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Glaucoma, the second leading cause of irreversible blindness worldwide, is associated with age and sensitivity to intraocular pressure (IOP). We have shown that elevated IOP causes an early increase in levels of reactive oxygen species (ROS) in the microbead occlusion mouse model. We also detected an endogenous antioxidant response mediated by Nuclear factor erythroid 2-Related Factor 2 (NRF2), a transcription factor that binds to the antioxidant response element (ARE) and increases transcription of antioxidant genes. Our previous studies show that inhibiting this pathway results in earlier and greater glaucoma pathology. In this study, we sought to determine if this endogenous antioxidant response is driven by the retinal ganglion cells (RGCs) or glial cells. We used Nrf2 fl/fl mice and cell-type specific adeno-associated viruses (AAVs) expressing Cre to alter Nrf2 levels in either the RGCs or glial cells. Then, we quantified the endogenous antioxidant response, visual function and optic nerve histology after IOP elevation. We found that knock-down of Nrf2 in either cell type blunts the antioxidant response and results in earlier pathology and vision loss. Further, we show that delivery of Nrf2 to the RGCs is sufficient to provide neuroprotection. In summary, both the RGCs and glial cells contribute to the antioxidant response, but treatment of the RGCs alone with increased Nrf2 is sufficient to delay onset of vision loss and axon degeneration in this induced model of glaucoma., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Oxidative stress contributes to degeneration of retinal ganglion cells and their axons in glaucoma, a leading cause of irreversible blindness worldwide, through sensitivity to intraocular pressure (IOP). Here, we investigated early elevations in reactive oxygen species (ROS) and a role for the NRF2-KEAP1-ARE endogenous antioxidant response pathway using microbead occlusion to elevate IOP in mice. ROS levels peaked in the retina at 1- and 2-wks following IOP elevation and remained elevated out to 5-wks. Phosphorylation of NRF2 and antioxidant gene transcription and protein levels increased concomitantly at 2-wks after IOP elevation, along with phosphorylation of PI3K and AKT. Inhibiting PI3K or AKT signaling prevented NRF2 phosphorylation and reduced transcription of antioxidant-regulated genes. Ocular hypertensive mice lacking Nrf2 had elevated ROS and a diminished increase in antioxidant gene expression. They also exhibited earlier axon degeneration and loss of visual function. In conclusion, the NRF2-KEAP1-ARE pathway is endogenously activated early in ocular hypertension due to phosphorylation of NRF2 by the PI3K/AKT pathway and serves to slow the onset of axon degeneration and vision loss in glaucoma. These data suggest that exogenous activation of this pathway might further slow glaucomatous neurodegeneration., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Wild-type erythropoietin (EPO) is promising for neuroprotection, but its therapeutic use is limited because it causes a systemic rise in hematocrit. We have developed an EPO-R76E derivative that maintains neuroprotective function without effects on hematocrit, but this protein has a short half-life in vivo. Here, we compare the efficacy and carrier-induced inflammatory response of two polymeric microparticle (MP) EPO-R76E sustained release formulations based on conventional hydrolytically degradable poly(lactic-co-glycolic acid) (PLGA) and reactive oxygen species (ROS)-degradable poly(propylene sulfide) (PPS). Both MP types effectively loaded EPO-R76E and achieved sustained release, providing detectable levels of EPO-R76E at the injection site in the eye in vivo for at least 28 days. Testing in an in vitro oxidative stress assay and a mouse model of blast-induced indirect traumatic optic neuropathy (bITON) showed that PPS and PLGA MP-mediated delivery of EPO-R76E provided therapeutic protection. While unloaded PLGA MPs inherently increase levels of pro-inflammatory cytokines in the bITON model, drug-free PPS MPs have innate antioxidant properties that provide therapeutic benefit both in vitro and in vivo. Both PLGA and PPS MPs enabled sustained release of EPO-R76E, providing therapeutic benefits including reduction in inflammation and axon degeneration, and preservation of visual function as measured by electroretinogram. The PPS-based MP platform is especially promising for further development, as the delivery system provides inherent antioxidant benefits that can be harnessed to work in complement with EPO-R76E or other drugs for neuroprotection in the setting of traumatic eye injury., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo . First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro . Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 ( Pcsk9 ) mRNA and secreted protein 5-fold in vitro . The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo . This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo ., (© 2020 The Authors.)

