cAMP-regulated Phosphoprotein Research Articles

Quercetin inhibits truncated isoform of dopamine- and cAMP-regulated phosphoprotein as adjuvant treatment for trastuzumab therapy resistance in HER2-positive breast cancer

Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer (BC). The truncated isoform of dopamine- and cAMP-regulated phosphoprotein (t-DARPP) has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression. To evaluate the t-DARPP expression in BC, paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+ BC tumor tissues. We established 2 patient-derived xenografts (PDX) mice models to test the efficacy of trastuzumab, named model 1 (non-responder) and model 2 (responder). t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays. Instead, there is no response from the responder. Furthermore, mechanistic studies using transwell and Western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins, enhance p95-HER2 expression and promote cell migration. We found that quercetin effectively reduced t-DARPP expression in HER2+ BC cells. In t-DARPP ShRNA-suppressed cells, quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest. In conclusion, the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+ BC patients has the potential as a biomarker for mitigating drug resistance.

Darpp-32 is regulated by dopamine and is required for the formation of GABAergic neurons in the developing telencephalon

DARPP-32 (dopamine and cAMP-regulated phosphoprotein Mr. 32 kDa) is a phosphoprotein that is modulated by multiple receptors integrating intracellular pathways and playing roles in various physiological functions. It is regulated by dopaminergic receptors through the cAMP/protein kinase A (PKA) pathway, which modulates the phosphorylation of threonine 34 (Thr34). When phosphorylated at Thr34, DARPP-32 becomes a potent protein phosphatase-1 (PP1) inhibitor. Since dopamine is involved in the development of GABAergic neurons and DARPP-32 is expressed in the developing brain, it is possible that DARPP-32 has a role in GABAergic neuronal development. We cloned the zebrafish darpp-32 gene (ppp1r1b) gene and observed that it is evolutionarily conserved in its inhibitory domain (Thr34 and surrounding residues) and the docking motif (residues 7–11 (KKIQF)). We also characterized darpp-32 protein expression throughout the 5 days post-fertilization (dpf) zebrafish larval brain by immunofluorescence and demonstrated that darpp-32 is mainly expressed in regions that receive dopaminergic projections (pallium, subpallium, preoptic region, and hypothalamus). We demonstrated that dopamine acutely suppressed darpp-32 activity by reducing the levels of p-darpp-32 in the 5dpf zebrafish larval brain. In addition, the knockdown of darpp-32 resulted in a decrease in the number of GABAergic neurons in the subpallium of the 5dpf larval brain, with a concomitant increase in the number of DAergic neurons. Finally, we demonstrated that darpp-32 downregulation during development reduced the motor behavior of 5dpf zebrafish larvae. Thus, our observations suggest that darpp-32 is an evolutionarily conserved regulator of dopamine receptor signaling and is required for the formation of GABAergic neurons in the developing telencephalon.

An exploratory study on the ability of manganese to supplement rotenone neurotoxicity in rats

Parkinson’s disease (PD) is a complex disorder, primarily of idiopathic origin, with environmental stressors like rotenone and manganese linked to its development. This study explores their potential interaction and resulting neurotoxicity, aiming to understand how environmental factors contribute to PD. In an eight-day experiment, male Wistar rats weighing 280–300 g were subjected to rotenone, manganese, or a combination of both. Various parameters were assessed, including body weight, behavior, serum markers, tissue damage, protein levels (tyrosine hydroxylase, Dopamine- and cAMP-regulated neuronal phosphoprotein –DARPP-32-, and α-synuclein), and mitochondrial function. Manganese heightened rotenone’s impact on reducing food intake without causing kidney or liver dysfunction. However, the combined exposure intensified neurotoxicity, which was evident in augmented broken nuclei and decreased tyrosine hydroxylase and DARPP-32 levels in the striatum. While overall mitochondrial function was preserved, co-administration reduced complex IV activity in the midbrain and liver. In conclusion, our findings revealed a parallel toxic effect induced by rotenone and manganese. Notably, while these substances do not target the same dopaminergic regions, a notable escalation in toxicity is evident in the striatum, the brain region where their toxic effects converge. This study highlights the need for further exploration regarding the interaction of environmental factors and their possible impact on the etiology of PD.

