In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy

Significance

In mammals, the brain is the organ with the highest level of sialic acids, a family of negatively charged monosaccharides that are commonly expressed as outer terminal residues of cell-surface glycans. Brain sialoglycans play essential roles in brain development, cognition, and disease progression; however, in vivo visualization of the sialoglycan biosynthesis in the mouse brain has been impossible. Here, we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for metabolic labeling and visualization of brain sialoglycans in living mice. Applying LABOR, we visualized the biosynthesis of brain sialoglycans by in vivo fluorescence imaging and histological analysis, and identified important sialylated glycoproteins in the brain by glycoproteomics. We discovered that the turnover of sialoglycans is spatially regulated in distinct brain regions.

Abstract

Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood−brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse−chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation.

Sialic acids are a family of negatively charged monosaccharides that are commonly expressed as outer terminal residues of cell surface glycans and widely distributed throughout mammalian tissues (1). Intriguingly, the brain is the organ with the highest level of sialylated glycans and the only organ, in mammals, with more sialic acids carried by glycolipids than glycoproteins (2). Accumulating evidence indicates that sialic acids are an essential nutrient for brain development and cognition (3). Gangliosides (i.e., glycosphingolipids containing α2,3-linked sialic acids) undergo dramatic changes in both structural complexity and expression density as the brain develops and matures (4). Polysialic acid (PSA), a linear α2,8-linked polymer of sialic acid, is predominantly attached to the N-glycans of neural cell adhesion molecule, which regulates neuronal differentiation and migration (5). In addition, α2,3-linked sialic acids and, less commonly, α2,6-linked sialic acids terminate N-glycans and O-glycans on synaptic proteins, mediating neural transmission and synaptic plasticity (6, 7). Aberrant sialylation has been implicated in cancer cell metastasis to the brain (8), lysosomal storage disorders (9), and neurodegenerative diseases (10).

Sialic acid metabolism can be probed in vivo using the recently emerged bioorthogonal chemical reporter strategy, in which analogs of sialic acid or its biosynthetic precursor N-acetylmannosamine (ManNAc) containing a chemical reporter (e.g., the azide) are used as metabolic tracers for labeling sialoglycans in live cells and in living animals (11). To label sialoglycans in living mice or rats, peracetylated N-azidoacetylmannosamine (Ac₄ManNAz) was intraperitoneally (i.p.) injected into living animals and metabolically converted to the corresponding azido sialic acid, which was incorporated into the sialoglycans in a panel of organs, including the heart, kidney, and liver (12). Reacting the azides with an alkyne-containing fluorescent probe via bioorthogonal chemistry (e.g., copper-free click chemistry) enabled imaging of cardiac sialoglycans in intact rat hearts and revealed the up-regulation of sialylation during cardiac hypertrophy (13). However, sialoglycans in the brain cannot be labeled or visualized by using this strategy, presumably due to the inability of azidosugars to cross the blood−brain barrier (BBB).

Herein, we report the development of a liposome-assisted bioorthogonal reporter (LABOR) strategy for metabolic labeling of brain sialoglycans with 9-azido sialic acid (9AzSia), a sialic acid reporter, in living mice. In vivo copper-free click chemistry conjugated the incorporated azides with fluorescent probes and allowed for visualization of brain sialoglycans in living mice. Further, the LABOR labeling is compatible with histochemistry on brain sections, which revealed the distribution of newly synthesized sialoglycans in the brain. Click-labeling of the azide-incorporated brain with affinity tags enabled proteomic profiling of sialylated glycoproteins in the brain. Finally, we demonstrated that LABOR can be used to probe dynamic sialylation in distinct brain regions by performing pulse−chase experiments.

Results

LABOR-Mediated Delivery and Metabolism of 9AzSia in the Brain.