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Glaucoma is a complex neurodegeneration and a leading cause of blindness worldwide. Current therapeutic strategies, which are all directed towards lowering the intraocular pressure (IOP), do not stop progression of the disease. We have demonstrated that recombinant adeno-associated virus (rAAV) gene delivery of a form of erythropoietin with attenuated erythropoietic activity (EpoR76E) can preserve retinal ganglion cells, their axons, and vision without decreasing IOP. The goal of this study was to determine if modulation of neuroinflammation or oxidative stress played a role in the neuroprotective activity of EPO.R76E., Methods: Five-month-old DBA/2J mice were treated with either rAAV.EpoR76E or a control vector and collected at 8 months of age. Neuroprotection was assessed by quantification of axon transport and visual evoked potentials. Microglia number and morphology and cytokine and chemokine levels were quantified. Message levels of oxidative stress-related proteins were assessed., Results: Axon transport and visual evoked potentials were preserved in rAAV.EpoR76E-treated mice. The number of microglia was decreased in retinas from 8-month-old rAAV.EpoR76E-treated mice, but proliferation was unaffected. The blood-retina barrier was also unaffected by treatment. Levels of some pro-inflammatory cytokines were decreased in retinas from rAAV.EpoR76E-treated mice including IL-1, IL-12, IL-13, IL-17, CCL4, and CCL5. TNFα messenger RNA (mRNA) was increased in retinas from 8-month-old mice compared to 3-month-old controls regardless of treatment. Expression of several antioxidant proteins was increased in retinas of rAAV.EpoR76E-treated 8-month-old mice., Conclusions: Treatment with rAAV.EpoR76E preserves vision in the DBA/2J model of glaucoma at least in part by decreasing infiltration of peripheral immune cells, modulating microglial reactivity, and decreasing oxidative stress.

The widespread distribution of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the adult brain suggests its role in a broad range of brain functions. Here we show evidence supporting a physical interaction of PTEN with a region in the third intracellular loop (3L4F) of the serotonin 5-HT2C receptor (5-HT2cR, formerly 5-HT1c receptor) in cell cultures. PTEN limits agonist-induced phosphorylation of 5-HT2cR through its protein phosphatase activity. We showed the probable existence of PTEN:5-HT2cR complexes in putative dopaminergic neurons in the rat ventral tegmental area (VTA), a brain region in which virtually all abused drugs exert rewarding effects by activating its dopamine neurons. We synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F or the 5-HT2cR agonist Ro600175 suppressed the increased firing rate of VTA dopaminergic neurons induced by delta9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Using behavioral tests, we found that Tat-3L4F or Ro600175 blocks conditioned place preference of THC or nicotine, and that Ro600175, but not Tat-3L4F, produces anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggest a potential strategy for treating drug addiction with the Tat-3L4F peptide.

The human serotonin 2C (5-HT2C) receptor undergoes extensive RNA editing, generating multiple isoforms; the most prominent isoform in the human brain is the extensively edited VSV isoform. In addition, a naturally occurring single nucleotide polymorphism (SNP) is found in the coding region of the 5-HT2C receptor gene, which converts cysteine to serine at the 23rd amino acid (C23S). To elucidate the functional consequences, pharmacological properties were evaluated in cells expressing C23 or S23 in the nonedited, INI, or edited, VSV, isoform. Confocal imaging of HEK293 cells expressing the C23 and S23 variants revealed no apparent difference in cellular localization, which was confirmed in NIH-3T3 fibroblasts by surface biotinylation. Competition binding experiments revealed comparable high-affinity agonist binding for the C23 and S23 receptors and no difference in ligand affinities in either the INI or VSV backbones. The dose-response functions for 5-HT and (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI) to elicit phosphoinositide hydrolysis did not differ in either HEK293 or NIH-3T3 fibroblasts expressing the receptor variants. Constitutive activity, evaluated in COS-7 and HEK293 cells, also was not different. Lastly, fluorescence resonance energy transfer demonstrated homodimerization of C23 receptors, which was reproduced in cells expressing the S23 variant. We conclude that the C23S SNP in the 5-HT2C receptor has no functional consequences, even when evaluated in the most common, edited receptor backbone. Therefore, positive associations between this polymorphism and disease states may be a consequence of linkage disequilibrium with another SNP that is involved in the disease.

