Reliable and sensitive detection of glycosaminoglycan chains with immunoblots.

Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.

The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a member of a family of lipid phosphatase related proteins, integral membrane proteins characterized by six transmembrane domains. This family of proteins is enriched in the brain and recent data indicate potential pleiotropic functions in several different contexts. An inherent ability of this family of proteins is to induce morphological changes, and we have previously reported that members of this family interact with each other and may function co-operatively. However, the function of PLPPR1 is not yet understood. Here we show that the expression of PLPPR1 reduces the inhibition of neurite outgrowth of cultured mouse hippocampal neurons by chondroitin sulfate proteoglycans and the retraction of neurites of Neuro-2a cells by lysophosphatidic acid (LPA). Further, we show that PLPPR1 reduces the activation of Ras homolog family member A (RhoA) by LPA in Neuro-2a cells, and that this is because of an association of PLPPR1with the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI1). These results establish a novel signaling pathway for the PLPPR1 protein.

Phospholipid phosphatase related 1 (PLPPR1) increases cell adhesion through modulation of Rac1 activity.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a six-transmembrane protein that belongs to the family of plasticity-related gene proteins, which is a novel brain-specific subclass of the lipid phosphate phosphatase superfamily. PLPPR1-5 have prominent roles in synapse formation and axonal pathfinding. We found that PLPPR1 overexpression in the mouse neuroblastoma cell line (Neuro2a) results in increase in cell adhesion and reduced cell migration. During migration, these cells leave behind long fibrous looking extensions of the plasma membrane causing a peculiar phenotype. Cells expressing PLPPR1 showed decreased actin turnover and decreased disassembly of focal adhesions. PLPPR1 also reduced active Rac1, and expressing dominant negative Rac1 produced a similar phenotype to overexpression of PLPPR1. The PLPPR1-induced phenotype of long fibers was reversed by introducing constitutively active Rac1. In summary, we show that PLPPR1 decreases active Rac1 levels that leads to cascade of events which increases cell adhesion.

Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents.

The accumulation of chondroitin sulfate proteoglycans (CSPGs) in the glial scar following acute damage to the central nervous system (CNS) limits the regeneration of injured axons. Given the rich diversity of CSPG core proteins and patterns of GAG sulfation, identifying the composition of these CSPGs is essential for understanding their roles in injury and repair. Differential expression of core proteins and sulfation patterns have been characterized in the brain and spinal cord of mice and rats, but a comprehensive study of these changes following optic nerve injury has not yet been performed. Here, we show that the composition of CSPGs in the optic nerve and retina following optic nerve crush (ONC) in mice and rats exhibits an increase in aggrecan, brevican, phosphacan, neurocan and versican, similar to changes following spinal cord injury. We also observe an increase in inhibitory 4-sulfated (4S) GAG chains, which suggests that the persistence of CSPGs in the glial scar opposes the growth of CNS axons, thereby contributing to the failure of regeneration and recovery of function.

Identification of novel binding sites for heparin in receptor protein-tyrosine phosphatase (RPTPσ): Implications for proteoglycan signaling.

Receptor protein-tyrosine phosphatase RPTPσ has important functions in modulating neural development and regeneration. Compelling evidence suggests that both heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs) bind to a series of Lys residues located in the first Ig domain of RPTPσ. However, HS promotes and CS inhibits axonal growth. Mutation of these Lys residues abolished binding and signal transduction of RPTPσ to CS, whereas HS binding was reduced, and signaling persisted. This activity was mediated through novel heparin-binding sites identified in the juxtamembrane region. Although different functional outcomes of HS and CS have been previously attributed to the differential oligomeric state of RPTPσ upon GAG binding, we found that RPTPσ was clustered by both heparin and CS GAG rich in 4,6-O-disulfated disaccharide units. We propose an additional mechanism by which RPTPσ distinguishes between HS and CS through these novel binding sites.

Parkin targets NOD2 to regulate astrocyte endoplasmic reticulum stress and inflammation.

