Mol Neurobiol (2013) 47:220–227 DOI 10.1007/s12035-012-8346-x

Protein Tyrosine Phosphatase σ in Proteoglycan-Mediated Neural Regeneration Regulation Pham Ngoc Chien & Seong Eon Ryu

Received: 6 July 2012 / Accepted: 27 August 2012 / Published online: 7 September 2012 # Springer Science+Business Media, LLC 2012

Abstract The receptor-type protein tyrosine phosphatase PTPσ mediates neural development and regeneration. Early studies on the ligands of PTPσ identified heparan sulfate proteolycan (HSPG) as a ligand. Binding of HSPG to PTPσ plays a critical role in axon guidance and synapse formation. PTPσ is also a receptor for chondroitin sulfate proteoglycan (CSPG). CSPG is deposited in high concentration at sites of neural injury. The deposited CSPG inhibits neural regeneration and axonal growth via PTPσ. The crystal structure of N-terminal immunoglobulin-like domains of PTPσ shows that the glycan binding site forms an elliptical surface patch of ∼35 by 24 Å, which interacts with sulfate groups of HSPG and CSPG. In this review, we focus on the structural and functional mechanisms for the neural regeneration regulation by different types of proteoglycans. We also discuss recent results on induction of neural regeneration in the stroke model and neural transplantation. The mechanistic understanding of relationships between proteoglycans and PTPσ provides new therapeutic opportunities against diseases with impaired neural regeneration. Keywords Protein tyrosine phosphatase σ . Proteoglycan . Neural regeneration . Chondroitin sulfate . Heparan sulfate . Structure and function

Introduction Protein-tyrosine phosphorylation plays a crucial role in proliferation, differentiation, migration, and transformation of P. N. Chien : S. E. Ryu (*) Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Korea e-mail: [email protected]

eukaryotic cells [1, 2]. The coordinated actions of proteintyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) control these processes. PTPs are divided into two categories based on their subcellular localization: receptorlike PTPs (RPTPs) and soluble PTPs [3]. Soluble PTPs usually have a single catalytic domain in conjunction with additional domains such as SH2 and PDZ domains. RPTPs are type I membrane proteins that have one membrane spanning region with its N- and C-termini in the extracellular and intracellular spaces, respectively. PTPσ belongs to the type IIa subfamily of RPTPs that include LAR, PTPσ, and PTPδ [1, 4, 5]. The extracellular region of PTPσ, which is highly reminiscent of cell adhesion molecules (CAM) in its domain structure, comprises three immunoglobulin-like (Ig) domains and four to nine fibronectin type III (FN) domains [6] (Fig. 1a). There are two intracellular catalytic domains (D1 and D2) in which only D1 has PTP activity and D2 has a regulatory role [7, 8]. The type IIa RPTPs play a vital role in neural development and regeneration [4]. Mice lacking PTPσ show various defects in neurological developments including hippocampal dysgenesis, late expression of the growth-associated protein (GAP) 43, reduction in brain size and abnormalities of hypothalamus [9–11]. PTPδ knockout mice exhibit learning impairment due to dysfunction of long-term potential and learning processes [12]. Mice with double mutations in PTPσ and PTPδ show severe muscle dysgenesis and loss of motor neurons in the spinal cord leading to paralysis [13]. LAR localizes to neurites and neuronal growth cones and regulates cell adhesion, synaptic formation and learning and memory [14–16]. Early studies showed that PTPσ is a receptor for heparan sulfate proteoglycan (HSPG) in neural development [17]. Proteoglycans are proteins with glucosaminoglycan (GAG) chains attached to the proteins' serine residues within the motif of Ser-Gly-X-Gly [18, 19]. The GAG chain, which

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b CNS injuries (glial scar, periinfarct)