We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development.

Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.

Cortical serotonin(2A) receptors are hypothesized to be involved in the pathology and treatment of schizophrenia. Light microscopic studies in the rat prefrontal cortex have localized serotonin(2A) receptors to the dendritic shafts of pyramidal and local circuit neurons. Electrophysiological studies have predicted that these receptors are also located on glutamate terminals, whereas neurochemical studies have hypothesized that they are located on dopamine terminals in this area. The present study sought to determine the ultrastructural localization of immunoperoxidase labeling for serotonin(2A) receptors in the middle layers of the prelimbic portion of the rat prefrontal cortex. Serotonin(2A) receptor immunoreactivity was observed in 325 identifiable structures. Of these, 73% were postsynaptic profiles that were composed of either dendritic shafts (58%) or dendritic spine heads and necks (42%). Twenty-four percent of the labeled profiles were presynaptic axons and varicosities; most of these had morphological features that were characteristic of monoamine axons: thin diameter, lack of myelination, occasional content of dense-cored vesicles, and infrequent formation of synapses in single sections. The remainder of the labeled profiles (4%) were glial processes. These findings suggest that serotonin(2A) receptor-mediated effects within the rat prelimbic prefrontal cortex are primarily postsynaptic in nature, affecting both the spines of pyramidal cells and the dendrites of pyramidal and local circuit neurons in this area. The results further suggest that serotonin acts presynaptically via this receptor subtype, most likely at receptors on monoamine fibers, and only rarely directly on glutamate axons., (Copyright 2003 IBRO)

Treatment with conventional antipsychotic drugs (APDs) is accompanied by extrapyramidal side effects (EPS), which are thought to be due to striatal dopamine D(2) receptor blockade. In contrast, treatment with atypical APDs is marked by a low incidence or absence of EPS. The reduced motor side effect liability of atypical APDs has been attributed to a high serotonin 5-HT(2A) receptor affinity coupled with a relatively low D(2) affinity. Despite the high density of 5-HT(2A) binding sites in the striatum, there are few detectable 5-HT(2A) mRNA-expressing neurons in the striatum. This suggests that most striatal 5-HT(2A) receptors are heteroceptors located on afferent axons. A combined retrograde tracer-immunohistochemistry method was used to determine the sites of origin of striatal 5-HT(2A)-like immunoreactive axons. 5-HT(2A)-like immunoreactive neurons in both the cortex and globus pallidus were retrogradely labeled from the striatum; very few nigrostriatal or thalamostriatal neurons expressed 5-HT(2A)-like immunoreactivity. Within the striatum, parvalbumin-containing interneurons displayed 5-HT(2A) immunolabeling; these neurons are the targets of cortical and pallidal projections. Our data indicate that cortico- and pallido-striatal neurons are the major source of 5-HT(2A) receptor binding in the striatum, and suggest that cortico- and pallido-striatal neurons are strategically positioned to reduce the motor side effects that accompany striatal D(2) receptor blockade or are seen in parkinsonism., (Copyright 2001 Wiley-Liss, Inc.)

