Botulinum neurotoxin A ameliorates depressive-like behavior in a reserpine-induced Parkinson's disease mouse model via suppressing hippocampal microglial engulfment and neuroinflammation.

Depression is one of the common non-motor symptoms of Parkinson's disease (PD). In the clinic, botulinum neurotoxin A (BoNT/A) has been used to treat depression. In this study, we investigated the mechanisms underlying the anti-depressive effect of BoNT/A in a PD mouse model. Mice were administered reserpine (3 µg/mL in the drinking water) for 10 weeks. From the 10th week, BoNT/A (10 U·kg-1·d-1) was injected into the cheek for 3 consecutive days. We showed that chronic administration of reserpine produced the behavioral phenotypes of depression and neurochemical changes in the substantia nigra pars compacta (SNpc) and striatum. BoNT/A treatment significantly ameliorated the depressive-like behaviors, but did not improve TH activity in SNpc of reserpine-treated mice. We demonstrated that BoNT/A treatment reversed reserpine-induced complement and microglia activation in the hippocampal CA1 region. Furthermore, BoNT/A treatment significantly attenuated the microglial engulfment of presynaptic synapses, thus ameliorating the apparent synapse and spine loss in the hippocampus in the reserpine-treated mice. Moreover, BoNT/A treatment suppressed microglia-mediated expression of pro-inflammatory cytokines TNF-α and IL-1ß in reserpine-treated mice. In addition, we showed that BoNT/A (0.1 U/mL) ameliorated reserpine-induced complement and microglia activation in mouse BV2 microglial cells in vitro. We conclude that BoNT/A ameliorates depressive-like behavior in a reserpine-induced PD mouse model through reversing the synapse loss mediated by classical complement induced-microglial engulfment as well as alleviating microglia-mediated proinflammatory responses. BoNT/A ameliorates depressive-like behavior, and reverses synapse loss mediated by classical complement pathway-initiated microglia engulfment as well as alleviates microglia-mediated proinflammatory response in the reserpine-induced Parkinson's disease mouse model.

C1q and SRPX2 regulate microglia mediated synapse elimination during early development in the visual thalamus but not the visual cortex.

The classical complement cascade mediates synapse elimination in the visual thalamus during early brain development. However, whether the primary visual cortex also undergoes complement-mediated synapse elimination during early visual system development remains unknown. Here, we examined microglia-mediated synapse elimination in the visual thalamus and the primary visual cortex of early postnatal C1q and SRPX2 knockout mice. In the lateral geniculate nucleus, deletion of C1q caused a persistent decrease in synapse elimination and microglial synapse engulfment, while deletion of SRPX2 caused a transient increase in the same readouts. In the C1q-SRPX2 double knockout mice, the C1q knockout phenotypes were dominant over the SRPX2 knockout phenotypes, a result which is consistent with SRPX2 being an inhibitor of C1q. We found that genetic deletion of either C1q or SRPX2 did not affect synapse elimination or microglial engulfment of synapses in layer 4 of the primary visual cortex in early brain development. Together, these results show that the classical complement pathway regulates microglia-mediated synapse elimination in the visual thalamus but not the visual cortex during early development of the central nervous system.

The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development.

Complement-mediated synapse elimination has emerged as an important process in both brain development and neurological diseases, but whether neurons express complement inhibitors that protect synapses against complement-mediated synapse elimination remains unknown. Here, we show that the sushi domain protein SRPX2 is a neuronally expressed complement inhibitor that regulates complement-dependent synapse elimination. SRPX2 directly binds to C1q and blocks its activity, and SRPX2-/Y mice show increased C3 deposition and microglial synapse engulfment. They also show a transient decrease in synapse numbers and increase in retinogeniculate axon segregation in the lateral geniculate nucleus. In the somatosensory cortex, SRPX2-/Y mice show decreased thalamocortical synapse numbers and increased spine pruning. C3-/-;SRPX2-/Y double-knockout mice exhibit phenotypes associated with C3-/- mice rather than SRPX2-/Y mice, which indicates that C3 is necessary for the effect of SRPX2 on synapse elimination. Together, these results show that SRPX2 protects synapses against complement-mediated elimination in both the thalamus and the cortex.

Sociability and synapse subtype-specific defects in mice lacking SRPX2, a language-associated gene.

