Double-stranded RNA-dependent protein kinase (PKR) is a component of signal transduction pathways mediating various stress signals including oxidative stress and endoplasmic reticulum (ER) stress and is suggested to be implicated in several neurodegenerative diseases. Cell death in neurodegenerative conditions has been linked to oxidative stress; however, the involvement of PKR in endogenous oxidative stress such as oxytosis and ferroptosis which is quite distinct from classical apoptosis remains unknown. We investigated here the effect of a PKR inhibitor C16 (an imidazole-oxindole derivative) on oxytosis and ferroptosis in cultured HT22 mouse hippocampal cells. C16 prevented glutamate- and erastin-induced cell death, reactive oxygen species accumulation, Ca2+ influx, phosphorylation of inositol-requiring enzyme 1 (IRE1), one of the three branches of ER stress signaling and its downstream signaling components. On the other hand, C16 did not prevent oxidative stress-induced heme oxygenase-1 expression; instead, C16 activated the extracellular signal-regulated kinase pathway. The protective effect of C16 is diminished in PKR knockout HT22 cells. Real time measurements of the oxygen consumption rate and extracellular acidification rate over a long period of time leading to cell death showed that C16 partially prevented erastin-induced mitochondrial and glycolytic dysfunction. These results suggest that PKR is an important component of oxytosis and ferroptosis and the inhibition of PKR is neuroprotective against endogenous oxidative stress-induced cell death and provide an effective strategy for neuroprotection.

A split NanoLuc assay system consisting of two fragments, large N-terminal and small C-terminal regions (NanoBiT), was developed to investigate protein-protein interactions within living cells. Interestingly, the replacement of five amino acids among 11 C-terminal amino acids dramatically increased affinity against the large N-terminal fragment, LgBiT, and the complex had NanoLuc luciferase activity. In this study, we first applied this small fragment, HiBiT, to elucidate the expression of ATF4 protein by transient overexpression of HiBiT-tagged ATF4. According to the regulation of intrinsic ATF4 protein, stabilization of HiBiT-tagged ATF4 with a proteasome inhibitor, MG132, was observed by detecting luciferase activity in cell lysate and after SDS-PAGE and transfer onto a PVDF membrane. Next, we knocked-in the HiBiT-epitope tag into the ATF4 gene using the CRISPR/Cas9 system and rapidly selected positive clones by measuring luciferase activity in an aliquot of each cell suspension. Using a selected clone, we observed that the expression of HiBiT-tagged ATF4 in the selected cells varied in response to treatment with protein synthesis inhibitors or proteasome inhibitors and tunicamycin. Altogether, this novel HiBiT tag is a useful tool to evaluate the endogenous expression levels of proteins of interest.

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1(-/-); Ngr2(-/-); Ngr3(-/-); which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1(-/-); Ngr3(-/-)), but not Ngr1 and Ngr2 (Ngr1(-/-); Ngr2(-/-)), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1(-/-); Ngr3(-/-) mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

The Unfolded Protein Response pathway is a conserved signaling mechanism having important roles in cellular physiology and is perturbed accompanying disease. We previously identified the novel UPR target gene CHAC1, a direct target of ATF4, downstream of PERK-EIF2A and activated by the UPR pathway. CHAC1 enzyme directs catalysis of γ-linked glutamate bonds within specific molecular targets. CHAC1 is the first enzyme characterized that can catalyze intracellular glutathione degradation in eukaryotes, having implications for regulation of oxidative stress. DDIT3 (CHOP) is a terminal UPR transcription factor, regulated by ATF4 and an output promoting cell death signaling. Herein we examine the relationship of CHOP controlling CHAC1 transcription in humans and mice. We note parallel induction of CHOP and CHAC1 in human cells after agonist induced UPR. Expanding upon previous reports, we define transcriptional induction of CHAC1 in humans and mice driven by ATF4 through a synergistic relationship with conserved ATF/CRE and CARE DNA sequences of the CHAC1 promoter. Using this system, we also tested effects of CHOP on CHAC1 transcription, and binding at the CHAC1 ATF/CRE using IM-EMSA. These data indicate a novel inhibitory effect of CHOP on CHAC1 transcription, which was ablated in the absence of the ATF/CRE control element. While direct binding of ATF4 to CHAC1 promoter sequences was confirmed, binding of CHOP to the CHAC1 ATF/CRE was not evident at baseline or after UPR induction. These data reveal CHAC1 as a novel CHOP inhibited target gene, acting through an upstream ATF/CRE motif via an indirect mechanism.