Phosphorylation-deficient serotonin 5-HT(2C) receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT(2C) receptors that lack the carboxyl-terminal PDZ recognition motif (Ser(458)-Ser-Val-COOH; DeltaPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of (32)P. Treatment of cells stably expressing DeltaPDZ or wild-type 5-HT(2C) receptors with serotonin produced identical maximal responses and EC(50) values for eliciting [(3)H]inositol phosphate formation. In calcium imaging studies, treatment of cells expressing DeltaPDZ or wild-type 5-HT(2C) receptors with 100 nm serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were approximately 5-fold lower with DeltaPDZ receptors relative to wild-type 5-HT(2C) receptors. After 10 min, responses of DeltaPDZ receptors recovered to wild-type 5-HT(2C) receptor levels. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT(2C) receptor phosphorylation and suggest that receptor phosphorylation at Ser(459) enhances resensitization of 5-HT(2C) receptor responses.

For more than 40 years the hallucinogen lysergic acid diethylamide (LSD) has been known to modify serotonin neurotransmission. With the advent of molecular and cellular techniques, we are beginning to understand the complexity of LSD's actions at the serotonin 5-HT2 family of receptors. Here, we discuss evidence that signaling of LSD at 5-HT2C receptors differs from the endogenous agonist serotonin. In addition, RNA editing of the 5-HT2C receptor dramatically alters the ability of LSD to stimulate phosphatidylinositol signaling. These findings provide a unique opportunity to understand the mechanism(s) of partial agonism.

The localization of 5-hydroxytryptamine2C receptors in the lumbosacral spinal cord of the rat was investigated using selective antibodies raised against the carboxyl-terminal part of the rat receptor. The distribution of immunoperoxidase labelling at the light microscope level revealed numerous labelled neurons in the gray matter, with a higher intensity in the sacral parasympathetic nucleus, the dorsal gray commissure and particularly the motoneurons of the ventral horn. Confocal microscope analysis showed that immunostaining was mainly intracellular (motoneurons), but could also be associated with the membrane of cell bodies and dendrites. Actually, electron microscope immunogold experiments demonstrated an exclusive staining of the cis-Golgi apparatus. Following pseudo-rabies virus transsynaptic retrograde labelling from the corpus cavernosum, labelled neurons were found in the sacral parasympathetic nucleus and the dorsal gray commissure of the L6-S1 segments. All virus-labelled neurons exhibited 5-hydroxytryptamine2C receptor immunoreactivity. These results indicate that all parasympathetic preganglionic neurons and their related interneurons which contribute to the innervation of cavernosal tissue bear 5-hydroxytryptamine2C receptors. In the sacral parasympathetic nucleus, most neurons which were retrogradely-labelled from the pelvic ganglion with Fast Blue also showed 5-hydroxytryptamine2C receptor immunoreactivity. In the ventral horn, motoneurons retrogradely labelled from the ischiocavernosus muscle and the bulbospongiosus muscle, both of which are involved in erection and ejaculation, were also 5-hydroxytryptamine2C receptor-immunopositive. The supraspinal serotoninergic control of erection at the lumbosacral level therefore appears to be strongly associated with the activation of 5-hydroxytryptamine2C receptors, consistent with the proerectile properties of 5-hydroxytryptamine2C agonists.

In this study, we demonstrate that clozapine and other atypical antipsychotic drugs induce a paradoxical internalization of 5-hydroxytryptamine-2A receptors in vitro and a redistribution of 5-hydroxytryptamine-2A receptors in vivo. We discovered that clozapine, olanzapine, risperidone and the putative atypical antipsychotic drug MDL 100,907 all induced 5-hydroxytryptamine-2A receptor internalization in fibroblasts stably expressing the 5-hydroxytryptamine-2A receptor in vitro. Two 5-hydroxytryptamine-2A antagonists (mianserin and ritanserin), which have been demonstrated to reduce negative symptoms in schizophrenia, also caused 5-hydroxytryptamine-2A receptor internalization. Four different drugs, each devoid of 5-hydroxytryptamine-2A antagonist activity, had no effect on the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro. Treatment of rats for seven days with clozapine induced an increase in intracellular 5-hydroxytryptamine-2A receptor-like immunoreactivity in pyramidal neurons, while causing a decrease in labeling of apical dendrites in the medial prefrontal cortex. This redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons was also seen when rats were chronically treated with another atypical antipsychotic drug, olanzapine. The typical antipsychotic drug haloperidol, however, did not induce a redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons in the medial prefrontal cortex. Taken together, these results demonstrate that several atypical antipsychotic drugs with high 5-hydroxytryptamine-2A receptor affinities induce a redistribution of 5-hydroxytryptamine-2A receptors both in vivo and in vitro. It is conceivable that the loss of 5-hydroxytryptamine-2A receptors from the apical dendrites of pyramidal neurons is important for the beneficial effects of atypical antipsychotic drugs and other 5-hydroxytryptamine-2A antagonists in schizophrenia.

