ACS Chemical Neuroscience最新文献

There currently are no medications proven to be effective for the treatment of stimulant-use disorder (SUD). Sigma-receptor (σR) antagonists block many effects of stimulant drugs but not the reinforcing effects assessed with self-administration in rats. However, a recent study suggests that σR antagonism combined with a dopamine (DA) transporter (DAT) blockade selectively attenuates stimulant self-administration. A compound with potential for dual DAT/σR inhibition, CM699, was synthesized and had the necessary ex vivo affinities of 311 and 14.1 nM at DAT and σ₁Rs, respectively. CM699 inhibited DA uptake ex vivo. Antagonist effects at σ₁Rs by CM699 were confirmed with a recently reported pharmacological assay: CM699 increased, whereas the σ₁R agonist, (+)-pentazocine, decreased σ₁R multimers detected in nondenaturing protein gels, and CM699 blocked the effects of (+)-pentazocine. CM699 after intravenous administration (5.0 mg/kg) in rats had an elimination half-life of 4.4 h. In rats, CM699 after intraperitoneal administration blunted the stimulatory effects of cocaine on DA levels in the nucleus accumbens and insurmountably blocked cocaine self-administration, indicating efficacy as a cocaine antagonist in vivo. When given alone, CM699 was not self-administered nor had significant effects on nucleus accumbens DA, suggesting minimal, if any, abuse potential. Further, in a biochemical assay designed to probe the conformation of DAT, (+)-pentazocine potentiated cocaine-induced cysteine accessibility of DAT transmembrane domain 6a, suggesting a shift in the conformational equilibrium of DAT toward outward-facing, whereas CM699 blocked this effect. The results provide preclinical proof of concept for dual DAT/σR inhibition as a novel DAT-conformational approach for the development of medications to treat SUD.

Cathepsin B has been shown to contribute to deficits in traumatic brain injury (TBI), an important risk factor for Alzheimer’s disease (AD). Cathepsin B is elevated in TBI and AD patients, as well as in animal models of these conditions. Knockout of the cathepsin B gene results in amelioration of TBI-induced motor dysfunction and improvement of AD memory deficit in mice. The mechanism of cathepsin B pathogenesis in these brain disorders has been hypothesized to involve its translocation to the cytosol from its normal lysosomal location. This study, therefore, evaluated brain cytosolic cathepsin B activity in the controlled cortical impact (CCI) mouse model of TBI. CCI-TBI resulted in motor deficits demonstrated by the rotarod assay, brain tissue lesions, and disorganization of the hippocampus. Significantly, CCI-TBI increased cytosolic cathepsin B activity in the brain cortex in the ipsilateral brain hemisphere that received the CCI-TBI injury, with a concomitant decrease in the lysosomal fraction. Cathepsin B activity was monitored using the substrate Z-Nle-Lys-Arg-AMC which specifically detects cathepsin B activity but not other cysteine proteases. The normal lysosomal distribution of cathepsin B was observed by its discrete localization in brain cortical cells. CCI-TBI resulted in a more diffuse cellular distribution of cathepsin B consistent with translocation to the cytosol. Further studies utilized the novel neutral pH-selective inhibitor, Z-Arg-Lys-AOMK, that specifically inhibits cathepsin B at neutral pH 7.2 of the cytosol but not at acidic pH 4.6 of lysosomes. Daily administration of Z-Arg-Lys-AOMK (ip), beginning 1 day before CCI-TBI, resulted in the reduction of the increased cytosolic cathepsin B activity induced by CCI-TBI. The inhibitor also reduced cathepsin B activities in homogenates of the brain cortex and hippocampus which were increased by CCI-TBI. Furthermore, the Z-Arg-Lys-AOMK inhibitor resulted in the reduction of motor function deficit resulting from CCI-TBI. These findings demonstrate the activation of cytosolic cathepsin B activity in CCI-TBI mouse brain injury.

Recently, we disclosed VU0467319, an M₁ positive allosteric modulator (PAM) clinical candidate that had successfully completed a phase I single ascending dose clinical trial. Pharmacokinetic assessment revealed that, in humans upon increasing dose, a circulating, inactive metabolite constituted a major portion of the total drug-related area under the curve (AUC). One approach the team employed to reduce inactive metabolite formation in the back-up program was the kinetic isotope effect, replacing the metabolically labile C-H bonds with shorter, more stable C-D bonds. The C-D dipole afforded VU6045422, a more potent M₁ PAM (human EC₅₀ = 192 nM, 80% ACh Max) than its proteocongener VU0467319 (human EC₅₀ = 492 nM, 71% ACh Max), and retained the desired profile of minimal M₁ agonism. Overall, the profile of VU6045422 supported advancement, as did greater in vitro metabolic stability in both microsomes and hepatocytes than did VU0467319. In both rat and dog in vivo, low doses proved to mirror the in vitro profile; however, at higher doses in 14-day exploratory toxicology studies, the amount of the same undesired metabolite derived from VU6045422 was equivalent to that produced from VU0467319. This unexpected IVIVC result, coupled with less than dose-proportional increases in exposure and no improvement in solubility, led to discontinuation of VU0467319/VU6045422 development.