Manganese neurotoxicity leads to Parkinson-like symptoms associated with the apoptotic cell death of dopaminergic neurons. Protein kinase R (PKR) is a serine/threonine-specific protein kinase that has been implicated in several cellular signal transduction pathways, including the induction of apoptosis. Here, we investigated the role of PKR in the manganese-induced apoptosis of dopamine-producing pheochromocytoma PC12 cells. Manganese (0.5 mM) induced the proteolytic cleavage of PKR and caspase-3, DNA fragmentation, and cell death, which were prevented by the co-treatment of PC12 cells with a PKR specific inhibitor, C16 in a concentration-dependent manner. C16 did not affect the manganese-induced activation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) pathway, indicating that PKR functions downstream of JNK and p38 MAPK. In contrast, C16 triggered the activation of the p44/42 MAPK (ERK1/2) pathway and induced hemoxygenase-1, both in the absence and presence of manganese. PKR is reportedly involved in endoplasmic reticulum (ER) stress-induced apoptosis. Manganese activated all three branches of the unfolded protein response in PC12 cells; however, this effect was very weak compared with the ER stress induced by the well-known ER stress inducers thapsigargin and tunicamycin. Moreover, C16 did not affect manganese-induced ER stress at concentrations that almost prevented caspase-3 activation and DNA fragmentation. These results suggest that PKR is involved in manganese-induced apoptotic cell death and stress response, such as the activation of the p44/42 MAPK pathway and the induction of hemoxygenase-1. Although manganese induced a faint, but typical, ER stress, these events contributed little to manganese-induced apoptosis.

Oxidative stress has been implicated in the aging process and the progression of many neurodegenerative disorders. We previously reported that a novel oxindole compound, GIF-0726-r, effectively prevents endogenous oxidative stress, such as oxytosis/ferroptosis, an iron-dependent form of non-apoptotic cell death, in mouse hippocampal cells. In this study, using two hundred compounds that were developed based on the structure-activity relationship of GIF-0726-r, we screened for the most potent compounds that prevent glutamate- and erastin-induced oxytosis and ferroptosis. Using submicromolar concentrations, we identified nine neuroprotective compounds that have N,N-dimethylaniline as a common structure but no longer contain an oxindole ring. The most potent derivatives, GIF-2114 and GIF-2197-r (the racemate of GIF-2115 and GIF-2196), did not affect glutathione levels, had no antioxidant activity in vitro, or ability to activate the Nrf2 pathway, but prevented oxytosis/ferroptosis via reducing reactive oxygen production and decreasing ferrous ions. Furthermore, we developed fluorescent probes of GIF-2114 and GIF-2197-r to image their distribution in live cells and found that they preferentially accumulated in late endosomes/lysosomes, which play a central role in iron metabolism. These results suggest that GIF-2114 and GIF-2197-r protect hippocampal cells from oxytosis/ferroptosis by targeting late endosomes and lysosomes, as well as decreasing ferrous ions.

Oxidative stress is the central pathomechanism in multiple cell death pathways, including ferroptosis, a form of iron-dependent programmed cell death. Various phytochemicals, which include the inducers of the nuclear factor erythroid-2-related factor 2-antioxidant response element (Nrf2-ARE) transcription pathway, prevent ferroptosis. We recently reported that several compounds, such as the potent Nrf2-ARE inducer curcumin, protect mouse hippocampus-derived HT22 cells against ferroptosis independently of Nrf2-ARE activity. The present study characterized the anti-ferroptotic mechanisms of two additional Nrf2-ARE inducers, quercetin and resveratrol. Both compounds prevented erastin- and RSL3-induced ferroptosis of wild-type HT22 cells, and also blocked the exacerbated erastin- and RSL3-induced ferroptosis of Nrf2-knockdown HT22 cells. In both HT22 cells, quercetin and resveratrol blocked erastin- and RSL3-induced elevation in reactive oxygen species. These results suggest that the Nrf2-ARE pathway does protect against ferroptosis, but quercetin and resveratrol act by reducing oxidative stress independently of Nrf2-ARE induction. Quercetin and resveratrol also reduced Fe2+ concentrations in HT22 cells and in cell-free reactions. Thus, quercetin and resveratrol likely protect against erastin- and RSL3-induced ferroptosis by inhibiting the iron-catalyzed generation of hydroxyl radicals. Unlike quercetin, resveratrol cannot form a chelate structure with Fe2+ but the density functional theory computation demonstrates that resveratrol can form stable monodentate complexes with the alkene moiety and the electron-rich A ring.