The mechanism by which antagonists down-regulate 5-HT2A receptors in unknown. We here report that a variety of 5-HT2A antagonists induce a change in the subcellular distribution of 5-HT2A receptors both in vitro and in vivo. In a stably transfected NIH 3T3 cell-line, brief exposure to 1 muM clozapine caused a 2.5-fold increase in intracellular 5-HT2A-like immunoreactivity, as measured by confocal microscopy. Confirmatory studies utilizing a biotin-trap technique, demonstrated that the increase in intracellular immunoreactivity results from internalization of receptor from the cell surface. Exposure of transfected cells to other 5-HT2A receptor antagonists produced similar increases in intracellular 5-HT2A-like immunoreactivity. In vivo administration of clozapine (20 mg/kg, sc, X 7 days) caused a greater than twofold increase in intracellular immunoreactivity in cell bodies of cortical pyramidal neurons. Additionally, chronic clozapine administration was associated with decrease in labeling of apical dendrites on pyramidal cells. These results show that clozapine causes a change in subcellular distribution of 5-HT2A receptors in vitro and in vivo.

Anti-peptide antibodies were generated against several 13-17 amino acid regions of rat serotonin 5-HT2A and 5-HT2C receptors. Peptides containing terminal cysteine residues were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) with the cross-linking reagent sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate). Both the carrier protein and the number of peptide molecules per carrier molecule were changed during the immunization schedule. For the early immunizations, immunogens were BSA-peptides at ratios of 8-27 mol peptide per mol BSA. For the later boosts, immunogens were OVA-peptides at ratios of 1-2 mol peptide per mol OVA. The peptide constructs were used to immunize rabbits and chickens. Anti-peptide antibodies were purified from sera (rabbits) or egg yolks (hens) using peptide matrices. Cell lines expressing similar densities of rat 5-HT2A or 5-HT2C receptors were used to monitor the specificity of purified antibodies on immunoblots and in immunocytochemistry. A total of five out of the six rabbit antibodies were positive on immunoblots (three anti-5-HT2A and two anti-5-HT2C) and four were also positive in immunocytochemistry (three anti-5-HT2A and one anti-5-HT2C). None of the anti-peptide chicken antibodies were useful on immunoblots or in immunocytochemistry. Since there is a paucity of high affinity reagents selective for 5-HT2A or 5-HT2C receptors, these rabbit antibodies will be useful tools. The methods used to generate site-directed antibodies specific for 5-HT2A or 5-HT2C receptors should be applicable to other proteins.

We reported earlier that the levels of Ca2+-dependent metalloproteinases are increased in Alzheimer's disease (AD) specimens, relative to control specimens. Here we show that these enzymes are forms of the matrix metalloproteinase MMP-9 (EC3.4.24. 35) and are expressed in the human hippocampus. Affinity-purified antibodies to MMP-9 labeled pyramidal neurons, but not granular neurons or glial cells. MMP-9 mRNA is expressed in pyramidal neurons, as determined with digoxigenin-labeled MMP-9 riboprobes, and the presence of this mRNA is confirmed with reverse transcriptase PCR. The cellular distribution of MMP-9 is altered in AD because 76% of the total 100 kDa enzyme activity is found in the soluble fraction of control specimens, whereas only 51% is detectable in the same fraction from AD specimens. The accumulated 100 kDa enzyme from AD brain is latent and can be converted to an active form with aminophenylmercuric acetate. MMP-9 also is detected in close proximity to extracellular amyloid plaques. Because a major constituent of plaques is the 4 kDa beta-amyloid peptide, synthetic Abeta1-40 was incubated with activated MMP-9. The enzyme cleaves the peptide at several sites, predominantly at Leu34-Met35 within the membrane-spanning domain. These results establish that neurons have the capacity to synthesize MMP-9, which, on activation, may degrade extracellular substrates such as beta-amyloid. Because the latent form of MMP-9 accumulates in AD brain, it is hypothesized that the lack of enzyme activation contributes to the accumulation of insoluble beta-amyloid peptides in plaques.

