RESUMO
Neuropathic pain is a form of chronic pain that develops because of damage to the nervous system. Treatment of neuropathic pain is often incompletely effective, and most available therapeutics have only moderate efficacy and present side effects that limit their use. Opioids are commonly prescribed for the management of neuropathic pain despite equivocal results in clinical studies and significant abuse potential. Thus, neuropathic pain represents an area of critical unmet medical and novel classes of therapeutics with improved efficacy and safety profiles are urgently needed. The cannabidiol (CBD) structural analogue and novel antagonist of GPR55, KLS-13019, was screened in rat models of neuropathic pain. Tactile sensitivity associated with chemotherapy exposure was induced in rats with once daily 1mg/kg paclitaxel injections for 4 days or 5 mg/kg oxaliplatin every third day for one week. Rats were then administered KLS-13019 or comparator drugs on day 7 in an acute dosing paradigm or days 7-10 in a chronic dosing paradigm and mechanical or cold allodynia was assessed. Allodynia was reversed in a dose-dependent manner in the rats treated with KLS-13019, with the highest dose reverting the response to pre-paclitaxel injection baseline levels with both I.P. and P.O. administration after acute dosing. In the chronic dosing paradigm, 4 consecutive doses of KLS-13019 completely reversed allodynia for the duration of the phenotype in control animals. Additionally, co-administration of KLS -13019 with paclitaxel prevented the allodynic phenotype from developing. Together, these data suggest that KLS-13019 represents a potential new drug for the treatment of neuropathic pain. Significance Statement Chemotherapy-induced neuropathic pain (CIPN) is a common, debilitating side effect of cancer treatment with no known cure. GPR55 antagonist KLS-13019 represents a novel class of drug for this condition that is a potent, durable inhibitor of allodynia associated with CIPN in rats in both prevention and reversal dosing paradigms. This novel therapeutic approach addresses a critical area of unmet medical need.
RESUMO
KLS-13019 was reported previously to reverse paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). Recent studies demonstrated that paclitaxel-induced increases in inflammatory markers (GPR55, NLRP3, and IL-1ß) of dorsal root ganglion (DRG) cultures were shown to be reversed by KLS-13019 treatment. The mechanism of action for KLS-13019-mediated reversal of paclitaxel-induced neuroinflammation now has been explored using GPR55 siRNA. Pre-treatment of DRG cultures with GPR55 siRNA produced a 21% decrease of immunoreactive (IR) area for GPR55 in cell bodies and a 59% decrease in neuritic IR area, as determined by high-content imaging. Using a 24-h reversal treatment paradigm, paclitaxel-induced increases in the inflammatory markers were reversed back to control levels after KLS-3019 treatment. Decreases in these inflammatory markers produced by KLS-13019 were significantly attenuated by GPR55 siRNA co-treatment, with mean IR area responses being attenuated by 56% in neurites and 53% in cell bodies. These data indicate that the percentage decreases in siRNA-mediated attenuation of KLS-13019-related efficacy on the inflammatory markers were similar to the percentage knockdown observed for neuritic GPR55 IR area. Similar studies conducted with cannabidiol (CBD), the parent compound of KLS-13019, produced low efficacy (25%) reversal of all inflammatory markers that were poorly attenuated (29%) by GPR55 siRNA. CBD was shown previously to be ineffective in reversing paclitaxel-induced mechanical allodynia. The present studies indicated significant differences between the anti-inflammatory properties of KLS-13019 and CBD which may play a role in their observed differences in the reversibility of mechanical allodynia in a mouse model of CIPN.
Assuntos
Canabidiol , Animais , Camundongos , RNA Interferente Pequeno/genética , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Hiperalgesia/tratamento farmacológico , Anti-Inflamatórios , Modelos Animais de Doenças , Paclitaxel/toxicidade , Receptores de Canabinoides/genéticaRESUMO
KLS-13019 was reported previously to reverse paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). Recent studies demonstrated that paclitaxel-induced increases in inflammatory markers (GPR55, NLRP3 and IL-1b) of dorsal root ganglion (DRG) cultures were shown to be reversed by KLS-13019 treatment. The mechanism of action for KLS-13019-mediated reversal of paclitaxel-induced neuroinflammation now has been explored using GPR55 siRNA. Pretreatment of DRG cultures with GPR55 siRNA produced a 21% decrease of immunoreactive (IR) area for GPR55 in cell bodies and a 59% decrease in neuritic IR area, as determined by high content imaging. Using a 24-hour reversal treatment paradigm, paclitaxel-induced increases in the inflammatory markers were reversed back to control levels after KLS-3019 treatment. Decreases in these inflammatory markers produced by KLS-13019 were significantly attenuated by GPR55 siRNA co-treatment, with mean IR area responses being attenuated by 56% in neurites and 53% in cell bodies. These data indicate that the percentage decreases in siRNA-mediated attenuation of KLS-13019-related efficacy on the inflammatory markers were similar to the percentage knockdown observed for neuritic GPR55 IR area. Similar studies conducted with cannabidiol (CBD), the parent compound of KLS-13019, produced low efficacy (25%) reversal of all inflammatory markers that were poorly attenuated (29%) by GPR55 siRNA. CBD was shown previously to be ineffective in reversing paclitaxel-induced mechanical allodynia. The present studies indicated significant differences between the anti-inflammatory properties of KLS-13019 and CBD which may play a role in their observed differences in the reversibility of mechanical allodynia in a mouse model of CIPN.