Glutathione (GSH), which is particularly important for antioxidant defenses, is synthesized in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine ligase (GCL) and GSH synthase. GCL comprises catalytic (GCLC) and regulatory subunits and catalyzes the rate-limiting step in de novo GSH synthesis. Accumulating evidence suggests that substances that stimulate GSH synthesis are therapeutic modalities for neurodegenerative disorders and schizophrenia, in which a deficit in brain GSH content has been observed. In the present study, we attempted to develop small organic compounds that increase GCLC transcription. Using HT22 cells stably expressing a luciferase reporter that contains rat GCLC promoter region (-1764 to +2), we assessed the effects of the novel neuroprotective compound oxindole and related compounds on GCLC promoter activity. Among approximately 220 synthesized compounds, five compounds increased GCLC promoter activity by >200% at a concentration of 50 μM, and 16 compounds increased promoter activity by approximately 150%. The most effective compound oxindole-curcumin hybrid GIF-2165X-G1 increased GCLC mRNA levels in HT22 mouse hippocampal cells, PC12 rat pheochromocytoma cells, and C6 rat glioma cells. Although GIF-2165X-G1 potently induced antioxidant response element (ARE)-driven transcription, the compound increased GCLC transcriptional activity through Sp1 pathway in a Keap1-Nrf2-ARE-independent manner. These results suggest that GIF-2165X-G1 itself and further modification of the compound are useful interventions for promoting neuronal survival by augmenting resistance to oxidative stress.

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-β (TGF-β) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-β regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-β 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-β 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-β-treated astrocytes. Our data implicate TGF-β Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.

Recently, we identified cysteine-rich with EGF-like domains 2 (CRELD2) as a novel endoplasmic reticulum (ER) stress-inducible gene and characterized its transcriptional regulation by ATF6 under ER stress conditions. Interestingly, the CRELD2 and asparagine-linked glycosylation 12 homolog (ALG12) genes are arranged as a bidirectional (head-to-head) gene pair and are separated by less than 400 bp. In this study, we characterized the transcriptional regulation of the mouse CRELD2 and ALG12 genes that is mediated by a common bidirectional promoter.

Parkinson's disease is characterized by degeneration of dopaminergic neurones in the substantia nigra. Chronic manganese poisoning shares many features of Parkinson's disease, and also induces extrapyramidal syndromes that resemble those of Parkinson's disease due to dopamine depletion in the central nervous system. This study was undertaken to develop novel neuroprotective drugs via the identification of compounds that inhibit manganese-induced apoptosis. Here, we report that (arylthio)cyclopentenone derivatives, which are synthetic analogs of cyclopentenone prostaglandins, prevent manganese-induced apoptosis in PC12 cells. A highly sensitive assay of caspase-3/7 activity was used for screening newly synthesized prostaglandin analogs. The results showed that some cyclopentenone derivatives (GIF-0642, GIF-0643, GIF-0644, GIF-0745, and GIF-0747) inhibit manganese-induced caspase-3/7 activation in a concentration-dependent manner. Effective compounds all have an arylthio group, indicating that this structure plays an important role in the anti-apoptotic effects of (arylthio)cyclopentenone derivatives. The anti-apoptotic effects of these compounds were confirmed by verifying their ability to inhibit the DNA fragmentation and caspase-9 activation induced by manganese. Furthermore, GIF-0747 prevented manganese-induced cytochrome c release from mitochondria. These results suggest that (arylthio)cyclopentenone derivatives may be good candidates for treating neurodegenerative diseases.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.