The actions of the neurotransmitter 5-hydroxytryptamine (5-HT) (serotonin) are mediated by multiple receptor subtypes. One of the prominent serotonin receptors in the brain is the 5-HT2C receptor (5-HT2C-R). We report the occurrence of a second 5-HT2C-R transcript, first identified using S1 nuclease protection of total RNA isolated from the choroid plexus. Analyses of the distribution of these two RNAs revealed that the short form is expressed in the same structures as the 5-HT2C-R mRNA, including choroid plexus, striatum, hippocampus, hypothalamus, olfactory tubercles, and spinal cord. Cloning and sequence analyses revealed a second cDNA with a 95-nt deletion in the region coding for the putative second intracellular loop and the fourth transmembrane domain of the 5-HT2C-R. This deletion leads to a frameshift in the coding sequence and the introduction of a premature stop codon. The predicted truncated protein (5-HT2C-tr) contains 172 amino acids, with 153 residues at the amino terminus, identical to the 5-HT2C-R, and 19 carboxyl-terminal amino acids that are unique. Although antibodies specific to the 5-HT2C-tr protein showed that the truncated form is expressed in a transfected fibroblast cell model system, there was no serotonergic ligand binding activity or phosphoinositide hydrolysis. Analyses of the 5-HT2C-R gene revealed that the two transcripts arise from a single gene by differential splicing using alternative donor sites and a common 3'-splice acceptor. Polymerase chain reaction amplification of mouse and human brain cDNAs demonstrated the occurrence of the same splicing patterns in these species. Although this study demonstrates tissue-specific expression of two 5-HT2C mRNA splice variants in rat, mouse, and human, the significance of the truncated form in these three species remains to be established.

Matrix metalloproteinases (MMPs) were analyzed by immunohistochemistry and zymography in amyotrophic lateral sclerosis (ALS) and control brain and spinal cord specimens. Three major bands of enzyme activity (70, 100, and 130 kDa) were consistently observed and were subsequently identified as MMP-2 (70 kDa; also known as EC 3.4.24.24 or gelatinase A) and MMP-9 (100 and 130 kDa; also known as EC 3.4.24.35 or gelatinase B). Immunohistochemical studies established the presence of MMP-2 in astrocytes and MMP-9 in pyramidal neurons in the motor cortex and motor neurons in the spinal cord of ALS patients. Although a significant decrease in MMP-2 activity was noticed in the ALS motor cortex, statistically significant increases in MMP-9 (100-kDa) activity were observed in ALS frontal and occipital cortices (BA10 and 17) and all three spinal cord regions when compared with control specimens. The highest MMP-9 (100-kDa) activities in ALS were found in the motor cortex and thoracic and lumbar cord specimens. The abnormally high amount of MMP-9 and its possible release at the synapse may destroy the structural integrity of the surrounding matrix, thereby contributing to the pathogenesis of ALS.

Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.