In search for a means to shuttle sialic acid chemical reporters into the brain, we were inspired by the brain delivery of small-molecule drugs using liposomes (14). We hypothesized that liposomes encapsulating azidosugars would cross the BBB and thus enable metabolic labeling of the brain sialoglycans with azides (Fig. 1A). To test this hypothesis, we evaluated 9AzSia and ManNAz, in their free, globally protected (i.e., Ac₄Me9AzSia and Ac₄ManNAz), and liposome-encapsulated forms, for brain labeling (Fig. 1B and SI Appendix, Scheme S1). Using a previously developed procedure (15), we prepared liposomes encapsulating 9AzSia (LP-9AzSia) and liposomes encapsulating ManNAz (LP-ManNAz) with a diameter of ∼200 nm and an azidosugar to lipid molar ratio of ∼1.3:1 (SI Appendix, Table S1). The liposome surface is PEGylated (i.e., functionalized with polyethylene glycol) by using the liposomal formulation made of dioleoylphosphatidylcholine, cholesterol, and PEGylated distearoylphosphatidylethanolamine at a molar ratio of 50:50:5. PEGylation is known to sterically stabilize liposomes and prolong the liposome half-life in the circulation, thus facilitating brain uptake and accumulation (16, 17).

Fig. 1.

LABOR enables metabolic incorporation of 9AzSia into the brain sialoglycans in living mice. (A) LP-9AzSia is injected i.v. into the tail vein of living mice. After crossing BBB and entering the brain tissues, LP-9AzSia is internalized into brain cells and releases 9AzSia. The sialic acid biosynthetic machinery uses 9AzSia and metabolically incorporates it into cellular sialoglycans. (B) Evaluation of six labeling protocols for metabolic incorporation of azides into brain sialoglycoproteins. Mice (n = 3 per treatment group) were administered daily with 0.70 mmol/kg LP-9AzSia [i.v. injection (i.v.), LP as the negative control (NC)], 9AzSia (i.v., PBS as the NC), Ac₄Me9AzSia [i.p. injection (i.p.), 70% (vol/vol) DMSO as the NC], LP-ManNAz (i.v., LP as the NC), ManNAz (i.v., PBS as the NC), or Ac₄ManNAz [i.p., 70% (vol/vol) DMSO as the NC] for 7 d. After a whole-body perfusion, the brain tissues were collected and the tissue lysates were reacted with alkyne-biotin, followed by anti-biotin Western blot analysis. Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) blot was used as the loading control.

BALB/c male mice were administered daily for 7 d by intravenous (i.v.) injection with free azidosugars or liposomal azidosugars dissolved in PBS, or by i.p. injection with Ac₄Me9AzSia or Ac₄ManNAz dissolved in 70% (vol/vol) DMSO. The dosage for all six labeling methods was kept the same, at 0.70 mmol/kg (calculated based on the azidosugars). After a whole-body perfusion, the brain tissues were harvested, homogenized, and reacted with an alkyne-functionalized biotin probe (alkyne-biotin) via Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC, also termed click chemistry) (18), followed by anti-biotin Western blot analysis, which showed that LP-9AzSia administration resulted in the incorporation of 9AzSia into brain sialoglycoproteins (Fig. 1B). In contrast, all other five methods did not lead to significant incorporation.