Endoplasmic reticulum (ER) stress and oxidative stress lead to protein misfolding, and the resulting accumulation of protein aggregates is often associated with the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion disease. Small molecules preventing these pathogenic processes may be effective interventions for such neurodegenerative disorders. In this paper, we identify several novel oxindole compounds that can prevent ER stress- and oxidative stress-induced cell death. Among them, derivatives of the lead compound GIF-0726-r in which a hydrogen atom at the oxindole ring 5 position is substituted with a methyl (GIF-0852-r), bromine (GIF-0854-r), or nitro (GIF-0856-r) group potently suppressed global ER stress. Furthermore, GIF-0854-r and -0856-r prevented protein aggregate accumulation in vitro and in cultured hippocampal HT22 neuronal cells, indicating that these two compounds function effectively as chemical chaperones. In addition, GIF-0852-r, -0854-r, and -0856-r prevented glutamate-induced oxytosis and erastin-induced ferroptosis. Collectively, these results suggest that the novel oxindole compounds GIF-0854-r and -0856-r may be useful therapeutics against protein-misfolding diseases as well as valuable research tools for studying the molecular mechanisms of ER and oxidative stress.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a newly emerging viral disease with fatal outcomes. However, no MERS-CoV-specific treatment is commercially available. Given the absence of previous structure-based drug discovery studies targeting MERS-CoV fusion proteins, this set of compounds is considered the first generation of MERS-CoV small molecule fusion inhibitors. After a virtual screening campaign of 1.56 million compounds followed by cell-cell fusion assay and MERS-CoV plaques inhibition assay, three new compounds were identified. Compound numbers 22, 73, and 74 showed IC50 values of 12.6, 21.8, and 11.12 μM, respectively, and were most effective at the onset of spike-receptor interactions. The compounds exhibited safe profiles against Human embryonic kidney cells 293 at a concentration of 20 μM with no observed toxicity in Vero cells at 10 μM. The experimental results are accompanied with predicted favorable pharmacokinetic descriptors and drug-likeness parameters. In conclusion, this study provides the first generation of MERS-CoV fusion inhibitors with potencies in the low micromolar range.

Propolis, a compound produced by honeybees, has long been used in food and beverages to improve health and prevent diseases. We previously reported that the ethanol extracts of Brazilian green propolis and its constituents artepillin C, kaempferide, and kaempferol mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. Here, we investigated the potential of Brazilian green propolis and its constituents to protect against endoplasmic reticulum stress in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis, artepillin C, and kaempferol attenuated tunicamycin-induced unfolded protein response and cell death. Interestingly, artepillin C inhibited both tunicamycin-induced protein aggregation in HT22 cells and the spontaneous protein aggregation of mutant canine superoxide dismutase 1 (E40K-SOD1-EGFP) in Neuro2a cells. These findings indicate that in addition to oxidative stress, the ethanol extracts of Brazilian green propolis help prevent endoplasmic reticulum stress-related neuronal cell death, which is proposedly involved in several neurodegenerative diseases. Moreover, artepillin C, a major constituent of Brazilian green propolis, may exhibit chemical chaperone-like properties.

Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5' untranslated region (5'UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5'UTR. The proteasome pathway predominantly participated in the stability of the Chac1 protein; however, its expression was remarkably up-regulated by co-transfection with ubiquitin genes. Using an immunoprecipitation assay, we revealed that ubiquitin molecule was directly conjugated to Chac1, and that mutated Chac1 with all lysine residues replaced by arginine was also ubiquitinated. Finally, we showed that WT Chac1 but not ΔChac1 reduced the intracellular level of glutathione. Taken together, our results suggest that the Chac1 protein expression is regulated in translational and post-translational fashion due to the Kozak-like sequence in the 5'UTR and the ubiquitin-mediated pathways. The bidirectional roles of ubiquitination in regulating Chac1 stabilization might give us a new insight into understanding the homeostasis of glutathione under pathophysiological conditions.