RESUMO
KLS-13019, a novel devised cannabinoid-like compound, was explored for anti-inflammatory actions in dorsal root ganglion cultures relevant to chemotherapy-induced peripheral neuropathy (CIPN). Time course studies with 3 µM paclitaxel indicated > 1.9-fold increases in immunoreactive (IR) area for cell body GPR55 after 30 min as determined by high content imaging. To test for reversibility of paclitaxel-induced increases in GPR55, cultures were treated for 8 h with paclitaxel alone and then a dose response to KLS-13019 added for another 16 h. This "reversal" paradigm indicated established increases in cell body GPR55 IR areas were decreased back to control levels. Because GPR55 had previously reported inflammatory actions, IL-1ß and NLRP3 (inflammasome-3 marker) were also measured in the "reversal" paradigm. Significant increases in all inflammatory markers were produced after 8 h of paclitaxel treatment alone that were reversed to control levels with KLS-13019 treatment. Accompanying studies using alamar blue indicated that decreased cellular viability produced by paclitaxel treatment was reverted back to control levels by KLS-13019. Similar studies conducted with lysophosphatidylinositol (GPR55 agonist) in DRG or hippocampal cultures demonstrated significant increases in neuritic GPR55, NLRP3 and IL-1ß areas that were reversed to control levels with KLS-13019 treatment. Studies with a human GPR55-ß-arrestin assay in Discover X cells indicated that KLS-13019 was an antagonist without agonist activity. These studies indicated that KLS-13019 has anti-inflammatory properties mediated through GPR55 antagonist actions. Together with previous studies, KLS-13019 is a potent neuroprotective, anti-inflammatory cannabinoid with therapeutic potential for high efficacy treatment of neuropathic pain.
Assuntos
Canabinoides , Neuralgia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Canabinoides/uso terapêutico , Gânglios Espinais/metabolismo , Hipocampo/metabolismo , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neuralgia/tratamento farmacológico , Paclitaxel/farmacologia , Receptores de Canabinoides/metabolismoRESUMO
BACKGROUND AND PURPOSE: Cannabidiol (CBD) is a non-euphorigenic component of Cannabis sativa that prevents the development of paclitaxel-induced mechanical sensitivity in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). We recently reported that the CBD structural analogue KLS-13019 shows efficacy in an in vitro model of CIPN. The present study was to characterize the behavioural effects of KLS-13019 compared to CBD and morphine in mouse models of CIPN, nociceptive pain and reinforcement. EXPERIMENTAL APPROACH: Prevention or reversal of paclitaxel-induced mechanical sensitivity were assessed following intraperitoneal or oral administration of CBD, KLS-13019 or morphine. Antinociceptive activity using acetic acid-induced stretching and hot plate assay, anti-reinforcing effects on palatable food or morphine self-administration and binding to human opioid receptors were also determined. KEY RESULTS: Like CBD, KLS-13019 prevented the development of mechanical sensitivity associated with paclitaxel administration. In contrast to CBD, KLS-13019 was also effective at reversing established mechanical sensitivity. KLS-13019 significantly attenuated acetic acid-induced stretching and produced modest effects in the hot plate assay. KLS-13019 was devoid of activity at µ-, δ- or κ-opioid receptors. Lastly, KLS-13019, but not CBD, attenuated the reinforcing effects of palatable food or morphine. CONCLUSIONS AND IMPLICATIONS: KLS-13019 like CBD, prevented the development of CIPN, while KLS-13019 uniquely attenuated established CIPN. Because KLS-13019 binds to fewer biological targets, this will help to identifying molecular mechanisms shared by these two compounds and those unique to KLS-13019. Lastly, KLS-13019 may possess the ability to attenuate reinforced behaviour, an effect not observed in the present study with CBD.