Gangliosides are the most abundant sialoglycoconjugates in the brain (2). To assess whether LP-9AzSia can metabolically label glycolipids in addition to glycoproteins, we extracted brain gangliosides from mice administered with LP-9AzSia using an established procedure (19). The isolated gangliosides were reacted with an alkyne-Cy5 conjugate (alkyne-Cy5) and analyzed by fluorometry, which indicated that brain gangliosides were incorporated with 9AzSia in the LP-9AzSia-treated mice (SI Appendix, Fig. S1A). Furthermore, the extracted gangliosides were resolved by high-performance thin layer chromatography (HPTLC). Staining of the HPTLC plates with the resorcinol reagent, which reacts specifically with sialic acid-containing molecules, exhibited the presence of GM1, GD1a, and GD1b as the major gangliosides in mouse brains (SI Appendix, Fig. S1B). To detect the incorporated azides on the HPTLC plates, we subjected the plates to a triphenylphosphine (PPh₃) solution to reduce the azides to primary amines, followed by staining with ninhydrin, which revealed two isoforms of GM1 as the major gangliosides that were metabolically incorporated with 9AzSia. Nevertheless, we could not rule out the possibility that other gangliosides were labeled with 9AzSia at a level lower than the detection sensitivity of PPh₃/ninhydrin staining. Collectively, these data establish that the LABOR method using LP-9AzSia enables in vivo metabolic labeling of brain sialoglycoconjugates, including both glycoproteins and glycolipids, with 9AzSia.

Mechanistic Studies and Condition Optimization of LABOR.

To investigate the mechanism of liposome-mediated metabolic labeling of brain sialoglycans, we used two in vitro models of BBB established with Madin-Darby canine kidney (MDCK) and Caco-2 cells (SI Appendix, Fig. S2A) (20). The cells were cultured on the transwell insert until the transendothelial electrical resistance (TEER) was above 500 Ω·cm², which indicated the successful establishment of tight interendothelial juntions. LP-9AzSia (250 μM) was added to the upper chamber and incubated for 8 h. LP-9AzSia with no apparent change in the diameter was detected in the lower chamber, indicating that at least some of LP-9AzSia remained intact upon crossing the model BBB (SI Appendix, Fig. S2B). Nevertheless, decrease of TEER was observed upon treating the model BBB with LP-9AzSia at high concentrations for 24 h, indicating that the BBB permeability might be increased (SI Appendix, Fig. S2 C and D). We therefore assayed whether LP-9AzSia administration could increase the BBB permeability in mice. Sodium fluorescein, as a fluorescent tracer (SI Appendix, Fig. S3A) (21), was tail vein injected into the mice treated with LP-9AzSia. Increased BBB permeability to sodium fluorescein was observed in LP-9AzSia-treated mice, but not in mice administered with 9AzSia or Ac₄ManNAz (SI Appendix, Fig. S3B). Based on these results and the possible transport routes for liposomes to cross the BBB (14), we hypothesize that both transcytosis and increase of BBB permeability may contribute to the brain delivery of LP-9AzSia. It should be noted, however, that further in vivo investigations are needed to confirm the proposed mechanism.

We then evaluated the parameters for LP-9AzSia administration. We first measured the 9AzSia accumulation profile in the brain upon one injection. At varying time points after the injection of LP-9AzSia, the total 9AzSia present in the brain including both the free and ketosidically bound forms was collected and quantified by high-pH anion exchange chromatography, followed by pulsed amperometric detection (HPAEC-PAD). The presence of 9AzSia in the brain was detected at 0.5 h after the injection and gradually increased, reaching saturation after 6 h (Fig. 2A). Because one single injection of LP-9AzSia was able to maintain the maximum concentration of 9AzSia in the brain until at least 24 h, multiple injections on a daily basis were performed for 3 d, 5 d, and 7 d. Anti-biotin Western blot analysis indicated that 9AzSia was incorporated into a variety of brain sialoglycoproteins in a time-dependent manner (Fig. 2B). Furthermore, we injected the mice with LP-9AzSia at varying concentrations. In-gel fluorescence scanning of the brain lysates reacted with alkyne-biotin showed that LP-9AzSia metabolically labeled brain sialoglycoproteins in a concentration-dependent manner (Fig. 2C). Based on these results, daily i.v. injection of LP-9AzSia at 0.70 mmol/kg for 7 d was chosen for the following experiments.

Fig. 2.