Parkinson’s disease (PD) is one of the most common progressive neurodegenerative pathologies that leads to dopaminergic deficiency and motor manifestations. Alpha-synuclein aggregation is a characteristic hallmark of PD pathogenesis. These aggregates facilitate the formation of Lewy bodies and degeneration. The epidemiological evidence demonstrates a definitive association of diabetes with PD risk. Considering this, many antidiabetic agents such as GLP-1 agonists and DPP-4 inhibitors are being explored as alternative PD therapeutics. This study evaluated the neuroprotective effect of the DPP-4 inhibitor sitagliptin mediated by the PI3K/AKT and Nrf2 pathways in PD models. In silico studies were conducted to determine the binding affinity, stability, and ADMET properties of DPP-4 inhibitors with target proteins. Sitagliptin (15 mg/kg p.o.) was administered in rotenone (30 mg/kg p.o. for 28 days)-induced and MPTP/P (25 mg/kg i.p. MPTP and 100 mg/kg probenecid i.p. twice a week for 5 weeks)-induced PD mouse (C57/BL6) models. Neurobehavioral assessments were carried out throughout the study. Biochemical (GSH, MDA), molecular estimations (AKT, Nrf2, PI3K, GSK-3β, GLP1, CREB, BDNF, NF-κB, alpha-synuclein), histopathological studies, and immunohistochemistry were carried out at the end of the study. The in silico studies demonstrate better binding, stability, and ADMET profile of sitagliptin with both target proteins. Sitagliptin restored cognitive and motor deficits in both rotenone- and MPTP/P-induced mouse models. There was upregulation of PI3K, AKT, Nrf2, CREB, and BDNF levels and downregulation of GSK-3β, NF-κB, and alpha-synuclein levels in both models after treatment with sitagliptin. However, GLP1 levels were not significantly restored, indicating a GLP1-independent mechanism. It also restored histopathological alterations and TH+ neuronal loss induced by rotenone and MPTP/P. These findings demonstrate that sitagliptin exhibits neuroprotective action mediated by upregulation of the PI3K/AKT and Nrf2 pathways in rotenone and MPTP/P mouse models of PD.

Prolonged exposure to corticosteroids (CORTs) triggers depression and anxiety symptoms either endogenously or exogenously via stimulating endoplasmic reticulum stress (ERS). The study assessed the therapeutic implications of hydrogen sulfide (H₂S) versus sertraline (SERT) in alleviating anxiety and depression induced by CORTs through the modulation of ERS and its inflammatory, oxidative, and apoptotic consequences. Rats were subdivided into four groups: control, CORT (20 mg/kg), NaHS (100 μmol/kg), and SERT (10 mg/kg) for 21 days. Behavioral and histological examinations of the cerebral cortex were performed. The levels of CHOP, GADD34, EIF2AK3, GRP78, caspase 3, and miR-146a were analyzed using qRT-PCR. The levels of CORTs, serotonin, BDNF, TNF-α, BCL2, NRF2, and ATF4 were measured using ELISA, whereas those of IL-1β and BAX were measured using immunohistochemical techniques. Total and phosphorylated PERK were assessed via western blotting, whereas GSH and MDA were assessed via a colorimetric assay. In the present study, CORTs upregulated the gene expression of CHOP, GADD34, EIF2AK3, GRP78, and Caspase 3, whereas it downregulated that of miR-146a. The levels of serotonin, BDNF, BCL2, GSH, and NRF2 were decreased, whereas those of ATF4, TNF-α, IL-1β, BAX, and MDA were elevated. On the contrary, NaHS and SERT reversed all the above-mentioned changes. H₂S shows promise in counteracting anxiety and depression symptoms induced by CORTs by targeting ERS cascades, mitigating inflammation, oxidative insults, and apoptosis in the cerebral cortex. H₂S elicits neuroprotective effects by targeting the miR-146a-3p/GRP78/CHOP/PERK/ATF4/GADD34 signaling pathway and regulating apoptotic markers BAX/BCL2 and inflammatory markers TNF-α and/IL-1β. Compared with SERT, H₂S exhibited superior anxiolytic and antidepressive effects.