The FoxP2 transcription factor and its target genes have been implicated in developmental brain diseases with a prominent language component, such as developmental verbal dyspraxia and specific language impairment. How FoxP2 affects neural circuitry development remains poorly understood. The sushi domain protein SRPX2 is a target of FoxP2, and mutations in SRPX2 are associated with language defects in humans. We have previously shown that SRPX2 is a synaptogenic protein that increases excitatory synapse density. Here we provide the first characterization of mice lacking the SRPX2 gene, and show that these mice exhibit defects in both neural circuitry and communication and social behaviors. Specifically, we show that mice lacking SRPX2 show a specific reduction in excitatory VGlut2 synapses in the cerebral cortex, while VGlut1 and inhibitory synapses were largely unaffected. SRPX2 KO mice also exhibit an abnormal ultrasonic vocalization ontogenetic profile in neonatal pups, and reduced preference for social novelty. These data demonstrate a functional role for SRPX2 during brain development, and further implicate FoxP2 and its targets in regulating the development of vocalization and social circuits.

Sulfated fucoidan FP08S2 inhibits lung cancer cell growth in vivo by disrupting angiogenesis via targeting VEGFR2/VEGF and blocking VEGFR2/Erk/VEGF signaling.

Fucoidan may inhibit angiogenesis. However, its functional target molecule and the underlying mechanism are still vague. In the present study, we showed that sulfated fucoidan FP08S2 from Sargassum fusiforme inhibited tube formation as well as migration and invasion of human microvascular endothelial cells (HMEC-1). In addition, FP08S2 was confirmed to disrupt VEGF-induced angiogenesis both in vitro and in vivo. Further study indicated that FP08S2 could bind to both VEGF and VEGFR2 to interfere with VEGF-VEGFR2 interaction. Moreover, VEGFR2/Erk/VEGF signaling pathway was blocked by FP08S2 in HMEC-1 cells. Importantly, FP08S2 impeded the growth and microvessel formation of A549 cancer cell xenograft in nude mice. These results suggested that FP08S2 presented remarkable anti-angiogenic activity via blocking VEGF signaling and could be a potential novel leading compound to inhibit lung cancer cell growth.

A fucoidan from Nemacystus decipiens disrupts angiogenesis through targeting bone morphogenetic protein 4.

A sulfated and acetylated fucoidan, named NDH01, was extracted from seaweed Nemacystus decipiens. NDH01 was composed of mannose, glucuronic acid, fucose, sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-d-1,2-Manp and ß-d-1,4-GlcpA disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-d-1,2-Manp. The sidechain was composed of α-l-1,3,4-Fucp, α-l-1,4-Fucp, α-l-1,3-Fucp and α-l-1,4-GlcpA. The sulfate group was linked to C4 of α-l-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-l-1,2,3-Fucp. NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells. Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4 expression. The results suggested that NDH01 might be an angiogenesis inhibitor through targeting bone morphogenetic protein 4.

Structural elucidation of a pectin from flowers of Lonicera japonica and its antipancreatic cancer activity.

To investigate polysaccharide structure from Lonicera japonica, and study its effects on behavior of pancreatic cells, a homogenous polysaccharide, LJ-02-1, was extracted and purified from flowers of L. japonica by DEAE-cellulose and Sephacryl S-200HR column. The molecular weight was estimated to be 54kDa. Monosaccharide composition was determined to be rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 10.77:7.88:15.45:65.89 by analyzing the PMP derivatives of the monosaccharides from 2M trifluoracetic acid hydrolysis via HPLC. Based on methylation analysis, partial acid hydrolysis, and NMR spectra, the polysaccharide was elucidated to be a rhamnogalacturonan backbone and substituted partly at C-4 of rhamnose. The branches were determined to be T- and 1,4,6-linked ß-d-Galp, T- and 1,5-linked α-l-Araf. The polysaccharide might inhibit BxPC-3 and PANC-1 pancreatic cancer cells growth at the concentration of 1mg/mL with inhibitory ratio of 66.7% and 52.1%, respectively.

Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme.

A fucoidan FP08S2 was isolated from the boiling-water extract of Sargassum fusiforme, purified by CaCl2 precipitation and chromatography on DEAE-cellulose and Sephacryl S-300. FP08S2 contained fucose, xylose, galactose, mannose, glucuronic acid, and 20.8% sulfate. The sulfate groups were attached to diverse positions of fucose, xylose, mannose, and galactose residues. The backbone of FP08S2 consisted of alternate 1,2-linked α-D-Manp and 1,4-linked ß-D-GlcpA. Sugar composition analysis and ESI-MS revealed that the oligosaccharides from branches contained fucose, xylose, galactose, glucuronic acid and sulfate. FP08S2 could significantly inhibit tube formation and migration of human microvascular endothelial cells (HMEC-1) dose-dependently. These results suggested that the fucoidan FP08S2 from brown seaweeds S. fusiforme could be a potent anti-angiogenic agent.