Optimization of the LABOR conditions. (A) HPAEC-PAD detection of the presence of 9AzSia in the brain. After the mice were administered with 0.70 mmol/kg LP-9AzSia or 9AzSia, the brain tissues were isolated at varying time points, followed by acid hydrolysis to release the bound 9AzSia. The total 9AzSia was collected and subjected to quantitative analysis using HPAEC-PAD. The concentration of 9AzSia in the brain was calculated based on a standard curve generated by measuring 9AzSia solutions at a series of concentrations and expressed as micrograms per gram of tissue. Error bars are SD from three replicate experiments. (B) Time dependence of LABOR-mediated incorporation of 9AzSia into brain sialylated glycoproteins. The mice were injected daily with 0.70 mmol/kg LP-9AzSia for 3 d, 5 d, and 7 d. The brain tissues were collected and the tissue lysates were reacted with alkyne-biotin, followed by anti-biotin Western blot analysis. (C) Concentration dependence of metabolic incorporation of 9AzSia into brain sialylated glycoproteins. The mice were administered with LP-9AzSia or LP at varying concentrations for 7 d. The brain lysates were reacted with alkyne-Cy5, resolved by SDS/PAGE, and the gel was directly scanned in a fluorescence imager. Anti-GAPDH blot was used as the loading control in B and C. Three animals (n = 3) were administered in each treatment group in A–C.

In Vivo Fluorescence Imaging of Brain Sialoglycans.

Next, we sought to image brain sialoglycans in vivo by using the LABOR strategy. The mice administered with LP-9AzSia were i.v. injected with an aza-dibenzocyclooctyne-Cy5 conjugate (DBCO-Cy5; 0.14 nmol/g) at the eighth day (Fig. 3A). Copper-free click chemistry has been performed in living mice and rats and exhibited no apparent toxicity (13, 22, 23). Using mice treated with empty liposomes (LP), we determined that DBCO-Cy5 had been cleared from the bloodstream 3 h after the injection (SI Appendix, Fig. S4A). In contrast, the fluorescence signal in the brain of LP-9AzSia-treated mice remained for 12 h, indicating the covalent conjugation of DBCO-Cy5 with azides incorporated in the brain sialoglycans (SI Appendix, Fig. S4B). We therefore chose 3 h after DBCO-Cy5 administration as the time point for performing in vivo imaging on the LP-9AzSia-treated mice by whole-body fluorescence imaging (Fig. 3 B and C). Robust fluorescence was observed in the brain, corresponding to the newly synthesized sialoglycans during the course of LP-9AzSia administration. In addition to the brain, strong fluorescence was also observed in the area of kidney, liver, and spleen, which is presumably due to metabolic labeling in these organs during the renal and reticuloendothelial clearance of LP-9AzSia. As two negative controls, the mice administered with 9AzSia or LP by i.v. injection exhibited no significant fluorescence in the brain (Fig. 3 B and C). In addition, minimal labeling of brain was observed in mice administered with Ac₄ManNAz by i.p. injection and DBCO-Cy5 by i.v. injection, confirming that the widely used Ac₄ManNAz is incapable of labeling brain sialoglycans in vivo.

Fig. 3.

In vivo fluorescence imaging of the sialoglycans in the mouse brain. (A) Living mice that have been metabolically labeled with LP-9AzSia are i.v. injected with DBCO-Cy5, a cyclooctyne-functionalized far-red fluorophore. In vivo copper-free click chemistry allows chemoselective conjugation of Cy5 onto the brain sialoglycans that have been newly synthesized and incorporated with 9AzSia. (B) Whole-body fluorescence imaging of living mice (n = 3 per treatment group) administered daily with LP-9AzSia, 9AzSia, LP, Ac₄ManNAz or 70% (vol/vol) DMSO for 7 d, followed by injection with 0.14 nmol/g DBCO-Cy5 at day 8. Three hours after the injection, the mice were imaged using an in vivo imaging system. Shown are representative images in each group. The color bar indicates the fluorescence radiant efficiency, multiplied by 10⁹. (Scale bar, 1 cm.) (C) Quantitative analysis of the brain signal-to-background ratio (BBR). BBR: the contrast-to-background ratio (CBR) of the brain divided by the CBR of nearby normal tissue. Error bars are SD from three animals in each treatment group. **P < 0.01; n.s., not significant (one-way ANOVA).