Lasting mesothalamic dopamine imbalance and altered exploratory behavior in rats after a mild neonatal hypoxic event.

Adversities during the perinatal period can decrease oxygen supply to the fetal brain, leading to various hypoxic brain injuries, which can compromise the regularity of brain development in different aspects. To examine the catecholaminergic contribution to the link between an early-life hypoxic insult and adolescent behavioral aberrations, we used a previously established rat model of perinatal hypoxia but altered the hypobaric to normobaric conditions. Exploratory and social behavior and learning abilities were tested in 70 rats of both sexes at adolescent age. Inherent vertical locomotion, sensory-motor functions and spatial learning abilities were explored in a subset of animals to clarify the background of altered exploratory behavior. Finally, the concentrations of dopamine (DA) and noradrenaline in midbrain and pons, and the relative expression of genes for DA receptors D1 and D2, and their down-stream targets (DA- and cAMP-regulated phosphoprotein, Mr 32 kDa, the regulatory subunit of protein kinase A, and inhibitor-5 of protein phosphatase 1) in the hippocampus and thalamus were investigated in 31 rats. A lesser extent of alterations in exploratory and cognitive aspects of behavior in the present study suggests that normobaric conditions mitigate the hypoxic injury compared to the one obtained under hypobaric conditions. Increased exploratory rearing was the most prominent consequence, with impaired spatial learning in the background. In affected rats, increased midbrain/pons DA content, as well as mRNA levels for DA receptors and their down-stream elements in the thalamus, but not the hippocampus, were found. We can conclude that a mild hypoxic event induced long-lasting disbalances in mesothalamic DA signaling, contributing to the observed behavioral alterations. The thalamus was thereby indicated as another structure, besides the well-established striatum, involved in mediating hypoxic effects on behavior through DA signaling.

Changes in the dopaminergic circuitry and adult neurogenesis linked to reinforcement learning in corvids.

The caudolateral nidopallium (NCL, an analog of the prefrontal cortex) is known to be involved in learning, memory, and discrimination in corvids (a songbird), whereas the involvement of other brain regions in these phenomena is not well explored. We used house crows (Corvus splendens) to explore the neural correlates of learning and decision-making by initially training them on a shape discrimination task followed by immunohistochemistry to study the immediate early gene expression (Arc), a dopaminoceptive neuronal marker (DARPP-32, Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) to understand the involvement of the reward pathway and an immature neuronal marker (DCX, doublecortin) to detect learning-induced changes in adult neurogenesis. We performed neuronal counts and neuronal tracing, followed by morphometric analyses. Our present results have demonstrated that besides NCL, other parts of the caudal nidopallium (NC), avian basal ganglia, and intriguingly, vocal control regions in house crows are involved in visual discrimination. We have also found that training on the visual discrimination task can be correlated with neurite pruning in mature dopaminoceptive neurons and immature DCX-positive neurons in the NC of house crows. Furthermore, there is an increase in the incorporation of new neurons throughout NC and the medial striatum which can also be linked to learning. For the first time, our results demonstrate that a combination of structural changes in mature and immature neurons and adult neurogenesis are linked to learning in corvids.

SUZ domain–containing proteins have multiple effects on nonsense-mediated decay target transcripts

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.

LncRNA HCG11 Promotes Colorectal Cancer Cell Malignant Behaviors via Sponging miR-26b-5p.