CSPG upregulation

acute

Protection of damaged area beneficial

chronic Inhibition of regeneration

adverse

PTPσ knockout or chondroitinase treatment Neural regeneration

Fig. 1 The role of PTPσ in CNS injuries with the upregulated CSPG. a Domain structure of PTPσ. Among the three Ig-like domains in Nterminus, the first domain (Ig1) has the glucoseaminoglycan (GAG) binding site. SP, signal peptide; Ig, immunoglobulin-like domains; FN, fibronectin type III domains; TM, transmembrane region; Phosphatase, protein tyrosine phosphatase domains. Depending on isotypes, PTPσ can have either five or nine FN domains. b Effects of the CSPG upregulation. The upregulated CSPG due to CNS injuries has both beneficial and adverse effects. In the early stage of stroke, the glial scar in the periinfarct region prohibits toxic materials from crossing the boundary of infarct regions. However, in the chronic stage, the glial scar becomes a barrier to the regenerating neurons. The upregulated CSPG plays a key role in the inhibition of neural regeneration and the inhibition can be overcome by either blocking PTPσ or removing chondroitin sulfates

often contains sulfate groups, is attached to a tetrasaccharide linker that connects the GAG chain with a serine, resulting in GAG-(GlcA-Gal-Gal-Xyl)-Ser. Proteoglycans fill the extracellular matrix (ECM) and play structural and regulatory roles such as molecular movement, protein activity regulation, and intercellular signal regulation [20, 21]. In addition to the structural role in the connective tissues, proteoglycans such as chondroitin sulfate proteoglycan (CSPG) and heparan sulfate proteoglycan (HSPG) play central roles in the development and regeneration of neurons in the central nervous system (CNS) [22]. During the injury of CNS, CSPG rapidly accumulates in the glial scar tissue mainly made of reactive astrocytes [23, 24] (Fig. 1b). The accumulated CSPG prohibits regeneration of the injured neurons such as axon regeneration through the damaged area and regeneration of corticospinal tract axons. The regeneration-inhibitory function of CSPG was proved by treatment of the chondroitin-degrading enzyme, chondroitinase ABC, which resulted in improvement of the neural regeneration [25–27]. In comparison, HSPG promotes

neuronal growth [28, 29]. The elimination of HSPG during brain development results in severe abnormalities. For example, the glypican-1 (a HSPG)-deficient mice showed a significantly reduced brain size, indicating that HSPG plays a vital role in the growth of neurons [30]. Despite the overlapping role of PTPσ and proteoglycans in neural functions, their relationship was not clear. Recent studies suggest that PTPσ is a receptor for both HSPG and CSPG [31]. The relative amount of HSPG/CSPG in the region of axon determines the promoting/inhibitory roles of the proteoglycans, respectively [32, 33]. This review focuses on the structural and functional mechanisms of activity regulation of PTPσ through interaction of proteoglycans. We also describe intracellular signaling pathway mediated by dimerization of PTPσ. The understanding of mechanisms on the regulation of neuronal growth and regeneration should contribute to the development of therapeutics for efficient regeneration of damaged neurons.

Role of PTPσ in Neural Regeneration PTPσ regulates neuronal growth and guidance in axonal growth cones [5, 34, 35]. Early studies on the ligands of PTPσ identified HSPGs agrin and collagen XVIII as binding partners for avian PTPσ cPTPσ [17]. Agrin and collagen XVIII bound to cPTPσ with high affinity in vitro, and the binding was dependent on the sulfate groups in the HSPG molecules. The mutagenesis of positively charged residues on the first Ig-like domain of cPTPσ entirely abolished the binding of the HSPGs, indicating that HSPGs are the ligands of PTPσ. CSPG is deposited in high concentration at sites of neural injury [23, 24] (Fig. 1b). Regeneration of the corticospinal tract (CST) axon after spinal hemisection or contusion injury is inhibited by CSPG, and enzymatic degradation of CS chains by chondroitinase ABC relieves the inhibition [25–27, 36]. However, the similar lesion or injury in PTPσ knockout mice does not show the inhibition, resulting in the regeneration of long distance axons [36–39] (Fig. 1b). In addition, cerebella granule neurons from the PTPσ knockout mice are less sensitive to the inhibitory effects of CSPG, which may also explain the efficient growth of PTPσ knockout CST axons through the CSPG-rich glial scar. These results indicate that PTPσ is likely involved in axonal growth inhibition after spinal cord injury. Studies using the alkaline phosphate (AP)-tagged neurocan and the Fc-fusion of PTPσ ectodomain showed the direct binding of neurocan (CSPG3) to PTPσ ectodomain [31]. The cell free binding assay indicated that neurocan binds to PTPσ with an affinity of 11 nM with a concentrationdependent manner, suggesting that their interaction is a ligand–receptor-type interaction. Pretreatment of neurocan-AP