To further rule out the possibility that the observed fluorescence in the brain was due to nonspecific binding of DBCO-Cy5, we isolated the brains from mice administered with LP-9AzSia and DBCO-Cy5. Ex vivo fluorescence imaging at the tissue level showed strong fluorescence in the brain of LP-9AzSia-treated mice, but not in the brain treated with LP (SI Appendix, Fig. S5A). Moreover, the labeled brain tissue was homogenized and subjected to in-gel fluorescence analysis (SI Appendix, Fig. S5B). A diverse repertoire of glycoproteins were resolved and fluorescently visualized on the gel. These results confirm that the 9AzSia-incorporated brain sialoglycans are covalently conjugated with DBCO-Cy5 in vivo.

Sialoglycans of Distinct Mouse Brain Regions Are Labeled.

The brain is the most complex organ, with distinct regions that are functionally specialized. To evaluate LP-9AzSia labeling in distinct brain regions, we coupled LABOR with histochemistry. After fasting the LP-9AzSia-treated mice for 24 h, the brains were isolated, and tissue sections from four distinct brain regions including the pyriform cortex, septo-diencephalic region, caudal diencephalon, and rostral cerebellum were prepared (Fig. 4A). After reacting 9AzSia with alkyne-biotin and staining the nucleus with DAPI, the tissue sections were imaged using a slide fluorescence scanner. Throughout the brain, we observed robust Cy5 fluorescence in all of the four brain regions (Fig. 4B). In contrast, only minimal Cy5 labeling was observed in brain tissue sections from mice treated with LP, 9AzSia, or Ac₄ManNAz (SI Appendix, Fig. S6). These results demonstrate that LP-9AzSia metabolically labels sialoglycans throughout the entire brain.

Fig. 4.

LP-9AzSia metabolically labels distinct brain regions. (A) Representative picture of the intact mouse brain. The dashed lines indicate four distinct brain regions, from which the coronal sections were made: 1, pyriform cortex; 2, septo-diencephalic; 3, caudal diencephalon; and 4, rostral cerebellum. (Scale bar, 2 mm.) (B) Representative fluorescence images of brain tissue sections. Mice (n = 3) were administered with 0.70 mmol/kg LP-9AzSia daily for 7 d. The tissue sections with a thickness of 20 μm were prepared, reacted with alkyne-Cy5, and stained with 4’,6-diamidino-2-phenylindole (DAPI), followed by imaging with a slide fluorescence scanner. (Scale bar, 1 mm.)

Cellular Distribution of the Newly Synthesized Sialoglycans in the Hippocampus.

The compatibility of LABOR with histological examination prompted us to analyze the cellular distribution of sialoglycans by using confocal fluorescence microscopy with a higher spatial resolution. Tissue sections from the caudal diencephalon were prepared and immunostained with the synaptic marker synaptophysin and the marker for astrocytes glial fibrillary acidic protein (GFAP), followed by reaction with DBCO-Cy5 and staining with DAPI (SI Appendix, Fig. S7). We focused the confocal imaging experiments on the dentate gyrus area of hippocampus within the caudal diencephalon, given that sialylation has been implicated in regulating hippocampal function (2, 24). In particular, sialoglycans have been shown to play an important role in synaptic plasticity and neurotransmission (25, 26). To better resolve the spatial distribution of 9AzSia, we zoomed in to the granule cell layer of the dentate gyrus of the hippocampus (Fig. 5). The 9AzSia labeling exhibited a fluorescence pattern that is exclusive of nucleus, indicating the labeling of cell surface sialoglycans. The punctate staining of synapses appeared to localize over the region with dense 9AzSia labeling, suggesting that many of the nascent sialoglycans are distributed in synapses (Fig. 5). In addition, 9AzSia fluorescence, although at a relatively lower level, was also observed at the locations of astrocytes, suggesting cell surface sialoglycans on astrocytes were metabolically labeled. As expected, confocal fluorescence imaging on the hippocampus of the control mice exhibited minimal Cy5 fluorescence (SI Appendix, Fig. S8). These results suggest that the turnover and biosynthesis of sialic acids are active in the brain, resulting in the incorporation of 9AzSia into the newly synthesized sialoglycans located on cell surfaces, including the synaptic sites.