Colorectal cancer (CRC) is a type of gastrointestinal cancer with an increasing incidence. Long noncoding RNAs (lncRNAs) have raised great concern because of wide participation in human diseases, including cancers. However, whether lncRNA HLA complex group 11 (HCG11) played a functional role in CRC remained to be elucidated. Herein, we utilized qRT-PCR to analyze the expression of HCG11 and found that HCG11 was highly expressed in CRC cells. Besides, HCG11 knockdown suppressed cell proliferation, migration, and invasion but facilitated cell apoptosis. Furthermore, supported by bioinformatics analyses and mechanism assays, HCG11, mainly located in cell cytoplasm, was confirmed to competitively bind to miR-26b-5p to modulate the expression of the target messenger RNA (mRNA), namely, cAMP-regulated phosphoprotein 19 (ARPP19). ARPP19 was detected to be upregulated in CRC cells, and ARPP19 silence was verified to inhibit the malignant behaviors of CRC cells. Rescue experiments validated that miR-26b-5p inhibition or ARPP19 overexpression could countervail the inhibitory influences of HCG11 silence on CRC cell biological behaviors in vitro. To conclude, HCG11, upregulated in CRC cells, could promote cell proliferation, migration, and invasion and inhibit cell apoptosis via targeting miR-26b-5p/ARPP19 axis.

PLPP/CIN inhibits dopamine D1 receptor-mediated seizure activity via DARPP-32 serine 97 dephosphorylation in the mouse hippocampus

Dopamine plays a central role in the regulation of psychomotor functions in the brain. Furthermore, the dopaminergic system is involved in the ictogenesis in human patients and animal models of epilepsy. Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) plays an important role in the regulation of interactions between dopamine and glutamate receptors in neurons. Indeed, SKF 83822 (a specific D1 receptor agonist) facilitates DARPP-32-mediated protein phosphatase 1 (PP1) inhibition leading to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activities and currents and thereby generates seizure activity. In the present study, we found that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN), a selective phosphatase for serine (S) residues, attenuated seizure susceptibility in response to SKF 83822 by dephosphorylating DARPP-32 S97 site. Similarly, inhibition of DARPP-32 S97 phosphorylation by 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective casein kinase 2 inhibitor) attenuated SKF 83822-induced seizure activity. These inhibitory effects of PLPP/CIN and TMCB were relevant to the regulations of DARPP-32-PP1-AMPAR signaling pathway. Therefore, our findings suggest that PLPP/CIN may be a modulator in dopaminergic neurotransmission as well as glutamatergic systems, and that the PLPP/CIN-mediated DARPP-32 regulation may be one of the potential therapeutic targets for medication of seizure or epilepsy induced by D1 receptor hyperactivation.

P-CREB and p-DARPP-32 orchestrating the modulatory role of cAMP/PKA signaling pathway enhanced by Roflumilast in rotenone-induced Parkinson's disease in rats

Recently, phosphodiesterases (PDEs) have gained great attention due to their implication in Parkinson's disease (PD) pathogenesis. Noteworthy, the PDE4 enzyme is highly expressed in the striatum and selectively degrades cyclic adenosine monophosphate (cAMP). The cAMP was shown to play a vital role in dopamine (DA) signaling besides maintaining the plasticity of dopaminergic neurons as well as protecting them from inflammation and oxidative stress-mediated death. Thus, PDE4 inhibition could be a promising strategy for treating PD. Accordingly, the present study investigated the neuroprotective efficacy of roflumilast, a PDE4 inhibitor, in abolishing neurodegeneration in the rotenone-induced PD model. Rotenone (1.5 mg/kg, s.c) was delivered via 11 injections on matching days. Roflumilast treatment (0.5 mg/kg, p.o) was given daily after the fifth rotenone injection. Roflumilast significantly reversed rotenone's adverse effects, as it enhanced trophic factors expression and abrogated inflammation as well as oxidative stress. Thus, promoting dopaminergic neuronal plasticity and survival, as well as restoring striatal DA level and function, which resulted in enhanced motor performance. The beneficial effect of roflumilast was mediated through inhibition of striatal PDE4 with consequent activation of cAMP-dependent protein kinase A (PKA) signaling pathways, including the cAMP response element-binding protein (CREB) pathway and dopamine and cAMP-regulated phosphoprotein 32,000 (DARPP-32) pathway that is essential for maintaining dopaminergic function. Therefore, the present work sheds light on the substantial neuroprotective potential of roflumilast in treating PD through the activation of the cAMP/PKA cascade.

