein complex of Cas9 nuclease and a single-guide RNA, enable safe and efficient gene editing in vitro and in vivo.

Ischemic stroke is a major cause of long-term disabilities, including vision loss. Neuronal and blood vessel maturation can affect the susceptibility of and outcome after ischemic stroke. Although we recently reported that exposure of neonatal mice to hypoxia–ischemia (HI) severely compromises the integrity of the retinal neurovasculature, it is not known whether juvenile mice are similarly impacted. Here we examined the effect of HI injury in juvenile mice on retinal structure and function, in particular the susceptibility of retinal neurons and blood vessels to HI damage. Our studies demonstrated that the retina suffered from functional and structural injuries, including reduced b-wave, thinning of the inner retinal layers, macroglial remodeling, and deterioration of the vasculature. The degeneration of the retinal vasculature associated with HI resulted in a significant decrease in the numbers of pericytes and endothelial cells as well as an increase in capillary loss. Taken together, these findings suggest a need for juveniles suffering from ischemic stroke to be monitored for changes in retinal functional and structural integrity. Thus, there is an emergent need for developing therapeutic approaches to prevent and reverse retinal neurovascular dysfunction with exposure to ischemic stroke.

Purpose Aging is a critical risk factor for the development of retinal diseases, but how aging perturbs ocular homeostasis and contributes to disease is unknown. We identified transmembrane protein 135 (Tmem135) as a gene important for regulating retinal aging and mitochondrial dynamics in mice. Overexpression of Tmem135 causes mitochondrial fragmentation and pathologies in the hearts of mice. In this study, we examine the eyes of mice overexpressing wild-type Tmem135 (Tmem135 TG) and compare their phenotype to Tmem135 mutant mice. Methods Eyes were collected for histology, immunohistochemistry, electron microscopy, quantitative PCR, and Western blot analysis. Before tissue collection, electroretinography (ERG) was performed to assess visual function. Mouse retinal pigmented epithelium (RPE) cultures were established to visualize mitochondria. Results Pathologies were observed only in the RPE of Tmem135 TG mice, including degeneration, migratory cells, vacuolization, dysmorphogenesis, cell enlargement, and basal laminar deposit formation despite similar augmented levels of Tmem135 in the eyecup (RPE/choroid/sclera) and neural retina. We observed reduced mitochondria number and size in the Tmem135 TG RPE. ERG amplitudes were decreased in 365-day-old mice overexpressing Tmem135 that correlated with reduced expression of RPE cell markers. In Tmem135 mutant mice, RPE cells are thicker, smaller, and denser than their littermate controls without any signs of degeneration. Conclusions Overexpression and mutation of Tmem135 cause contrasting RPE abnormalities in mice that correlate with changes in mitochondrial shape and size (overfragmented in TG vs. overfused in mutant). We conclude proper regulation of mitochondrial homeostasis by TMEM135 is critical for RPE health.

Background Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease yet with no effective treatment available. The sigma-1 receptor (S1R), a ligand-regulated chaperone, emerges as a potential retina-protective therapeutic target. In particular, pharmacological activation of S1R was recently shown to rescue cones in the rd10 mouse, a rod Pde6b mutant that recapitulates the RP pathology of autonomous rod degeneration followed by secondary death of cones. The mechanisms underlying the S1R protection for cones are not understood in detail. Methods By rearing rd10/S1R−/− and rd10/S1R+/+ mice in dim light to decelerate rapid rod/cone degeneration, we were able to compare their retinal biochemistry, histology and functions throughout postnatal 3–6 weeks (3 W–6 W). Results The receptor-interacting protein kinases (RIP1/RIP3) and their interaction (proximity ligation) dramatically up-regulated after 5 W in rd10/S1R−/− (versus rd10/S1R+/+) retinas, indicative of intensified necroptosis activation, which was accompanied by exacerbated loss of cones. Greater rod loss in rd10/S1R−/− versus rd10/S1R+/+ retinas was evidenced by more cleaved Caspase3 (4 W) and lower rod electro-retinographic a-waves (4 W–6 W), concomitant with reduced LC3-II and CHOP (4 W–6 W), markers of autophagy and endoplasmic reticulum stress response, respectively. However, the opposite occurred at 3 W. Conclusion This study reveals previously uncharacterized S1R-associated mechanisms during rd10 photoreceptor degeneration, including S1R’s influences on necroptosis and autophagy as well as its biphasic role in rod degeneration upstream of cone death. Electronic supplementary material The online version of this article (doi:10.1186/s13024-017-0202-z) contains supplementary material, which is available to authorized users.

The KCNJ13 gene encodes the inwardly rectifying potassium channel, Kir7.1. Mutations in this gene cause childhood blindness, in which the a- and b-wave responses of electroretinogram (ERG) are abolished. The ERG a-wave is the light-induced hyperpolarization of retinal photoreceptors, and the b-wave is the depolarization of ON-bipolar cells. The Kir7.1 channel is localized to the apical aspects of retinal pigment epithelium (RPE) cells and contributes to a delayed c-wave response. We sought to understand why a defect in an RPE ion-channel result in abnormal electrophysiology at the level of the retinal neurons. We have established the expression of Kir7.1 channels in the mouse RPE. ERGs recorded after mice Kir7.1 suppression by shRNA, or by blocking with VU590, showed reduced a-, b- and c-wave amplitudes. In contrast, the Kir7.1 blocker had no effect on the ex-vivo isolated mouse retina ERG where the RPE is not attached to the isolated retina preparation. Finally, we confirmed the specificity of VU590 action by inhibition of native mouse RPE Kir7.1 current in patch-clamp experiment. We propose that mutant RPE Kir7.1 channels contribute directly to the abnormal ERG associated with blindness via alterations in sub-retinal space K+ homeostasis in the vicinity of the photoreceptor outer segment.

