Regular articleAntibody recognizing 4-sulfated chondroitin sulfate proteoglycans restores memory in tauopathy-induced neurodegeneration
Introduction
Memory loss is a largely unmet medical challenge. Halting brain degeneration would be the most effective form of treatment, but it has yet to be achieved. An alternative is to enable the brain to function despite the degenerative damage. Plasticity enables the brain to bypass damage, but it is limited in adults by inhibitory proteoglycans in the brain extracellular matrix (ECM) situated in perineuronal nets (PNNs), which surround the key regulatory neurons.
PNNs are dense pericellular ECM structures with a structure similar to cartilage that develop at the time of closure of critical periods for plasticity. PNNs are a highly organized complex, mainly composed of hyaluronan, chondroitin sulfate proteoglycans (CSPGs), link proteins, and tenascin-R. The form of PNN most studied is those surrounding parvalbumin-positive (PV) interneurons recognized by the lectin Wisteria floribunda (WFA), although other forms of PNN exist around pyramidal and other neurons (Matthews et al., 2002). In the cortex, PNNs surround PV interneurons which are known to influence plasticity and excitability, and PNNs are involved in the termination of plasticity at the end of critical periods (Fawcett, 2015, Pizzorusso et al., 2002, Sugiyama et al., 2009). PNNs are also involved in memory. Depletion of chondroitin sulfate (CS) with the CS-digesting enzyme, chondroitinase ABC (ChABC) prolonged object recognition (OR) memory in normal mice (Romberg et al., 2013), restored lost memory in a neurodegeneration model in tauopathy mice and Abeta transgenic mice (Vegh et al., 2014, Yang et al., 2015), and affected fear memory extinction (Gogolla et al., 2009), demonstrating that CSPGs are the key molecules in regulating this form of memory and that the CS glycosaminoglycan (GAG) chains digested by ChABC are their effectors. Animals deficient in the PNN component Crtl1 link protein have very attenuated PNNs and show the same changes in memory and plasticity as animals treated with ChABC despite having unaltered CSPGs in the central nervous system (CNS), demonstrating that it is CSPGs in PNNs that are controllers of plasticity and memory (Carulli et al., 2010, Romberg et al., 2013). However, in order to reactivate plasticity, ChABC has to be injected directly into the brain parenchyma where it digests a region of up to 1 mm across, and the effect lasts for around 3 weeks. ChABC is therefore not a practicable treatment for memory restoration in neurodegenerative diseases. This study investigates antibody treatment as an alternative.
CSPGs and PNNs are implicated in neurodegenerative conditions in various ways. CSPGs are abundant in amyloid and neurofibrillary tangles in Alzheimer's disease (AD; reviewed in DeWitt et al., 1993, Kwok et al., 2011) and differentially sulfated CS such as chondroitin 4-sulfate (C4S), chondroitin 6-sulfate (C6S), and unsulfated chondroitin were detected (DeWitt et al., 1993). A reduction of PNNs in brains affected by AD was shown by staining with lectin WFA (Baig et al., 2005, Kobayashi et al., 1989), although many markers were not affected in AD patients and animal models of AD (Morawski et al., 2010b, Morawski et al., 2012b). PV interneurons are largely unaffected by neurofibrillary tangles (Baig et al., 2005, Bruckner et al., 1999, Hartig et al., 2001, Morawski et al., 2010a, Morawski et al., 2012b). This suggests that PNNs may be protective, and there is evidence for regulation of ion homeostasis (Suttkus et al., 2014) and neuroprotection against oxidative stress (Cabungcal et al., 2013, Suttkus et al., 2016). Treatment with chondroitinase does not affect hyperphosphorylated or filamentous tau (Yang et al., 2015), but an effect on Abeta quantity has been reported (Howell et al., 2015).