Association of PPP1R1B polymorphisms with working memory in healthy Han Chinese adults.

The dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is encoded by the PPP1R1B gene, plays a converging regulatory role in the central nervous system by mediating the actions of dopamine, serotonin, and glutamate. Previous studies have demonstrated that variations in genes related to the dopamine system influence working memory. The present study thus investigated whether polymorphisms in PPP1R1B gene were associated with working memory. A sample of 124 healthy Han Chinese were genotyped for three single nucleotide polymorphisms of PPP1R1B gene, namely rs12601930C/T, rs879606A/G, and rs3764352A/G, using polymerase chain reaction and restriction fragment length polymorphism analysis. Working memory performance was assessed using the Wisconsin Card Sorting Test (WCST). Significant differences were observed in the Total Correct (TC), Total Errors (TE), and Conceptual Level Responses (CLR) scores of the WCST among the three rs12601930C/T genotypes (p = 0.044, 0.044, and 0.047, respectively); in TC, TE, Non-Perseverative Errors (NPE), and CLR scores between participants with the CC and (CT + TT) rs12601930C/T polymorphism genotypes (p = 0.032, 0.032, 0.019, and 0.029, respectively); in TC, TE, Perseverative Errors (PE), NPE, and CLR scores between participants with the (CT + CC) and TT rs12601930C/T polymorphism genotypes (p = 0.001, 0.001, 0.011, 0.004, and 0.001, respectively); and in NPE and CLR scores between participants with the GG and (AG + AA) genotypes of the rs3764352A/G polymorphism (p = 0.011 and 0.010). Furthermore, for males only, there were significant differences in TC, TE, PE, NPE, and CLR scores among the rs12601930C/T genotypes (p = 0.020, 0.020, 0.037, 0.029, and 0.014, respectively) and NPE and CLR scores among the rs3764352 genotypes (p = 0.045 and 0.042). PPP1R1B gene polymorphisms rs12601930C/T and rs3764352A/G might be associated with working memory assessed by the WCST in healthy Chinese adults, especially among males.

PLPP/CIN-mediated DARPP-32 serine 97 dephosphorylation delays the seizure onset in response to kainic acid in the mouse hippocampus

Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32)-mediated protein phosphatase 1 (PP1) inhibition leads to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activity and current and thereby may facilitate seizure activity. In the present study, we found that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) transiently dephosphorylated DARPP-32 serine (S) 97 site in the early time window, and casein kinase 2 (CK2) subsequently phosphorylated this site in the later time points after kainic acid (KA) injection, which increased the latency of seizure onset in response to KA, but exacerbated the intensity (severity), duration and progression of seizures. TMCB (a CK2 inhibitor) delayed the seizure onset in response to KA, concomitant with the reduced DARPP-32 S97 phosphorylation. Therefore, our findings suggest that PLPP/CIN may play an important role in the latency of seizure onset via DARPP-32-PP1-AMPAR signaling pathway, and may be one of the potential therapeutic targets for medication of seizure or epilepsy.

Elevation of Extracellular Glutamate by Blockade of Astrocyte Glutamate Transporters Inhibits Cocaine Reinforcement in Rats via a NMDA-GluN2B Receptor Mechanism.