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2–5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 μg). No rabbit or mouse eye irritation was observed over 12–17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 μM, PEDF 336 (100 μM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G- Y - D - L-Y-R-V ) was identified, variants (adipic-Sar- Y - N - L-Y-R-V ) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Background Hyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs. Methods We performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon. Results SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations. Conclusions HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

Mutations in the KCNJ13 gene that encodes the inwardly rectifying potassium channel Kir7.1 cause snowflake vitreoretinal degeneration (SVD) and leber congenital amaurosis (LCA). Kir7.1 controls the microenvironment between the photoreceptors and the retinal pigment epithelium (RPE) and also contributes to the function of other organs such as uterus and brain. Heterologous expressions of the mutant channel have suggested a dominant-negative loss of Kir7.1 function in SVD, but parallel studies in LCA16 have been lacking. Herein, we report the identification of a novel nonsense mutation in the second exon of the KCNJ13 gene that leads to a premature stop codon in association with LCA16. We have determined that the mutation results in a severe truncation of the Kir7.1 C-terminus, alters protein localization, and disrupts potassium currents. Coexpression of the mutant and wild-type channel has no negative influence on the wild-type channel function, consistent with the normal clinical phenotype of carrier individuals. By suppressing Kir7.1 function in mice, we were able to reproduce the severe LCA electroretinogram phenotype. Thus, we have extended the observation that Kir7.1 mutations are associated with vision disorders to include novel insights into the molecular mechanism of disease pathobiology in LCA16.

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P(1,3,4,5) agonist) and SEW 2871 (an S1P(1) agonist) had no effects on pacemaker activity. Suramin (an S1P(3) antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P(2) antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P(2) in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH(2)-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca(2+) ([Ca(2+)](i)) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca(2+)](i) oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P(2) via regulation of external and internal Ca(2+) and mitogenactivated protein kinase activation.

Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs.We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine.LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4(+/+) mice was not present in TLR4(-/-) mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K(+) channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E(2) (PGE(2))-EP(2) receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs.LPS can activate ICCs to release NO and PGE(2) through TLR4 activation. The released NO and PGE(2) inhibit pacemaker currents by activating ATP-sensitive K(+) channels. The LPS actions are mediated by tyrosine kinase signaling pathways.

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na+- free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca2+-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca2+ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca2+-release through neurotensin receptor1.

AIM: To investigate lipopolysaccharide (LPS) related signal transduction in interstitial cells of Cajal (ICCs) from mouse small intestine. METHODS: For this study, primary culture of ICCs was prepared from the small intestine of the mouse. LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique. Immunocytochemistry was used to examine the expression of the proteins in ICCs. RESULTS: LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809, a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-L-arginine Methyl Ester, an inhibitor of nitric oxide (NO) synthase. Toll-like receptor 4, inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs. Catalase (antioxidant agent) had no action on LPS-induced action in ICCs. LPS actions were blocked by nuclear factor κB (NF-κB) inhibitor, actinomycin D (a gene transcription inhibitor), PD 98059 (a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases (MAPK) inhibitor]. SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO. CONCLUSION: LPS inhibit the pacemaker currents in ICCs via prostaglandin E2- and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-κB are implicated in these actions.

Background The bromodomain and extraterminal domain (BET) family proteins (BET2, BET3, and BET4) “read” (bind) histone acetylation marks via two distinct bromodomains (Brom1 and Brom2) facilitating transcriptional activation. These epigenetic “readers” play crucial roles in pathogenic processes such as inflammation. The role of BETs in influencing the degenerative process in the retina is however unknown. Methods We employed the rd10 mouse model (Pde6b rd10 mutation) of retinitis pigmentosa (RP) to examine the involvement of BET proteins in retinal neurodegeneration. Results Inhibition of BET activity by intravitreal delivery of JQ1, a BET-specific inhibitor binding both Brom1 and Brom2, ameliorated photoreceptor degeneration and improved electroretinographic function. Rescue effects of JQ1 were related to the suppression of retinal microglial activation in vivo, as determined by decreased immunostaining of activation markers (IBA1, CD68, TSPO) and messenger RNA (mRNA) levels of inflammatory cytokines in microglia purified from rd10 retinas. JQ1 pre-treatment also suppressed microglial activation in vitro, decreasing microglial proliferation, migration, and mRNA expression of inflammatory cytokines (TNFα, MCP-1, IL-1β, IL-6, and RANTES). Expression of BET2, but not BET3 and BET4, was significantly elevated during photoreceptor degeneration at postnatal day (PN)24 in retinas of rd10 mice relative to age-matched wild-type controls. siRNA knockdown of BET2 but not BET4, and the inhibitor of Brom2 (RVX208) but not of Brom1 (Olinone), decreased microglial activation. Conclusions These findings indicate that BET inhibition rescues photoreceptor degeneration likely via the suppression of microglial activation and implicates BET interference as a potential therapeutic strategy for the treatment of degenerative retinal diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0775-4) contains supplementary material, which is available to authorized users.