Phenazopyridine promotes RPS23RG1/Rps23rg1 transcription and ameliorates Alzheimer-associated phenotypes in mice.

Alzheimer's disease (AD) is the most common form of dementia with no effective treatment options. A complete elucidation of its underlying molecular mechanisms, including the transcription regulation of genes critically involved in AD, may shed light on new therapeutic development. RPS23RG1 is a newly identified AD-associated gene, whose expression is decreased in AD and restoration can attenuate AD-like phenotypes in animal models. However, the transcription regulation of RPS23RG1 remains unknown. In this study, we explored the promoter of RPS23RG1 and identified its transcription initiation site (TSS) at 1525 bp upstream of the ATG translation start codon. Progressive deletion analysis determined the presence of a negative regulatory region and a positive regulatory region within nucleotide positions +1127 to +1187 and +732 to +1127 relative to the TSS (+1), respectively. We conducted a reporter system to screen for compounds that increase RPS23RG1 expression through antagonizing its negative regulatory elements and identified phenazopyridine. Importantly, we demonstrated that phenazopyridine not only promoted RPS23RG1/Rps23rg1 expression, but also reduced AD-like pathologies and cognitive impairments in the APP/PS1 AD model mice. We also determined a critical negative regulatory domain of RPS23RG1 within nucleotide positions +1177 to +1187 and found that the transcription factor SMAD3 bound to this domain. Inhibition of SMAD3 promoted RPS23RG1 expression. Moreover, phenazopyridine reduced SMAD3 binding to the RPS23RG1 promoter without affecting SMAD3 phosphorylation and nuclear localization. Taken together, our results determine the transcription regulation mechanism of RPS23RG1 and show that phenazopyridine has potential for AD treatment through regulating RPS23RG1 transcription.

RPS23RG1 modulates tau phosphorylation and axon outgrowth through regulating p35 proteasomal degradation.

Tauopathies are a group of neurodegenerative diseases characterized by hyperphosphorylation of the microtubule-binding protein, tau, and typically feature axon impairment and synaptic dysfunction. Cyclin-dependent kinase5 (Cdk5) is a major tau kinase and its activity requires p35 or p25 regulatory subunits. P35 is subjected to rapid proteasomal degradation in its membrane-bound form and is cleaved by calpain under stress to a stable p25 form, leading to aberrant Cdk5 activation and tau hyperphosphorylation. The type Ib transmembrane protein RPS23RG1 has been implicated in Alzheimer's disease (AD). However, physiological and pathological roles for RPS23RG1 in AD and other tauopathies are largely unclear. Herein, we observed retarded axon outgrowth, elevated p35 and p25 protein levels, and increased tau phosphorylation at major Cdk5 phosphorylation sites in Rps23rg1 knockout (KO) mice. Both downregulation of p35 and the Cdk5 inhibitor roscovitine attenuated tau hyperphosphorylation and axon outgrowth impairment in Rps23rg1 KO neurons. Interestingly, interactions between the RPS23RG1 carboxyl-terminus and p35 amino-terminus promoted p35 membrane distribution and proteasomal degradation. Moreover, P301L tau transgenic (Tg) mice showed increased tau hyperphosphorylation with reduced RPS23RG1 levels and impaired axon outgrowth. Overexpression of RPS23RG1 markedly attenuated tau hyperphosphorylation and axon outgrowth defects in P301L tau Tg neurons. Our results demonstrate the involvement of RPS23RG1 in tauopathy disorders, and implicate a role for RPS23RG1 in inhibiting tau hyperphosphorylation through homeostatic p35 degradation and suppression of Cdk5 activation. Reduced RPS23RG1 levels in tauopathy trigger aberrant Cdk5-p35 activation, consequent tau hyperphosphorylation, and axon outgrowth impairment, suggesting that RPS23RG1 may be a potential therapeutic target in tauopathy disorders.

Overexpression of Human SNX27 Enhances Learning and Memory Through Modulating Synaptic Plasticity in Mice.

Abnormal synaptic transmission leads to learning and memory disorders and is the main feature of neurological diseases. Sorting nexin 27 (SNX27) is an endosomal adaptor protein associated with a variety of nervous system diseases, and it is mainly responsible for the trafficking of postsynaptic membrane receptors. However, the roles of SNX27 in regulating synaptic and cognitive function are not fully understood. Here, we first generated a neuron-specific human-SNX27 transgenic mouse model (hSNX27 Tg) that exhibited enhanced excitatory synaptic transmission and long-term potentiation (LTP). In addition, we found that the hSNX27 Tg mice displayed enhanced learning and memory, lower-level anxiety-like behavior, and increased social interaction. Furthermore, we found that SNX27 overexpression upregulated the expression of glutamate receptors in the cortex and hippocampus of hSNX27 Tg mice. Together, these results indicate that SNX27 overexpression promotes synaptic function and cognition through modulating glutamate receptors.

RPS23RG1 Is Required for Synaptic Integrity and Rescues Alzheimer's Disease-Associated Cognitive Deficits.