The protein DJ-1 appears to play a protective role in the development of Parkinson's disease (PD). Here, we show that endogenous neurotoxins of the 1,2,3,4-tetrahydroisoquinoline family (TIQs), formed upon reaction of various aldehydes such as methylglyoxal (MGO) with the neurotransmitter dopamine, act as irreversible inhibitors of the esterase activity of human DJ-1, with IC50 values between 15 and 57 μM. The presence of a catechol function appears to be essential for these inhibitory effects, which may be at the origin of the oxidation of cysteine 106, a crucial residue in the DJ-1 active site, thereby leading to DJ-1 inhibition. We also show that these endogenous neurotoxins inhibit the protective effects of DJ-1 against glycated guanosine diphosphate (GDP) formation and against alpha-synuclein (aSyn) aggregation induced by MGO. In total, the observed inhibition of DJ-1 by these endogenous neurotoxins may contribute to their damaging effects on the nervous system and, should be taken into account in therapeutic strategies for PD and related disorders.

Alzheimer's disease (AD) is a progressive pathology that is linked to abrupt aggregation of amyloid β_1-42 (Aβ_1-42) peptide in the central nervous system. Aβ_1-42 aggregation yields amyloid oligomers and fibrils, toxic protein aggregates that cause progressive neuronal degeneration in the frontal lobe of the brain. Although neurons remain the focus of AD for decades, a growing body of evidence suggests that the degeneration of immune cells in the brain can be the major cause of AD. However, the extent to which Aβ_1-42 aggregates are toxic to the major classes of immune cells in the brain remains unclear. In the current study, we examine the cytotoxic effects of Aβ_1-42 fibrils on macrophages, dendritic cells, and microglia. These cells play vitally important roles in development and homeostasis of the central nervous system. We found that Aβ_1-42 fibrils caused calcium release and enhanced levels of reactive oxygen species in macrophages, dendritic cells, and microglia as well as neurons. We also investigated the extent to which the lysozymes of these immune cells could alter the aggregation properties of Aβ_1-42. Our results showed that lysosomes extracted from macrophages, dendritic cells, and microglia drastically accelerated Aβ_1-42 aggregation as well as altered cytotoxicity of these protein aggregates. These results indicate that impairment of immune cells in the brain can be a critically important aspect of neurodegenerative processes that are taking place upon the onset of AD.

The ApoE4 allele of apolipoprotein E (ApoE4) is the strongest hereditary predisposition to Alzheimer's disease, even though ApoE4 only differs from the more common ApoE3 by a single amino acid substitution. Previous studies have shown that ApoE4 is more susceptible to proteolytic degradation than ApoE3. This is an important finding because of ApoE's role in cholesterol homeostasis and lipid transport in the brain. The molecular determinants of the increased susceptibility of ApoE4 to proteolysis are unknown. Here, we apply a combination of spectrometric and spectroscopic methods to show that amyloid-β (Aβ) peptides, including Aβ(1-40) and Aβ(pyroE3-42), differentially modulate the plasmin-dependent degradation of ApoE3 and ApoE4. In particular, our data reveal that while the Aβ peptides do not affect the proteolysis of ApoE3, the peptides enhance the degradation of ApoE4 significantly. Overall, this work motivates therapeutic development that targets the Aβ-induced dysregulation of ApoE4 homeostasis in individuals carrying the ApoE4 allele.

It has been reported that nicotine affects brain dopamine homeostasis. By binding to nicotinic acetylcholine receptors, including those expressed by dopaminergic neurons of the ventral tegmental area, nicotine stimulates dopamine release and signaling. Dopamine is taken up from the synaptic cleft by the dopamine transporter (DAT) into presynaptic neurons, where it is degraded by monoamine oxidase (MAO). Besides nicotine, other tobacco compounds play a role in dopamine modulation. To better understand the biological effects of nicotine and other tobacco compounds on dopamine regulation, we selected a group of tobacco compounds based on their potential affinity to bind human MAO-A and MAO-B enzymes using an in silico approach. Subsequently, we tested the putative compounds in an enzymatic assay to verify their ability to inhibit human MAO-A or MAO-B. The positive hits were harman, norharman, harmaline, and 1-ethyl-β-carboline. While harman and norharman have been extensively studied, both harmaline and 1-ethyl-β-carboline have not been described in the context of tobacco and MAO inhibition before. We investigated DAT activity in an overexpressing cell line and dopamine release and uptake in rat striatal synaptosomes. We clearly demonstrate that tested MAO-A inhibitors (MAO-AIs) significantly attenuated human DAT activity and consequent dopamine uptake, establishing a functional connection between MAOIs and dopamine uptake via DAT. Interestingly, the tested MAO-AIs elicited pronounced dopamine release in crude synaptosomal preparations. In summary, this in vitro study demonstrates that tested MAO-AIs found in cigarette smoke not only reduce MAO activity but also strongly impact dopamine homeostatic mechanisms via DAT. Further in vivo investigations would advance our understanding of the underlying mechanisms of dopamine regulation and homeostasis.