It is well established that glutamate plays an important role in drug-induced and cue-induced reinstatement of drug seeking. However, the role of glutamate in drug reward is unclear. In this study, we systemically evaluated the effects of multiple glutamate transporter (GLT) inhibitors on extracellular glutamate and dopamine (DA) in the nucleus accumbens (NAc), intravenous cocaine self-administration, intracranial brain-stimulation reward (BSR), and reinstatement of cocaine seeking in male and female rats. Among the five GLT inhibitors we tested, TFB-TBOA was the most potent. Microinjections of TFB-TBOA into the NAc, but not the ventral tegmental area (VTA), or dorsal striatum (DS), dose-dependently inhibited cocaine self-administration under fixed-ratio and progressive-ratio (PR) reinforcement schedules, shifted the cocaine dose-response curve downward, and inhibited intracranial BSR. Selective downregulation of astrocytic GLT-1 expression in the NAc by GLT-1 antisense oligonucleotides also inhibited cocaine self-administration. The reduction in cocaine self-administration following TFB-TBOA administration was NMDA GluN2B receptor dependent, and rats self-administering cocaine showed upregulation of GluN2B expression in NAc DA- and cAMP-regulated phosphoprotein 32 (DARPP-32)-positive medium-spiny neurons (MSNs). In contrast, TFB-TBOA, when locally administered into the NAc, VTA, or ventral pallidum (VP), dose-dependently reinstated cocaine-seeking behavior. Intra-NAc TFB-TBOA-evoked drug-seeking was long-lasting and NMDA/AMPA receptor dependent. These findings, for the first time, indicate that glutamate in the NAc negatively regulates cocaine's rewarding effects, while an excess of glutamate in multiple brain regions can trigger reinstatement of drug-seeking behavior.SIGNIFICANCE STATEMENT It is well known that glutamate plays an important role in relapse to drug seeking. However, the role of glutamate in drug reward is less clear. Here, we report that TFB-TBOA, a highly potent glutamate transporter (GLT) inhibitor, dose-dependently elevates extracellular glutamate and inhibits cocaine self-administration and brain-stimulation reward (BSR), when administered locally into the nucleus accumbens (NAc), but not other brain regions. Mechanistic assays indicate that cocaine self-administration upregulates NMDA-GluN2B receptor subtype expression in striatal dopaminoceptive neurons and activation of GluN2B by TFB-TBOA-enhanced glutamate inhibits cocaine self-administration. TFB-TBOA also reinstates cocaine-seeking behavior when administered into the NAc, ventral tegmental area (VTA), and ventral pallidum (VP). These findings demonstrate that glutamate differentially regulates cocaine reward versus relapse, reducing cocaine reward, while potentiating relapse to cocaine seeking.

MiRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

MicroRNA-802 Inhibits Cell Proliferation and Induces Apoptosis in Human Laryngeal Cancer by Targeting cAMP-Regulated Phosphoprotein 19 [Retraction

[This retracts the article DOI: 10.2147/CMAR.S228429.].

Sex Differences in the Ventral Tegmental Area and Nucleus Accumbens Proteome at Baseline and Following Nicotine Exposure.

Sex differences in behaviors relevant to nicotine addiction have been observed in rodent models and human subjects. Behavioral, imaging, and epidemiological studies also suggest underlying sex differences in mesolimbic dopamine signaling pathways. In this study we evaluated the proteome in the ventral tegmental area (VTA) and nucleus accumbens (NAc) shell in male and female mice. Experimental groups included two mouse strains (C3H/HeJ and C57BL/6J) at baseline, a sub-chronic, rewarding regimen of nicotine in C3H/HeJ mice, and chronic nicotine administration and withdrawal in C57BL/6J mice. Isobaric labeling with a TMT 10-plex system, sample fractionation, and tandem mass spectrometry were used to quantify changes in protein abundance. In C3H/HeJ mice, similar numbers of proteins were differentially regulated between sexes at baseline compared with within each sex after sub-chronic nicotine administration. In C57BL/6J mice, there were significantly greater numbers of proteins differentially regulated between sexes at baseline compared with within each sex after chronic nicotine administration and withdrawal. Despite differences by sex, strain, and nicotine exposure parameters, glial fibrillary acidic protein (GFAP) and dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32, Ppp1r1b) were repeatedly identified as significantly altered proteins, especially in the VTA. Further, network analyses showed sex- and nicotine-dependent regulation of a number of signaling pathways, including dopaminergic signaling. Sub-chronic nicotine exposure in female mice increased proteins related to dopaminergic signaling in the NAc shell but decreased them in the VTA, whereas the opposite pattern was observed in male mice. In contrast, dopaminergic signaling pathways were similarly upregulated in both male and female VTA after chronic nicotine and withdrawal. Overall, this study identifies significant sex differences in the proteome of the mesolimbic system, at baseline and after nicotine reward or withdrawal, which may help explain differential trajectories and susceptibility to nicotine addiction in males and females.