BACKGROUND: Although synaptic impairment is a prerequisite to cognitive deficiencies in Alzheimer's disease (AD), mechanisms underlying the dysregulation of essential synaptic scaffolding components and their integrity remain elusive. RPS23RG1 is a newly identified protein implicated in AD. However, the physiological function of RPS23RG1 has yet to be determined. METHODS: We investigated the role of RPS23RG1 in maintaining synaptic structure and function in cell cultures and in Rps23rg1 knockout mice and determined whether targeting RPS23RG1-mediated pathways has therapeutic potential in APP/PS1 AD model mice. RESULTS: Deletion of the Rps23rg1 gene resulted in severe memory deficits and impairment of postsynaptic structure and function, with marked reductions in postsynaptic densities-93 and -95 (PSD-93 and PSD-95) levels. RPS23RG1 interacted with PSD-93/PSD-95 through its intracellular domain, consequently sequestering PSD-93/PSD-95 from murine double minute 2-mediated ubiquitination and degradation, thereby maintaining synaptic function. Restoration of PSD-93/PS-D95 levels reversed synaptic and memory deficits in Rps23rg1 knockout mice. We further observed attenuated RPS23RG1 expression in human AD, which positively correlated with PSD-93/PSD-95 levels. Importantly, an RPS23RG1-derived peptide comprising a unique PSD-93/PSD-95 interaction motif rescued synaptic and cognitive defects in Rps23rg1 knockout and AD mouse models. CONCLUSIONS: Our results reveal a role for RPS23RG1 in maintaining synaptic integrity and function and provide a new mechanism for synaptic dysfunction in AD pathogenesis. This demonstrates that RPS23RG1-mediated pathways show good therapeutic potential in AD intervention.

[Effects of intrathecal administration of AM22-52 on mechanical allodynia and CCL2 expression in DRG in bone cancer rats].

The pain peptide adrenomedullin (AM) plays a pivotal role in pathological pain. The present study was designed to investigate the effect of blockade of AM receptor on bone cancer pain (BCP) and its mechanism. BCP was developed by inoculation of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity of Sprague Dawley rats. The selective AM receptor antagonist AM22-52 was administered intrathecally on 15 d after the inoculation. Quantitative real-time PCR was used to detect mRNA level of CC chemokine ligand 2 (CCL2) in dorsal root ganglion (DRG). Double immunofluorescence staining was used to analyze the localizations of CCL2 and AM in DRG of normal rats. The results showed that, from 6 to15 d after the inoculation, the animals showed significant reduction in the mechanical pain threshold in the ipsilateral hindpaw, companied by the decline in bone density of tibia bone. The expression of CCL2 mRNA in DRG of BCP rats was increased by 3 folds (P < 0.001 vs saline group). Intrathecal administration of AM22-52 abolished bone cancer-induced mechanical allodynia and increase of CCL2 mRNA level (P < 0.001). In normal rats, CCL2 was co-localized with AM in DRG neurons. These results suggest that AM may play a role in the pathogenesis of BCP. The increased AM bioactivity up-regulates CCL2 expression in DRG, which may contribute to the induction of pain hypersensitivity in bone cancer.

Adrenomedullin mediates tumor necrosis factor-α-induced responses in dorsal root ganglia in rats.

Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pain peptide. This study investigated the possible involvement of AM in tumor necrosis factor-alpha (TNF-α)-induced responses contributing to neuronal plasticity in the dorsal root ganglia (DRG). Exposure of the DRG explant cultures to TNF-α (5nM) for 48h upregulated the expression of AM mRNA. The treatment with TNF-α also increased the level of CGRP, CCL-2 and MMP-9 mRNA in the cultured DRG. This increase was attenuated by the co-treatment with the selective AM receptor antagonist AM22-52 (2µM). The blockade of AM receptors inhibited TNF-α-induced increase of the glial fibrillary acidic protein (GFAP), interleukin-1ß (IL-1ß), phosphorylated cAMP response element binding protein (pCREB) and nuclear factor kappa B (pNF-κB) proteins. On the other hand, the treatment with the AM receptor agonist AM1-50 (10nM) for 96h induced an increase in the level of GFAP, IL-1ß, pCREB and pNF-κB proteins. The inhibition of AM activity did not change TNF-α-induced phosphorylation of extracellular signal-related kinase (pERK) while the treatment with AM1-50 still increased the level of pERK in the cultured DRG. Immunofluorescence assay showed the colocalization of AM-like immunoreactivity (IR) with TNF-α-IR in DRG neurons. The present study suggests that the increased AM receptor signaling mediated the many, but not all, TNF-α-induced activities, contributing to peripheral sensitization in neuropathic pain.

Involvement of adrenomedullin in the attenuation of acute morphine-induced analgesia in rats.

Adrenomedullin (AM) is a member of calcitonin gene-related peptide (CGRP) family and a pain-related peptide. We have shown that chronic administration of morphine (20 µg) upregulates AM activity contributing to morphine tolerance. The present study investigated if AM is involved in acute morphine-induced analgesia. Single intrathecal (i.t.) injection of morphine at a dose of 5 µg increased the tail-flick latency (TFL). This analgesic effect was potentiated by the co-administration of the AM receptor antagonist AM22-52 (5 and 10 nmol). Exposure of sensory ganglion culture to morphine increased AM content in the ganglia in concentration (0.33-10 µM)- and time (10-240 min)-dependent manners. However, treatment with morphine (3.3 µM) for 30-240 min did not alter AM mRNA levels in the cultured ganglia. Furthermore, exposure of ganglion cultures to morphine (3.3 µM) for 30-240, but not 10 min induced an increase in AM content in the culture medium. These results reveal that a single morphine treatment potentiates post-translational change and the release of AM in sensory ganglia masking morphine-induced analgesia. Thus, targeting AM and its receptors should be considered as a novel approach to improve the analgesic potency of opiates during their acute use.