Fig. 5.

Cellular distribution of 9AzSia-incorporated sialoglycans in the granule cell layer of dentate gyrus in the hippocampus. The tissue sections (thickness, 10 μm) of the caudal diencephalon were made from mice (n = 3) treated with LP-9AzSia and immunostained with synaptophysin and GFAP, followed by reaction with DBCO-Cy5 and staining with DAPI. Images were recorded with a 63× objective lens on a confocal fluorescence microscope. (Scale bar, 20 μm.) Images with a lower magnification showing the dentate gyrus structure are shown in SI Appendix, Fig. S7.

Proteomic Analysis of Sialylated Glycoproteins in the Brain.

To assess protein sialylation in the brain, we performed large-scale proteomic profiling of sialylated glycoproteins on LABOR-labeled mice. Tissue lysates of mouse brains treated with LP-9AzSia were reacted with alkyne-biotin via CuAAC for enrichment of 9AzSia-incorporated proteins with streptavidin beads, followed by gel-based proteomic identification using tandem mass spectrometry (SI Appendix, Fig. S9). From three independent experiments, we selectively identified 140 proteins by LP-9AzSia labeling compared with control mice treated with LP (Fig. 6 A and B and SI Appendix, Table S2). The 140 proteins were selected using a high-confidence filter, that is, selecting proteins with ≥fivefold increases of the spectral counts in the LP-9AzSia-treated samples above the LP-treated control samples.

Fig. 6.

Proteomic profiling of newly synthesized sialylated glycoproteins in the brain of mice treated with LP-9AzSia. (A) Tissue lysates of mouse brain treated with LP-9AzSia or LP were reacted with alkyne-biotin, enriched using streptavidin beads, and subjected to gel-based proteomic identification by tandem mass spectrometry. For each protein, the total spectral counts of LP-9AzSia samples subtracted by the total spectral counts of LP samples was plotted. Several known proteins that are associated with synapses in the brain are shown in red. (B) Pearson correlation plot for overlapping proteins from LP-9AzSia experiments (140 proteins, comparing experiment 1 with experiment 2). (C) Brain lysates were enriched using alkyne-biotin and analyzed by Western blot. All lanes are cropped from the same gel. Anti-GAPDH blot was used as the loading control. (D) Cellular localizations enriched in the newly synthesized sialoglycoproteins identified in the LP-9AzSia-treated brain. The top six enriched localizations are shown.

To validate the proteins identified by mass spectrometry, we performed Western blot analysis on the LP-9AzSia-labeled and enriched brain lysates using antibodies against cell adhesion molecule 4 (CADM4) and contactin-associated protein-like 2 (CNTNAP2), confirming their robust and specific recovery dependent on 9AzSia incorporation (Fig. 6C). Among the list of identified sialylated glycoproteins, we noticed that there are a wealth of synapse-associated proteins, such as synaptotagmin 2 (Syt2), zinc transporter 3 (ZnT3), and leucine-rich, glioma-inactivated protein 1 (LGI1) (indicated by the red dots in Fig. 6A). Syt2 functions as a Ca²⁺ sensor for synchronous synaptic vesicle exocytosis (27, 28). ZnT3 is localized in clear synaptic vesicles of cortical glutamatergic terminals and involved in synaptic vesicle zinc uptake and release (29, 30). LGI1 is a secreted protein that is localized to synapses, where it modulates synaptic AMPA receptors (31, 32). We therefore performed the gene ontology analysis of the identified sialoglycoproteins, which revealed that synapse is one of the most enriched cellular localizations, indicating that many synaptic proteins are sialylated and the biosynthesis of these sialoglycoproteins is active (Fig. 6D). Notably, a list of sialylated proteins was also identified at several other cellular locations, in agreement with the imaging results.