Multi-cohort profiling reveals elevated CSF levels of brain-enriched proteins in Alzheimer's disease.

ObjectiveDecreased amyloid beta (Aβ) 42 together with increased tau and phospho‐tau in cerebrospinal fluid (CSF) is indicative of Alzheimer’s disease (AD). However, the molecular pathophysiology underlying the slowly progressive cognitive decline observed in AD is not fully understood and it is not known what other CSF biomarkers may be altered in early disease stages.MethodsWe utilized an antibody‐based suspension bead array to analyze levels of 216 proteins in CSF from AD patients, patients with mild cognitive impairment (MCI), and controls from two independent cohorts collected within the AETIONOMY consortium. Two additional cohorts from Sweden were used for biological verification.ResultsSix proteins, amphiphysin (AMPH), aquaporin 4 (AQP4), cAMP‐regulated phosphoprotein 21 (ARPP21), growth‐associated protein 43 (GAP43), neurofilament medium polypeptide (NEFM), and synuclein beta (SNCB) were found at increased levels in CSF from AD patients compared with controls. Next, we used CSF levels of Aβ42 and tau for the stratification of the MCI patients and observed increased levels of AMPH, AQP4, ARPP21, GAP43, and SNCB in the MCI subgroups with abnormal tau levels compared with controls. Further characterization revealed strong to moderate correlations between these five proteins and tau concentrations.InterpretationIn conclusion, we report six extensively replicated candidate biomarkers with the potential to reflect disease development. Continued evaluation of these proteins will determine to what extent they can aid in the discrimination of MCI patients with and without an underlying AD etiology, and if they have the potential to contribute to a better understanding of the AD continuum.

Selective Inhibition of Phosphodiesterase 7 Enzymes Reduces Motivation for Nicotine Use through Modulation of Mesolimbic Dopaminergic Transmission.

Approximately 5 million people die from diseases related to nicotine addiction and tobacco use each year. The nicotine-induced increase of corticomesolimbic dopaminergic (DAergic) transmission and hypodopaminergic conditions occurring during abstinence are important for maintaining drug-use habits. We examined the notion of reequilibrating DAergic transmission by inhibiting phosphodiesterase 7 (PDE7), an intracellular enzyme highly expressed in the corticomesolimbic circuitry and responsible for the degradation of cyclic adenosine monophosphate (cAMP), the main second messenger modulated by DA receptor activation. Using selective PDE7 inhibitors, we demonstrated in male rats that systemic PDE7 enzyme inhibition reduced nicotine self-administration and prevented reinstatement to nicotine seeking evoked by cues or by the pharmacological stressor yohimbine. The effect was also observed by direct application of the PDE7 inhibitors into the nucleus accumbens (NAc) shell but not into the core. Inhibition of PDE7 resulted in increased DA- and cAMP-regulated neuronal phosphoprotein and cAMP response element-binding protein and their phosphorylated forms in the NAc. It also enhanced the DA D1 receptor agonism-mediated effects, indicating potentiation of protein kinase A–dependent transmission downstream of D1 receptor activation. In electrophysiological recordings from DA neurons in the lateral posterior ventral tegmental area, the PDE7 inhibitors attenuated the spontaneous activity of DA neurons. This effect was exerted through the potentiation of D1 receptor signaling and the subsequent facilitation of γ-aminobutyric acid transmission. The PDE7 inhibitors did not elicit conditioned place preference and did not induce intravenous self-administration, indicating lack of reinforcing properties. Thus, PDE7 inhibitors have the potential to treat nicotine abuse.SIGNIFICANCE STATEMENT The World Health Organization estimates that there are 1.25 billion smokers worldwide, representing one-third of the global population over the age of 15. Nicotine-induced increase of corticomesolimbic DAergic transmission and hypodopaminergic conditions occurring during abstinence are critical for maintaining drug-use habits. Here, we demonstrate that nicotine consumption and relapse to nicotine seeking are attenuated by reequilibrating DAergic transmission through inhibition of PDE7, an intracellular enzyme responsible for the degradation of cAMP, the main second messenger modulated by DA receptor activation. PDE7 inhibition may represent a novel treatment approach to aid smoking cessation.