The functional effect and binding properties of CSPGs are heavily dependent on the patterns of sulfation of their GAG chains (Lin et al., 2011, Malavaki et al., 2008, Miyata and Kitagawa, 2015). The sugars in the GAG chain can be sulfated in the 4, 6, 2 + 6, and 4 + 6 positions on the CSs (C4S, C6S, C2,6S, and C4,6S, also known as CS-A, CS-C, CS-D, and CS-E), respectively, and different patterns of sulfation give motifs that can provide specific binding sites (Mikami and Kitagawa, 2013, Xu and Esko, 2014, Yabuno et al., 2015). C4S is inhibitory to neurite growth while C6S is more permissive to neurite growth and to plasticity (Lin et al., 2011, Miyata et al., 2012, Wang et al., 2008). Thus, mice with a knockout of chondroitin 6-sulfotransferase 1 showed worse regeneration and less plasticity than wild-type (WT) animals (Lin et al., 2011) while chondroitin 6-sulfotransferase 1 overexpressing mice demonstrated enhanced plasticity into adulthood (Miyata et al., 2012). During embryonic development C6S predominates, but at the end of the critical period in the PNN fraction only 4.9% of CS-GAG is 6-sulfated while 80.6 % is the inhibitory 4-sulfated form (Carulli et al., 2010, Deepa et al., 2006). We have recently demonstrated that this trend continues into aging when memory deficits start to appear. C6S was reduced almost to zero in aged rats (12 and 18 months old) when compared to young animals (Foscarin et al., 2017). Moreover, the sulfation pattern of PNN CS-GAGs (which make up 2% of total brain CS-GAG) is different from that of the general brain matrix, giving the PNN specific binding properties (Deepa et al., 2006). Thus, semaphorin 3A (Sema3A) and OTX2 bind specifically to PNNs but not to the general matrix (Beurdeley et al., 2012, Dick et al., 2013, Miyata et al., 2012, Vo et al., 2013), where they are positioned to affect synapse dynamics and PV cell maturation. Both these molecules bind most strongly to 4,6 disulfated CS-GAG. The major CSPG core protein of PNNs is aggrecan, which is necessary for PNN formation (Carulli et al., 2016, Dick et al., 2013, Giamanco et al., 2010, Morawski et al., 2012a) and contains specific glycan modifications in its linkage region (Yabuno et al., 2015). The CS-GAGs carried by aggrecan are probably the key component of PNNs that regulate memory and plasticity, because animals lacking aggrecan in the CNS show the same memory prolongation phenotype as animals with attenuated PNNs due to Crtl1 knockout or ChABC treatment (unpublished observations) (Romberg et al., 2013). Our hypothesis therefore was that masking C4S on aggrecan would make PNNs less inhibitory and would therefore have a similar effect as ChABC in restoration of memory to brains damaged by neurodegeneration or in prolonging memory in normal animals.
Seeking potential treatments for memory restoration, we have tested an inhibitory proteoglycan neutralizing antibody. In this manuscript, we have identified Cat316 as a C4S binding antibody that can modulate the inhibitory properties of the glycan, reduce Sema3A binding, prolong OR memory in normal animals, and restore lost memory in tauopathy animals.
Section snippets
Screening of PNN-blocking antibodies using PNN-HEK cells
PNN-human embryonic kidney (HEK) cells (∼12,000 cells) were plated on poly-L-lysine–coated coverslips for 2 hours and cultured in low-serum medium (0.5 % fetal bovine serum in Dulbecco's modified eagle medium [DMEM]) in the presence of 5% of CO2. Detached cells were then removed with a rinse in fresh warm medium. Potential PNN-blocking antibodies (Cat316 is from Millipore; HAPLN1 is from R&D; 6B4 and 7B7 are from CosmoBio) were diluted into 5 μg/mL in cultured medium before incubation with the
Results
The aim of our experiments was to select and evaluate an antibody that can reactivate plasticity and restore memory through blocking inhibitory proteoglycans in PNNs. We tested several candidates for their ability to block inhibition by PNN proteoglycans, from which we selected Cat316. The ability of Cat316 to bind to proteoglycans and PNNs and to compete with Sema3A binding to PNNs was then evaluated in more detail. We then asked whether Cat316 has the same effects on memory prolongation and
Discussion
PNNs have been implicated in memory through several types of experiment. In OR memory, transgenic attenuation of PNNs or their digestion with ChABC leads to memory prolongation (Romberg et al., 2013). In fear memory, ChABC digestion in the amygdala restores memory erasure (a form of learning) to the infantile pattern (Gogolla et al., 2009). Memory stimulated by environmental enrichment depends on the formation of new inhibitory synapses on PV neurons, and this synapse formation is facilitated
Disclosure statement
JWF is a paid consultant for Acorda Therapeutics Inc, which is involved in the commercial development of chondroitinase. The other authors declare no other competing financial interests.
Acknowledgements
This work was supported by the ERC advanced grant ECM Neuro (294502), by a fellowship to SY from Alzheimer's Research UK (ARUK-RF2016A-1), and by the NIHR Cambridge Biomedical Research Centre. The authors are grateful to Dr Michel Goedert (MRC Laboratory of Molecular Biology, Cambridge, UK) for providing the human P301S tau transgenic mice.
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