Brain Sialylation Is Dynamically and Spatiotemporally Regulated.

Finally, we sought to investigate the spatiotemporal regulation of sialylation dynamics in the brain. By taking advantage of the metabolic labeling nature of LABOR, we performed pulse−chase experiments to image the turnover of newly synthesized sialoglycans in the brain. After administration with LP-9AzSia for 7 d, the mice were chased for 0 h or 6 h, followed by brain isolation (Fig. 7A). Brain sections were prepared and reacted with alkyne-Cy5. We examined six encephalic regions by confocal fluorescence microscopy (Fig. 7B). In most of these regions, the 9AzSia labeling had been significantly decreased within 6 h, indicating sialylation is dynamic in the brain. Remarkably, the hippocampus exhibited a significantly lower decay of the 9AzSia-incorporated sialoglycans. These results suggest that the dynamics of sialoglycan biosynthesis may be regulated in a spatiotemporally controlled manner and that the hippocampal sialoglycans possess a slow turnover rate.

Fig. 7.

Pulse−chase analysis of sialoglycan turnover in distinct brain regions. (A) Schematic of the pulse−chase experimental procedures. Mice (n = 3 per treatment group) were injected with 0.70 mmol/kg LP-9AzSia daily for 7 d, followed by chasing for 0 h or 6 h. Brain sections with a thickness of 20 μm were prepared, reacted with alkyne-Cy5, and stained with DAPI. (B) Confocal fluorescence images of the olfactory area, corpus striatum, cerebral cortex, hippocampal formation, cerebellum, and medulla regions were recorded using a 10× objective lens. (Scale bar, 200 μm.)

Discussion

Despite the functional importance of brain sialylation, in vivo visualization of sialoglycans in the mouse brain has been impossible. Azidosugars have been used as tracers to report the sialic acid biosynthesis in living animals (12, 13), but the BBB impedes their access to the brain. The LABOR strategy developed herein overcomes this obstacle by exploiting liposomes to shuttle 9AzSia into the brain. Previous in vitro studies using neuron cell culture (33) and brain tissue culture (34) have shown that neurons can uptake azidosugars and metabolically incorporate them into sialoglycans. Our results demonstrate that LABOR-mediated delivery of 9AzSia into the brain results in robust metabolic incorporation of azides into brain sialoglycans in living mice. In vivo copper-free click chemistry enables whole-body fluorescence imaging of brain sialylation. This strategy may be further applied to visualize sialylation in mouse models of brain tumors and neurodegenerative diseases (8, 10).

The mouse brain, which is fluorescently labeled using the LABOR strategy, is compatible with immuofluorescent histochemistry. Multicolor confocal fluorescence imaging of brain sections revealed that the newly synthesized sialoglycans are widely distributed on cell surfaces of brain cells, including neurons and glial cells. Of particular interest, dense labeling was observed on synapses, indicating active biosynthesis and turnover of sialoglycans at the synaptic sites. Sialoglycans around the synaptic cleft have been proposed to modulate neurotransmission through interaction with Ca²⁺, which is essential in neuronal responses (26). Because neurotransmission and synaptic connectivity are dynamically regulated (28, 35), dynamic sialylation observed in this study may play an important role in these processes. Furthermore, glycoproteomic profiling can be performed on the brain tissues isolated from the LABOR-labeled mice. We have identified a list of sialylated proteins in the brain. The identified sialoglycoproteins include those involved in calcium signaling in synapses such as Syt2, supporting the hypothesis that sialoglycans regulate neurotransmission by interacting with Ca²⁺. Furthermore, the fact that ZnT3 is sialylated and actively synthesized in the synapse suggests that sialylation may also play a regulatory function in synaptic zinc signaling. The newly synthesized sialoglycoproteins associated with synapses contribute, at least partially, to the newly synthesized synaptic sialoglycans observed in the fluorescent histochemistry. In addition, sialoglycans can also be carried by sialylated glycolipids such as gangliosides (2). Our results indicate that those gangliosides are also metabolically labeled with 9AzSia.