Impact of Caffeine on Ethanol-Induced Stimulation and Sensitization: Changes in ERK and DARPP-32 Phosphorylation in Nucleus Accumbens.

Caffeine is frequently consumed with ethanol to reduce the impairing effects induced by ethanol, including psychomotor slowing or incoordination. Both drugs modulate dopamine (DA)-related markers in accumbens (Acb), and Acb DA is involved in voluntary locomotion and locomotor sensitization. The present study determined whether caffeine can affect locomotion induced by acute and repeated ethanol administration in adult male CD-1 mice. Acute administration of caffeine (7.5 to 30.0mg/kg) was evaluated for its effects on acute ethanol-induced (1.5 to 3.5g/kg) changes in open-field horizontal locomotion, supported rearing, and rearing not supported by the wall. DA receptor-dependent phosphorylation markers were assessed: extracellular signal-regulated kinase (pERK), and dopamine-and cAMP-regulated phosphoprotein Mr32kDa phosphorylated at threonine 75 site (pDARPP-32-Thr75) in Acb core and shell. Acutely administered caffeine was also evaluated in ethanol-sensitized (1.5g/kg) mice. Acute ethanol decreased both types of rearing. Caffeine increased supported rearing but did not block ethanol -induced decreases in rearing. Both substances increased horizontal locomotion in a biphasic manner, and caffeine potentiated ethanol-induced locomotion. Although ethanol administered repeatedly induced sensitization of locomotion and unsupported rearing, acute administration of caffeine to ethanol-sensitized mice in an ethanol-free state resulted in blunted stimulant effects compared with those seen in ethanol-naïve mice. Ethanol increased pERK immunoreactivity in both subregions of the Acb, but coadministration with caffeine blunted this increase. There were no effects on pDARPP-32(Thr75) immunoreactivity. The present results demonstrated that, after the first administration, caffeine potentiated the stimulating actions of ethanol, but did not counteract its suppressant or ataxic effects. Moreover, our results show that caffeine has less activating effects in ethanol-sensitized animals.

Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors.

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS—the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.

DARPP-32 40 years later.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with an apparent Mr of 32,000), now also known as phosphoprotein phosphatase 1 regulatory subunit 1B (PPP1R1B), is a potent inhibitor of protein phosphatase 1 (PP1, also known as PPP1) when phosphorylated at Thr34 by cAMP-dependent protein kinase (PKA). DARPP-32 exhibits a remarkable regional distribution in brain, roughly similar to that of dopamine innervation. Its discovery was a culmination of the long-standing effort of Paul Greengard to understand the mechanisms through which neurotransmitters such as dopamine exert their effects on target neurons. DARPP-32 is particularly enriched in striatal projection neurons where it is regulated by numerous signals through which it integrates and amplifies responses to many stimuli. Molecular studies of DARPP-32 have revealed that its regulation and function are more complex than anticipated. It is phosphorylated on multiple sites by several protein kinases that modulate DARPP-32 properties. Primarily, when phosphorylated at Thr34 DARPP-32 is a potent inhibitor of PP1, whereas when phosphorylated at Thr75 by Cdk5 it inhibits PKA. Phosphorylation at serine residues by CK1 and CK2 modulates its intracellular localization and its sensitivity to kinases or phosphatases. Modeling studies provide evidence that the signaling pathways including DARPP-32 are endowed of strong robustness and bistable properties favoring switch-like responses. Thus DARPP-32 combined with a set of other distinct signaling molecules enriched in striatal projection neurons plays a key role in the characteristic properties and physiological function of these neurons.