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with chondroitinase ABC abolished the binding. Mutations in the lysine cluster (Lys67, Lys68, Lys70, and Lys71) of Ig1 of PTPσ impaired binding of neurocan, indicating that the positively charged surface of PTPσ Ig1 is the binding site of CSPGs. Dorsal root ganglion (DRG) neurons normally express high levels of PTPσ [40]. The normal and the PTPσ knockout DRG neurons [10] exhibit differences in their abilities for neurite outgrowth [31]. An addition of a neural CSPG mixture in postnatal day 8 DRG neuron cultures inhibits neurite outgrowth in wildtype neurons. In comparison, the PTPσ-negative DRG neurons exhibit far less inhibitory effects in the same condition. The pretreatment of the CSPG mixture by chondroitinase ABC significantly reduced the inhibitory effect of CSPG mixture on the wildtype DRG neurons, whereas the treatment does not affect the function of PTPσ knockout neurons. The PTPσ-Fc fusion protein binds to spinal cord sections from a mouse injury model where the CSPG neurocan level increases, indicating that PTPσ is a receptor for neurocan [31]. In comparison, the fusion protein does not bind to normal and uninjured spinal cord. Treatment with chondroitinase ABC or the use of the mutant PTPσ with deletion on the Lys-rich region in Ig1 diminished binding of the fusion protein, indicating that PTPσ specifically binds to neurocan by using its positively charged Ig1. Together with molecular and cellular experiments, the interaction of neurocan with PTPσ indicates that PTPσ is a physiological receptor for CSPGs.

Proteoglycan Specific Molecular Switch In adult mouse dorsal root ganglion (DRG) sensory axons, the inhibition of neuronal growth by CSPG is mediated partly by PTPσ [31]. In comparison, PTPσ promotes neuronal growth in response to the basal lamina, which contains HSPGs [17]. Thus, PTPσ affects neuronal growth bimodally depending on the types of interacting proteoglycans. While PTPσ and HSPG colocalize in puncta on sensory neurons, CSPG occupies the extracellular matrix [38]. Site-directed mutagenesis studies indicated that both CSPG and HSPG bind a common site on PTPσ, indicating the competitive nature of the two proteoglycans [17, 31, 32]. Type IIa RPTPs including PTPσ, LAR, and PTPδ, have three Ig domains plus either five or nine FN domains in their ectodomain regions [3]. The N-terminal Ig domains contain the GAG-binding site [17, 31] (Fig. 1a). Among N-terminal domains of PTPσ, the first two Ig domains (Ig1-2) form a stable unit [32]. The crystal structure of the Ig1-2 of PTPσ shows a V-shape arrangement with extensive interdomain interactions including hydrophobic, salt bridge, and hydrogen bond interactions [32]. The residues involved in the