The turnover of sialic acids in the brain has been a subject of extensive studies. One commonly used method is to administer the animals with ManNAc or sialic acid monosaccharide substituted with radioactive isotopes such as ³H and ¹⁴C (36–39). Isotopic labeling enables pulse−chase experiments but suffers from limited spatial resolution. Alternatively, histological analysis can be performed using lectins (40) and antibodies (41) to provide spatial information on sialoglycans at the static state. The LABOR method allows for spatiotemporal visualization of the dynamic sialylation. Our results reveal spatially distinct turnover rates among different brain regions. Of particular interest, the turnover of sialic acids in the hippocampus appears to be uniquely slow. Sialylation in the hippocampus is important for axonal growth and synaptic activity-induced neuronal−glial plasticity (42). Induction of long-term potentiation and depression at the synapses was found to be impaired if PSA is removed, thus affecting spatial learning and memory function in hippocampal regions (43). Whether the overall slow dynamics of sialylation has functional implications in these processes is an interesting topic for future studies.

Although we focus on brain sialylation in this work, metabolic incorporation of 9AzSia in other organs, including the heart, liver, spleen, kidney, lung, and thymus, in mice treated with LP-9AzSia was also observed (SI Appendix, Figs. S10 and S11). In comparison with Ac₄ManNAz labeling, LP-9AzSia exhibits superior labeling efficiency in the heart, kidney, and thymus, whereas Ac₄ManNAz labels better in the intestine. These observations provide a foundation for choosing the appropriate labeling method for studying glycosylation in specific organs and suggest that LABOR will find broad applications, in addition to probing brain sialylation.

Receptor-mediated transcytosis has been explored for liposomal delivery into the brain (14). For future technical development, it will be interesting to evaluate whether ligand-targeted liposomes can be exploited for labeling brain glycosylation. For example, liposomes can also be modified with ligands such as transferrin (44), OX26 antibody (17), and short peptides (45), which target specific brain receptors. Given that the current method labels a panel of organs in addition to the brain, the ligand-targeted liposomes may improve the brain specificity.

Finally, the LABOR methodology may be further extended to probe other types of glycosylation in the mouse brain (46). Of particular interest is the protein O-GlcNAc modification, which has been implicated in neurodegenerative diseases (47). Bioorthogonal chemical reporters for O-GlcNAc have been developed for metabolically labeling and identifying O-GlcNAcylated proteins in live cells (48–50), which may be explored for in vivo LABOR labeling of brain O-GlcNAcylation.

Materials and Methods

The liposomes were prepared as described (15). BALB/c male mice (8 wk, 20–25 g) were administered daily for 7 d with 0.70 mmol/kg LP-9AzSia (i.v.), LP-ManNAz (i.v.), 9AzSia (i.v.), ManNAz (i.v.), Ac₄ManNAz (i.p.), or Ac₄Me9AzSia (i.p.). All animal experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Peking University accredited by Association for Assessment and Accreditation of Laboratory Animal Care International. Further details are provided in SI Appendix, which also includes detailed methods for whole-body fluorescence imaging, fluorescence histochemistry, glycoproteomic analysis, Western blot analysis, and in-gel fluorescence scanning.