Loss of substantia nigra dopaminergic neurons results in Parkinson disease (PD). Degenerative PD usually presents in the seventh decade whereas genetic disorders, including mutations in PARK2, predispose to early onset PD. PARK2 encodes the parkin E3 ubiquitin ligase which confers pleotropic effects on mitochondrial and cellular fidelity and as a mediator of endoplasmic reticulum (ER) stress signaling. Although the majority of studies investigating ameliorative effects of parkin focus on dopaminergic neurons we found that astrocytes are enriched with parkin. Furthermore, astrocytes deficient in parkin display stress-induced elevation of nucleotide-oligomerization domain receptor 2 (NOD2), a cytosolic receptor integrating ER stress and inflammation. Given the neurotropic and immunomodulatory role of astrocytes we reasoned that parkin may regulate astrocyte ER stress and inflammation to control neuronal homeostasis. We show that, in response to ER stress, parkin knockdown astrocytes exhibit exaggerated ER stress, JNK activation and cytokine release, and reduced neurotropic factor expression. In coculture studied we demonstrate that dopaminergic SHSY5Y cells and primary neurons with the presence of parkin depleted astrocytes are more susceptible to ER stress and inflammation-induced apoptosis than wildtype astrocytes. Parkin interacted with, ubiquitylated and diminished NOD2 levels. Additionally, the genetic induction of parkin ameliorated inflammation in NOD2 expressing cells and knockdown of NOD2 in astrocytes suppressed inflammatory defects in parkin deficient astrocytes and concurrently blunted neuronal apoptosis. Collectively these data identify a role for parkin in modulating NOD2 as a regulatory node in astrocytic control of neuronal homeostasis.

Extracellular matrix and traumatic brain injury.

The brain extracellular matrix (ECM) plays a crucial role in both the developing and adult brain by providing structural support and mediating cell-cell interactions. In this review, we focus on the major constituents of the ECM and how they function in both normal and injured brain, and summarize the changes in the composition of the ECM as well as how these changes either promote or inhibit recovery of function following traumatic brain injury (TBI). Modulation of ECM composition to facilitates neuronal survival, regeneration and axonal outgrowth is a potential therapeutic target for TBI treatment.

Astrocytes from the brain microenvironment alter migration and morphology of metastatic breast cancer cells.

Tumor cell metastasis to the brain involves cell migration through biochemically and physically complex microenvironments at the blood-brain barrier (BBB). The current understanding of tumor cell migration across the BBB is limited. We hypothesize that an interplay between biochemical cues and physical cues at the BBB affects the mechanisms of brain metastasis. We found that astrocyte conditioned medium(ACM) applied directly to tumor cells increased tumor cell velocity, induced elongation, and promoted actin stress fiber organization. Notably, treatment of the extracellular matrix with ACM led to even more significant increases in tumor cell velocity in comparison with ACM treatment of cells directly. Furthermore, inhibiting matrix metalloproteinases in ACM reversed ACM's effect on tumor cells. The effects of ACM on tumor cell morphology and migration also depended on astrocytes' activation state. Finally, using a microfluidic device, we found that the effects of ACM were abrogated in confinement. Overall, our work demonstrates that astrocyte-secreted factors alter migration and morphology of metastatic breast tumor cells, and this effect depends on the cells' mechanical microenvironment.-Shumakovich, M. A., Mencio, C. P., Siglin, J. S., Moriarty, R. A., Geller, H. M., Stroka, K. M. Astrocytes from the brain microenvironment alter migration and morphology of metastatic breast cancer cells. FASEB J. 31, 5049-5067 (2017). www.fasebj.org.

Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology.

The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members.

The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke.

BACKGROUND: Reactive astrogliosis is one of the significantly pathological features in ischemic stroke accompanied with changes in gene expression, morphology, and proliferation. KCa3.1 was involved in TGF-ß-induced astrogliosis in vitro and also contributed to astrogliosis-mediated neuroinflammation in neurodegeneration disease. METHODS: Wild type mice and KCa3.1-/- mice were subjected to permanent middle cerebral artery occlusion (pMCAO) to evaluate the infarct areas by 2,3,5-triphenyltetrazolium hydrochloride staining and neurological deficit. KCa3.1 channels expression and cell localization in the brain of pMCAO mice model were measured by immunoblotting and immunostaining. Glia activation and neuron loss was measured by immunostaining. DiBAC4 (3) and Fluo-4AM were used to measure membrane potential and cytosolic Ca2+ level in oxygen-glucose deprivation induced reactive astrocytes in vitro. RESULTS: Immunohistochemistry on pMCAO mice infarcts showed strong upregulation of KCa3.1 immunoreactivity in reactive astrogliosis. KCa3.1-/- mice exhibited significantly smaller infarct areas on pMCAO and improved neurological deficit. Both activated gliosis and neuronal loss were attenuated in KCa3.1-/- pMCAO mice. In the primary cultured astrocytes, the expressions of KCa3.1 and TRPV4 were increased associated with upregulation of astrogliosis marker GFAP induced by oxygen-glucose deprivation. The activation of KCa3.1 hyperpolarized membrane potential and, by promoting the driving force for calcium, induced calcium entry through TRPV4, a cation channel of the transient receptor potential family. Double-labeled staining showed that KCa3.1 and TRPV4 channels co-localized in astrocytes. Blockade of KCa3.1 or TRPV4 inhibited the phenotype switch of reactive astrogliosis. CONCLUSIONS: Our data suggested that KCa3.1 inhibition might represent a promising therapeutic strategy for ischemia stroke.