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interdomain interactions are conserved in other members of type IIa RPTPs. In the PTPσ Ig1-2 structure, residues implicated in the GAG binding (Lys67, Lys68, Lys70, Lys71, Arg96, and Arg99) reside in two loops between β-strands CD and EF forming an extended positively charged surface [32]. To confirm the GAG-binding site, the crystal structure of human LAR Ig1-2 in complex with sucrose octasulfate (SOS), a synthetic heparin-mimic was determined [32]. As expected, SOS bound to the surface patch made of the positively charged residues. In the complex structure, binding of SOS induced a movement of the region containing residues from Val72 to Phe77, a part of the Lys loop (residues Lys67-Phe77). The plasticity of the Lys loop seems to account for binding of diverse GAGs to the common type IIa RPTP GAG-binding site. The oligomerization studies of a PTPσ ectodomain of Ig1 to FN3 (six domains containing Ig1, Ig2, and Ig3 and FN1, FN2, and FN3) show that the oligomerization is dependent on the length of HS chains [32] (Fig. 2). Dp8 and dp10 can induce dimerization of PTPσ. Dp20 and dp30 can form trimeric and tetrameric complexes. However, CS does not mediate the PTPσ ectodomain oligomerization. In addition, excess CS competes with HS in the HS-induced oligomerization. Addition of CSPG to a DRG culture prohibited the HSPG-induced promotion of neuronal growth, indicating that the activity of PTPσ is regulated by the ratio of HSPG/CSPG. The PTPσ Ig1-2 structure shows that the glycan binding site of Ig1 forms an elliptical surface patch of ∼35 by 24 Å [32]. In solution, heparin forms a helical structure that has a repeat of four saccharides and a pitch of 17.5 Å [41]. Recently, the constrained X-ray scattering modeling by using synchrotron radiation revealed a flexible and mildly bent structure for purified heparin fragments dp6–dp36 [42]. The alignment of a heparin dp24 chain with the PTPσ glycan binding domain (Ig1-2) indicates that the HS chain of about dp10 can bind to two glycan binding domains of PTPσ (Fig. 2a). The two PTPσ ectodomains bound to the HS chain may form a dimer. Overall structures of CS and HS are similar, and both have repeating units of disaccharides with variable number of sulfate groups. However, the distribution of sulfate groups is different. CS has evenly distributed mono or di-sulfated glycan units, whereas HS does islands of highly sulfated glycans (triple sulfate groups) separated by intermediately sulfated glycans [43, 44] (Fig. 2b). These highly sulfated regions in HSPG may mediate close packing of PTPσ molecules. The HSPG-induced PTPσ clustering may inhibit PTP activity as shown in the case of PTPα [45] and prolongs kinase activity leading to neuronal growth in the region (Fig 2c). Otherwise, the oligomerization may generate regions lacking PTP activity by depletion of PTPσ in the regions lacking oligomerization (Fig. 2c). Thus, the molecules that promote the PTPσ

Mol Neurobiol (2013) 47:220–227 Fig. 2 Differential regulation mechanisms of PTPσ activity by proteoglycans. a Structural model of the interaction between a proteoglycan chain and PTPσ molecules. A heparin dp24 structure (PDB code: 3IRJ) [42] was aligned with four molecules of the PTPσ Ig1-2 (PDB code: 2YD3). The positively charged patch (blue, the GAG-binding region) of Ig1 domain was aligned to face the heparin chain to mimic the proteoglycan–PTPσ interaction. The heparin chain of dp24 can bind four molecules of PTPσ. For the dimerization, a proteoglycan of around dp10 is necessary, which is consistent with data discussed in the text. b Domain structure of HSPG. In heparan sulfate proteoglycan (HSPG), there are islands (S domain) of highly sulfated regions of dp4-16 (red). NAc, Nacetylated; GlcA-GlcNAc, (Glucuronic acid)-(N-acetylated glucosamine); GluNS(+/-6S)IdoA(2S), (N-sulfoglucosamine (+/-6S))-(Iduronic acid(2S)); NA/ NS, N-acetylated/N-sulfated. c PTPσ regulation by different domain structures of heparan sulfate (HS) and chondroitin sulfate (CS). PTPσ Ig1-2 molecules are represented as blue circles on the strips of proteoglycans HS and CS. The highly sulfated islands (red) in HS mediate an efficient oligomerization of PTPσ. The oligomerization of PTPσ molecules can generate uneven distribution of PTP activity either by dimerizationmediated enzyme inhibition or depletion of enzyme in the regions lacking oligomerization. The CS with even distribution of medium or low-sulfated disaccharide units cannot mediate efficient oligomerization of PTPσ

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a

97 Å

dp24

53 Å

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S domain size: dp4-dp16

NA cdomain [GlcA-GlcNAc]n S domain [GlcNS(+/-6S)-IdoA(2S)]n NA/NS domain [GlcNAc-GlcA-GlcNS-IdoA]n

c HS i) Uneven PTP activity ii) Neural regeneration

CS

oligomerization or inhibit the PTP enzyme activity may be beneficial to the neural regeneration after CNS injury.