The 5-hydroxytryptamine (5-HT)2C receptor is a G protein-coupled receptor that exhibits constitutive receptor activation, defined as agonist-independent receptor activation of the signal transduction pathway. The present studies were performed to determine whether NIH/3T3 fibroblasts expressing the 5-HT2C receptor exhibited desensitization of agonist-mediated phosphoinositide hydrolysis. Furthermore, 5-HT2C receptor-specific antibodies were used to determine whether the 5-HT2C receptor was phosphorylated in the absence of agonist and whether treatment with an agonist or an inverse agonist altered receptor phosphorylation. Time course studies of basal and serotonin-stimulated phosphoinositide hydrolysis demonstrated that basal values increased in a linear manner, whereas the response to serotonin plateaued within 60 min. In addition, pretreatment with serotonin resulted in a rightward shift of the subsequently determined serotonin dose-response curve. To determine the phosphorylation state of the 5-HT2C receptor, specific antibodies were used to immunoprecipitate the receptor from lysates prepared from 32P-labeled fibroblasts. Phosphorylation of the 5-HT2C receptor was evident under basal conditions, and serotonin treatment increased receptor phosphorylation. The inverse agonist mianserin had no detectable effect on 5-HT2C receptor phosphorylation when added alone but blocked the serotonin-stimulated increase in 5-HT2C receptor phosphorylation. The present study is the first to demonstrate that the 5-HT2C receptor is phosphorylated under basal conditions and phosphorylation is increased by agonist treatment conditions that result in desensitization of receptor signaling. Thus, these studies demonstrate that a cell line exhibiting a high level of constitutive 5-HT2C receptor activity has the ability to undergo agonist-mediated desensitization, consistent with current models of G protein-coupled receptor regulation.

Matrix metalloproteinase-9 (MMP-9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP-9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP-9 (94 kDa) was purified from the human cell line HL-60 and converted to an intermediary active form (84 kDa) with p-aminophenylmercuric acetate. This active form of MMP-9 degraded SP with a kcat/Km of 170 mM-1 min-1, which is 30-fold greater than the previously reported value for a representative collagen-derived peptide. The major digestion products were identified as SP and SP, which were derived from cleavage of the Gln6-Phe7 bond. Minor products were also generated from cleavage of the Gly9-Leu10 bond. The other representative tachykinin peptides were cleaved at rates > 10-fold slower than that of SP. The 84-kDa peptidase was also active as a gelatinase. Longer treatment of MMP-9 with p-aminophenylmercuric acetate caused the conversion of the 84-kDa enzyme to the established 68-kDa active form; however, the rate of SP degradation did not increase. Because MMP-9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in interacellular communication by altering the availability of bioactive peptides.

Three neutral proteinases from human hippocampal tissue have been identified and partially characterized using substrate gel electrophoresis. The proteinases showed activity when gelatin was used as the substrate, but had no detectable activity against casein. Based on the results of inhibition studies and the calcium requirements, it was concluded that the activities were due to calcium-dependent metalloproteinases. The apparent molecular weights were 130,000 (MP-130), 100,000 (MP-100), and 70,000 (MP-70). Half-maximal activities were observed with 20 microM Ca2+ for MP-130, 40 microM Ca2+ for MP-100, and 800 microM Ca2+ for MP-70. In the presence of Ca2+, Zn2+ reestablished the activities of the three metalloproteinases at a lower concentration than did either Co2+ or Mn2+. One millimolar Al3+ inhibited 67% of the MP-70 activity, but did not affect the MP-100 and MP-130 activities. An analysis of Alzheimer-affected hippocampal and control samples showed that the specific activity (in units per milligram of sodium dodecyl sulfate-soluble protein) of MP-70 varied less than the activities of MP-100 and MP-130 between the two groups. Although p-amino-phenylmercuric acetate (p-APMA) increased the activities of MP-70 by 70% in both groups of specimens, the resulting activities from Alzheimer samples were greater than those from control samples (p less than 0.01). A wide range of MP-100 specific activity was observed in both groups, and its mean activity was higher in Alzheimer-affected samples (p less than 0.05). Treatment with p-APMA increased the activity of MP-100 only 25% in both groups of tissue samples. MP-130 activity was detected predominantly in Alzheimer-affected hippocampal specimens, and treatment with p-APMA failed to increase its activity in both the control and the Alzheimer-affected specimens. The results demonstrate an elevated level of metalloproteinase activities, capable of degrading tissue matrix components, in the hippocampus from postmortem Alzheimer patients.