Structural characterization of a polysaccharide from Chrysanthemum morifolium flowers and its antioxidant activity.

The flowers of Chrysanthemum morifolium were extracted by boiling water, and a water-soluble polysaccharide (CMJA0S2) with a molecular weight of 6.5 kDa was isolated by anion-exchange chromatography on a DEAE-cellulose column and gel permeation chromatography on a Sephacryl S-300 HR column. Monosaccharide composition analysis indicated that CMJA0S2 was composed of galactose, glucose, mannose and arabinose in molar ratio of 4.1: 3.3: 1.0: 2.3. According to linkage analysis, partial acid hydrolysis and NMR spectra, the backbone was shown to contain 1, 4-linked ß-Galp, 1, 4-linked ß-Glcp and 1, 4-linked ß-Manp, with branches substituted at C-6 of 1, 4-linked ß-Manp by 1, 6-linked ß-Galp and at O-6 of partial 1, 4-linked ß-Galp substituted by T-linked α-Glcp. About 40% of 1, 6-linked ß-Galp with T-linked α-Glcp was substituted at O-3 by α-Araf-(1→[5)-α-Araf-(1]3. The anti-oxidative analysis showed that CMJA0S2 could scavenge the DPPH radicals and relieve the damage of PC12 cells caused by H2O2, thus it could be regarded as a potential natural antioxidant.

Structural and functional study of D-glucuronyl C5-epimerase.

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Structure and biological activities of an alginate from Sargassum fusiforme, and its sulfated derivative.

An alginate fraction, 04S2P, was isolated from the brown seaweed Sargassum fusiforme and was structurally characterized by the ratio (M/G) of ß-d-mannuronic acid residues (M) to α-l-guluronic acid residues (G) via (1)H and (13)C NMR spectroscopy. When compared to commercial alginate (Alg) and alginates from other brown algae, 04S2P has a higher M/G ratio of 9.0:1.0 as determined by a modified high-performance liquid chromatography method after pre-column derivatization with PMP. Furthermore, the sulfated polysaccharides 04S2P-S and Alg-S were prepared by the chlorosulfonic acid-pyridine method. Both C-2 and/or C-3 of M and G residues of 04S2P-S were substituted by sulfate groups, with C-3 of M residues preferentially substituted. Their effects on tube formation of HMEC-1 cells were examined, and the results indicated that the sulfated Alg, Alg-S, exhibited a strong anti-angiogenic effect on HMEC-1 cells. The anti-tumor activity of native and sulfated alginates was tested on five different tumor cell lines. Alg-S demonstrated significant anti-tumor effects on the Bel7402, SMMC7721, and HT-29 cell lines, whereas 04S2P-S showed a distinct anti-tumor effect only on the Bel7402 cell line.

Structure and activities of a novel heteroxylan from Cassia obtusifolia seeds and its sulfated derivative.

COB1B1S2 was isolated from an alkaline extract of Cassia obtusifolia seeds, and purified by anion-exchange and gel permeation chromatography. It contains arabinose, xylose, and glucuronic acid, in the molar ratio of 5:81:14, with an apparent molecular weight estimated to be 70.4 kDa. Elucidated by using chemical and spectroscopic methods, COB1B1S2 was shown to have a backbone consisting of 1,4-linked ß-D-Xylp, with one single-unit terminal α-D-GlcpA or α-L-Araf substituted at O-2 for nearly every five 1,4-linked Xylp. COB1B1S2 is structurally different from typical glucuronoxylans by its absence of methylation at O-4 of GlcA. The native COB1B1S2 showed no significant inhibition on the tube formation of human microvascular endothelial cells (HMEC) and on the growth of liver and colon cancer cells. On the contrary, COB1B1S2-Sul, prepared as the sulfated derivative of COB1B1S2, exhibited a significant inhibition on tube formation of HMEC in a dose-dependent manner, and on the growth of Bel7402 liver cancer cells. These results indicated that the introduction of sulfate groups significantly enhanced the biological activity of glucuronoxylan.