CSPG-Mediated Intracellular Signaling CSPGs target and lead to the dephosphorylation of tropomyosin-related kinase B (TrkB), the receptor of brain-derived neurotrophic factor (BDNF), in embryonic

i) Even PTP activity ii) Inhibition of neural regeneration

cortical neurons in vitro [46]. While BDNF promotes dendritic spine formation in embryonic cortical neurons [47–50], CSPGs abolish the effects of BDNF and eliminate existing dendritic spines [46]. CSPGs attenuate BDNFinduced phosphorylation of Shc, indicating that Shc is a downstream effector of TrkB [46]. Knockout of PTPσ in cells by the shRNA method abolishes downregulation of phosphorylated TrkB in response to CSPGs. In comparison, the PTPσ knockout cells do not affect phosphorylation

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levels of TrkB induced by BDNF, indicating that the response to CSPG signal is mediated by PTPσ. Earlier studies also indicated that PTPσ interacts with neurotrophin receptors (Trk proteins) and regulates their phosphorylation level [51]. PTPσ interacts with two (TrkA and C) of three Trk proteins, while it dephosphorylates all three Trk proteins (TrkA, B and C) in transfected HEK293 cells. In comparison, TrkA phosphorylation level is not affected by PTPα, indicating that the interaction of PTPσ with Trk proteins is specific. Consistent with the function of Trk proteins as receptors for neural growth factor neurotrophin, overexpression of PTPσ in primary sensory neurons blocks neurite outgrowth. Recent studies also indicated that postsynaptic TrkC interacts with presynaptic PTPσ to mediate bidirectional adhesion for excitatory synapse development [52]. Additional intracellular signaling partners for PTPσ whose relationship with proteoglycans are not reported yet include Liprin-α, N-cadherin, β-catenin, Trio, and p250GAP [53–55]. The structure of intracellular catalytic domains of PTPσ is known [56]. The structure contains tandem PTP catalytic domains (D1 and D2). D1 has an intact catalytic core including the catalytically crucial WPD and KNRY-loops, whereas D2 has substitutions of residues in both WPDloop (aspartate to glutamate) and KNRY-loop (tyrosine to leucine), resulting in the absence of catalytic activity in D2. Unlike the potential dimerization interaction found in the crystal lattice of PTPα [45], the crystal packing of PTPσ did not show any indication of dimerization interactions [56]. The purified D1D2 protein of PTPσ also did not form dimers in solution as judged by an analytical size exclusion chromatography [56]. The D1D2 protein of LAR existed as monomers in solution, and its crystal contacts did not have the dimeric interactions observed in PTPα [57]. Dimerization of RPTP intracellular domains in PTPα inhibits the PTP activity by blocking the active site through the dimeric interface [45, 58–60]. Although the in vitro biochemical studies could not find an indication of dimerization of PTPσ and other RPTPs, the dimerization was observed in cell studies of PTPσ and shown to be crucial in ligand binding [61]. Thus, the activity regulation of PTPσ by HSPGs and CSPGs are likely to be controlled by dimerization. Then, how would one resolve the discrepancy between the lack of dimerization in the biochemical analysis and the expected dimerization for functional regulation? One possibility is that the dimerization occurs through regions outside the D1D2 domains. Biochemical studies indicate that the dimeric interaction of PTPσ involves the membrane spanning and juxtamembrane regions [61]. One also has to answer for how the dimerization by regions outside the D1D2 domains can affect phosphatase activity. The initial dimerization by the outside regions may trigger

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dimerization of D1D2 domains. However, the detailed mechanism for the dimerization-induced signal transduction of PTPσ may be revealed by 3D-structure determination of PTPσ including more domains than the D1D2 domains.