KCa3.1 constitutes a pharmacological target for astrogliosis associated with Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia and is characterized by a progression from decline of episodic memory to a global impairment of cognitive function. Astrogliosis is a hallmark feature of AD, and reactive gliosis has been considered as an important target for intervention in various neurological disorders. We previously found in astrocyte cultures that the expression of the intermediate conductance calcium-activated potassium channel KCa3.1 was increased in reactive astrocytes induced by TGF-ß, while pharmacological blockade or genetic deletion of KCa3.1 attenuated astrogliosis. In this study, we sought to suppress reactive gliosis in the context of AD by inhibiting KCa3.1 and evaluate its effects on the cognitive impairment using murine animal models such as the senescence-accelerated mouse prone 8 (SAMP8) model that exhibits some AD-like symptoms. We found KCa3.1 expression was increased in reactive astrocytes as well as neurons in the brains of both SAMP8 mice and Alzheimer's disease patients. Blockade of KCa3.1 with the selective inhibitor TRAM-34 in SAMP8 mice resulted in a decrease in astrogliosis as well as microglia activation, and moreover an attenuation of memory deficits. Using KCa3.1 knockout mice, we further confirmed that deletion of KCa3.1 reduced the activation of astrocytes and microglia, and rescued the memory loss induced by intrahippocampal Aß1-42 peptide injection. We also found in astrocyte cultures that blockade of KCa3.1 or deletion of KCa3.1 suppressed Aß oligomer-induced astrogliosis. Our data suggest that KCa3.1 inhibition might represent a promising therapeutic strategy for AD treatment.

The phenotype of the musculocontractural type of Ehlers-Danlos syndrome due to CHST14 mutations.

The musculocontractural type of Ehlers-Danlos syndrome (MC-EDS) has been recently recognized as a clinical entity. MC-EDS represents a differential diagnosis within the congenital neuromuscular and connective tissue disorders spectrum. Thirty-one and three patients have been reported with MC-EDS so far with bi-allelic mutations identified in CHST14 and DSE, respectively, encoding two enzymes necessary for dermatan sulfate (DS) biosynthesis. We report seven additional patients with MC-EDS from four unrelated families, including the follow-up of a sib-pair originally reported with the kyphoscoliotic type of EDS in 1975. Brachycephaly, a characteristic facial appearance, an asthenic build, hyperextensible and bruisable skin, tapering fingers, instability of large joints, and recurrent formation of large subcutaneous hematomas are always present. Three of seven patients had mildly elevated serum creatine kinase. The oldest patient was blind due to retinal detachment at 45 years and died at 59 years from intracranial bleeding; her affected brother died at 28 years from fulminant endocarditis. All patients in this series harbored homozygous, predicted loss-of-function CHST14 mutations. Indeed, DS was not detectable in fibroblasts from two unrelated patients with homozygous mutations. Patient fibroblasts produced higher amounts of chondroitin sulfate, showed intracellular retention of collagen types I and III, and lacked decorin and thrombospondin fibrils compared with control. A great proportion of collagen fibrils were not integrated into fibers, and fiber bundles were dispersed into the ground substance in one patient, all of which is likely to contribute to the clinical phenotype. This report should increase awareness for MC-EDS.

Targeted inhibition of KCa3.1 attenuates TGF-ß-induced reactive astrogliosis through the Smad2/3 signaling pathway.

Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate-conductance calcium-activated potassium channel), a potassium channel protein, has been reported to be up-regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)-ß in primary cultures of mouse astrocytes. We found that TGF-ß increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF-ß was attenuated in KCa3.1(-/-) astrocytes. TRAM-34 also suppressed astrocytic proliferation. In addition, the TGF-ß-induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM-34 or in KCa3.1(-/-) astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries. Reactive astrogliosis is characterized by the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans. We demonstrate that either pharmacological blockade or knockout of KCa3.1 channels reduces reactive gliosis in cultured astrocytes caused by TGF-ß, and also reduces TGF-ß-induced phosphorylation of Smad2/3.

Correction: A novel cytoskeletal action of xylosides.

[This corrects the article DOI: 10.1371/journal.pone.0269972.].

Biomimetic polymer brushes containing tethered acetylcholine analogs for protein and hippocampal neuronal cell patterning.

This paper describes a method to control neuronal cell adhesion and differentiation with both chemical and topographic cues by using a spatially defined polymer brush pattern. First, biomimetic methacrylate polymer brushes containing tethered neurotransmitter acetylcholine functionalities in the form of dimethylaminoethyl methacrylate or free hydroxyl-terminated poly(ethylene glycol) units were prepared using the "grown from" method through surface-initiated atom transfer radical polymerization reactions. The surface properties of the resulting brushes were thoroughly characterized with various techniques and hippocampal neuronal cell culture on the brush surfaces exhibit cell viability and differentiation comparable to, or even better than, those on commonly used poly-l-lysine coated glass coverslips. The polymer brushes were then patterned via UV photolithography techniques to provide specially designed surface features with different sizes (varying from 2 to 200 µm) and orientations (horizontal and vertical). Protein absorption experiments and hippocampal neuronal cell culture tests on the brush patterns showed that both protein and neurons can adhere to the patterns and therefore be guided by such patterns. These results also demonstrate that, because of their unique chemical composition and well-defined nature, the developed polymer brushes may find many potential applications in cell-material interactions studies and neural tissue engineering.

Strength in the periphery: growth cone biomechanics and substrate rigidity response in peripheral and central nervous system neurons.

There is now considerable evidence of the importance of mechanical cues in neuronal development and regeneration. Motivated by the difference in the mechanical properties of the tissue environment between the peripheral (PNS) and central (CNS) nervous systems, we compare substrate-stiffness-dependent outgrowth and traction forces from PNS (dorsal root ganglion (DRG)) and CNS (hippocampal) neurons. We show that neurites from DRG neurons display maximal outgrowth on substrates with a Young's modulus of ∼1000 Pa, whereas hippocampal neurite outgrowth is independent of substrate stiffness. Using traction force microscopy, we also find a substantial difference in growth cone traction force generation, with DRG growth cones exerting severalfold larger forces compared with hippocampal growth cones. The traction forces generated by DRG and hippocampal growth cones both increase with increasing stiffness, and DRG growth cones growing on substrates with a Young's modulus of 1000 Pa strengthen considerably after 18-30 h. Finally, we find that retrograde actin flow is almost three times faster in hippocampal growth cones than in DRG. Moreover, the density of paxillin puncta is significantly lower in hippocampal growth cones, suggesting that stronger substrate coupling of the DRG cytoskeleton is responsible for the remarkable difference in traction force generation. These findings reveal a differential adaptation of cytoskeletal dynamics to substrate stiffness in growth cones of different neuronal types, and highlight the potential importance of the mechanical properties of the cellular environment for neuronal navigation during embryonic development and nerve regeneration.

Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.

A novel cytoskeletal action of xylosides.

Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.

Smad proteins differentially regulate transforming growth factor-ß-mediated induction of chondroitin sulfate proteoglycans.

Traumatic injury to the CNS results in increased expression and deposition of chondroitin sulfate proteoglycans (CSPGs) that are inhibitory to axonal regeneration. Transforming growth factor-ß (TGF-ß) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-ß regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-ß induction of CSPG expression in astrocytes is Smad-dependent. However, we find a differential dependence of the synthetic machinery on Smad2 and/or Smad3. TGF-ß induction of neurocan and xylosyl transferase 1 required both Smad2 and Smad3, whereas induction of phosphacan and chondroitin synthase 1 required Smad2 but not Smad3. Smad3 knockdown selectively reduced induction of chondroitin-4-sulfotransferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes. Additionally, Smad3 knockdown in astrocytes was more efficacious in promoting neurite outgrowth of neurons cultured on the TGF-ß-treated astrocytes. Our data implicate TGF-ß Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.