Oxidative stress contributes to myocardial ischemia-reperfusion injury, which causes cardiomyocyte death and precipitate life-threatening heart failure. Propofol has been proposed to protect cells or tissues against oxidative stress. However, the mechanisms underlying its beneficial effects are not fully elucidated. In the present study, we employed an in vitro oxidative injury model, in which rat cardiac H9c2 cells were treated with H2O2, and investigated roles of propofol against oxidative stress. Propofol treatment reduced H2O2-induced apoptotic cell death. While H2O2 induced expression of the antioxidant enzyme HO-1, propofol further increased HO-1 mRNA and protein levels. Propofol also promoted nuclear localization of Nrf2 in the presence of H2O2. Knockdown of Nrf2 using siRNA suppressed propofol-inducible Nrf2 and expression of Nrf2-downstream antioxidant enzyme. Knockdown of Nrf2 suppressed the propofol-induced cytoprotection. In addition, Nrf2 overexpression induced nuclear localization of Nrf2 and HO-1 expression. These results suggest that propofol exerts antioxidative effects by inducing nuclear localization of Nrf2 and expression of its downstream enzyme in cardiac cells. Finally, we examined the effect of propofol on cardiomyocytes using myocardial ischemia-reperfusion injury models. The expression level of Nrf2 protein was increased at 15 min after reperfusion in the ischemia-reperfusion and propofol group compared with ischemia-reperfusion group in penumbra region. These results suggest that propofol protects cells or tissues from oxidative stress via Nrf2/HO-1 cascade.

We have previously identified a novel endoplasmic reticulum (ER) stress-inducible protein, namely, cysteine-rich with EGF-like domains 2 (CRELD2), which is predominantly regulated by ATF6. However, few studies on intrinsic CRELD2 have been published. In the present study, we elucidated the expression of intrinsic CRELD2 in mouse tissues and ER stress- treated Neuro2a cells. Among nine tissues we tested, CRELD2 protein in the heart and skeletal muscles was negligible. CRELD2 expression in Neuro2a cells was induced at the late phase after treatment with tunicamycin (Tm) compared with rapid induction of growth arrest and DNA damage inducible gene 153 (GADD153). On the other hand, another ER stress inducer, thapsigargin, increased the intrinsic CRELD2 secretion from Neuro2a cells. We furthermore established CRELD2-deficient Neuro2a cells to evaluate their features. In combination with the NanoLuc complementary reporter system, which was designed to detect protein-protein interaction in living cells, CRELD2 interacted with not only CRELD2 itself but also with ER localizing proteins in Neuro2a cells. Finally, we investigated the responsiveness of CRELD2-deficient cells against Tm-treatment and found that CRELD2 deficiency did not affect the expression of genes triggered by three canonical ER stress sensors but rendered Neuro2a cells vulnerable to Tm-stimulation. Taken together, these findings provide the novel molecular features of CRELD2, and its further characterization would give new insights into understanding the ER homeostasis and ER stress-induced cellular dysfunctions.

The role of type IIA receptor protein tyrosine phosphatases (RPTPs), which includes LAR, RPTPσ and RPTPδ, in the nervous system is becoming increasingly recognized. Evidence supports a significant role for these RPTPs during the development of the nervous system as well as after injury, and mutations in RPTPs are associated with human disease. However, a major open question is the nature of the ligands that interact with type IIA RPTPs in the adult brain. Candidates include several different proteins as well as the glycosaminoglycan chains of proteoglycans. In order to investigate this problem, we used a receptor affinity probe assay with RPTPσ-AP fusion proteins on sections of adult mouse brain and to cultured neurons. Our results demonstrate that the major binding sites for RPTPσ in adult mouse brain are on neurons and are not proteoglycan GAG chains, as RPTPσ binding overlaps with the neuronal marker NeuN and was not significantly altered by treatments which eliminate chondroitin sulfate, heparan sulfate, or both. We also demonstrate no overlap of binding of RPTPσ with perineuronal nets, and a unique modulation of RPTPσ binding to brain by divalent cations. Our data therefore point to neuronal proteins, rather than CSPGs, as being the ligands for RPTPσ in the adult, uninjured brain.