Therapeutic Implications The role of CSPGs in growth inhibition of neurons was analyzed by a stroke model system [33]. Brain infarct due to blockage of cerebral artery creates glial scars in the periinfarct region [62]. The glial scar, which is made of reactive astrocytes and ECM proteins including CSPGs, plays a beneficial role in the acute phase of neural ischemia by confining toxic materials within the injured regions [63] (Fig. 1b). However, in chronic phases, the glial scar prohibits axonal growth and functional recovery of neurons, limiting the treatment options for the disease [62, 64]. Treatment of middle cerebral artery occlusion (MCAO) mouse with neurocan (a CSPG), glypican (a HSPG), and chondroitinase ABC revealed the relationship between neural regeneration and PGs [33]. When those proteins were infused into the 7-day MCAO-induced infarct area, glypican and chondroitinase ABC reduced glial fibrillary acidic protein and increased microtubule-associated protein-2 in the periinfarct area, resulting in behavioral improvement of the mouse. In comparison, neurocan did not show changes in either the protein levels in the periinfarct region or the behavior of the mouse. The beneficial effects of HSPG and chondroitinase ABC in stroke are consistent with the previous suggestion that HSPG and CSPG are competitive ligands for the function of PTPσ [32, 65]. Treatment of HSPG to the CSPG-upregulated glial scar appears to suppress the effects of CSPG on PTPσ and shifts the PTPσ switch towards the neural regeneration. The degradation of CS chains by chondroitinase ABS also promotes the neural regeneration. In addition to PTPσ, another Type IIa RPTP LAR also mediates inhibitory effects of CSPG on neural regeneration [66]. Structure of LAR shows the positively charged surface patch that can interact with sulfate groups of CSPGs [65]. The in vitro binding studies show that LAR binds CSPGs with high affinity [66]. Selective LAR blocking peptides show enhancement of neurite outgrowth into the region of enriched CSPGs [67, 68], indicating that LAR also may function as a receptor for CSPGs. Treatment of LAR blocking peptides to mice with thoracic spinal cord transection injuries enhances axon growth through the lesion area, resulting in locomotor function recovery [66]. However, LAR-deficient mice showed reduced neural regeneration, indicating that LAR may play multiple roles in neuronal growth regulation [69].

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Studies on the transplantation of neural restricted precursors (NRP) and glial restricted precursors (GRP) showed that expression levels of PTPσ and LAR determine sensitivity to the CSPG-mediated inhibition of neural regeneration [70]. In contrast to DRG neurons, the transplanted NRP could cross the glial scar that is rich with CSPGs such as brevican, phosphcan, aggrecan, and versican [71–73]. Growth cones of NRP were found to contain significantly low levels of PTPσ and LAR [70], consistent with previous results that disruption of PTPσ gene promotes axonal outgrowth into the CSPG-enriched regions.

showed a dimerization different from PTPα, suggesting that the dimerization model based on PTPα may not be general [74]. In comparison, cellular analyses indicated dimerization of PTPσ in vivo [61]. Thus, dimerization of PTPσ may be induced by regions other than the catalytic domains. It is also crucial to resolve the question of how the induced dimerization regulates the catalytic domains' enzyme activity. Future structural and functional studies on PTPσ involving more domains are needed to understand the detailed mechanism of the PG-mediated PTPσ activity regulation and neural regeneration.

Conclusions

References

The type IIa RPTP PTPσ, which comprises three Ig-like domains and 5–9 FN domains in the extracellular space, functions as a receptor for both HSPG and CSPG [31]. The Ig1 domain of PTPσ has a positively charged surface patch that functions as a PG-binding site and the two types of PGs (HSPG and CSPG) seem to compete with each other for the same binding site. The complex structure of a synthetic heparin-mimic SOS and the Ig1-2 of LAR indicates the flexible nature of the PG-binding site for accommodating two different PGs [32]. Despite the same binding mechanism, effects of HSPG and CSPG on neuronal growth are opposite, inhibitory and promoting, respectively. The dual effects of different PGs appear to originate from the different chemical structures of HSPG and CSPG. HSPG has regions of concentrated sulfate groups that can trigger oligomerization of PTPσ [43, 44]. The oligomerization may result in uneven distribution of PTP activity either by dimerization-mediated enzyme inhibition or depletion of enzyme in the regions lacking oligomerization. In comparison, sulfate groups of CSPG are less dense and evenly located, so that the CSPG-bound PTPσ molecules may have less chance of oligomerization [32]. Thus, CSPG-bound PTPσ efficiently dephosphorylates the substrate proteins and turns off the neuronal growth. The mechanistic understanding of relationships between PGs and PTPσ provides new therapeutic opportunities. Treatment of HSPG or CS-degrading enzyme promotes neurite outgrowth through the periinfarct region of chronic stroke, resulting in behavioral recovery [33]. The transplantation of NRP that has a low amount of expressed PTPσ, into the neural injury regions also induced an efficient neural regeneration through the CSPG-enriched scar regions [70, 71]. Although it is established that PTPσ is a receptor for both HSPG and CSPG, there remain several questions as to the activity regulation of intracellular PTP domains. Unlike PTPα, PTPσ and LAR did not show the activity regulating dimerization either in crystals or in solution [56, 57]. PTPγ

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