Assuntos
Canabidiol , Dor Nociceptiva , Animais , Canabidiol/farmacologia , Modelos Animais de Doenças , Camundongos , Morfina , Reforço PsicológicoRESUMO
We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimer's disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100x more potent than 1, such as 7 (5 nM K(i)).
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Quinazolinas/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Simulação por Computador , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Quinazolinas/síntese química , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Previous studies have shown that cathepsins control amyloid beta (Abeta) levels in chromaffin cells via a regulated secretory pathway. In the present study, this concept was extended to investigations in primary hippocampal neurons to test whether Abeta release was coregulated by cathepsins and electrical activity, proposed components of a regulated secretory pathway. Inhibition of cathepsin B (catB) activity with CA074Me or attenuation of catB expression through small interfering RNA produced decreases in Abeta release, similar to levels produced with suppression of beta-site APP-cleaving enzyme 1 (BACE1) expression. To test whether the catB-dependent release of Abeta was linked to ongoing electrical activity, neurons were treated with tetrodotoxin (TTX) and CA074Me. These comparisons demonstrated no additivity between decreases in Abeta release produced by TTX and CA074Me. In contrast, pharmacological inhibition of cathepsin L (catL) selectively elevated Abeta42 levels but not Abeta40 or total Abeta. Mechanistic studies measuring C-terminal fragments of amyloid precursor protein (APP) suggested that catL elevated alpha-secretase activity, thereby suppressing Abeta42 levels. The mechanism of catB-mediated regulation of Abeta release remains unclear but may involve elevation of beta-secretase. In summary, these studies provide evidence for a significant alternative pathway for APP processing that involves catB and activity-dependent release of Abeta in a regulated secretory pathway for primary neurons.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Catepsina B/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Hipocampo/fisiologia , Neurônios/fisiologia , Animais , Catepsina B/genética , Catepsina L , Catepsinas/genética , Cisteína Endopeptidases/genética , Hipocampo/enzimologia , Humanos , Neurônios/efeitos dos fármacos , RNA Interferente Pequeno/genética , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Tetrodotoxina/farmacologiaRESUMO
Treatment with cannabidiol (CBD) or KLS-13019 (novel CBD analog), has previously been shown to prevent paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). The mechanism of action for CBD- and KLS-13019-mediated protection now has been explored with dissociated dorsal root ganglion (DRG) cultures using small interfering RNA (siRNA) to the mitochondrial Na+ Ca2+ exchanger-1 (mNCX-1). Treatment with this siRNA produced a 50-55% decrease in the immunoreactive (IR) area for mNCX-1 in neuronal cell bodies and a 72-80% decrease in neuritic IR area as determined with high-content image analysis. After treatment with 100 nM KLS-13019 and siRNA, DRG cultures exhibited a 75 ± 5% decrease in protection from paclitaxel-induced toxicity; whereas siRNA studies with 10 µM CBD produced a 74 ± 3% decrease in protection. Treatment with mNCX-1 siRNA alone did not produce toxicity. The protective action of cannabidiol and KLS-13019 against paclitaxel-induced toxicity during a 5-h test period was significantly attenuated after a 4-day knockdown of mNCX-1 that was not attributable to toxicity. These data indicate that decreases in neuritic mNCX-1 corresponded closely with decreased protection after siRNA treatment. Pharmacological blockade of mNCX-1 with CGP-37157 produced complete inhibition of cannabinoid-mediated protection from paclitaxel in DRG cultures, supporting the observed siRNA effects on mechanism.
Assuntos
Canabidiol/farmacologia , Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Animais , Células Cultivadas , Hiperalgesia , Neurônios/metabolismo , Paclitaxel/toxicidade , Interferência de RNA , Ratos , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
Cannabidiol (CBD) exhibits neuroprotective properties in many experimental systems. However, development of CBD as a drug has been confounded by the following: (1) low potency; (2) a large number of molecular targets; (3) marginal pharmacokinetic properties; and (4) designation as a schedule 1 controlled substance. The present work compared the properties of CBD with a novel molecule (KLS-13019) that has structural similarities to CBD. The design strategy for KLS-13019 was to increase hydrophilicity while optimizing neuroprotective potency against oxidative stress toxicity relevant to hepatic encephalopathy. The protective responses of CBD and KLS-13019 were compared in dissociated rat hippocampal cultures co-treated with toxic levels of ethanol and ammonium acetate. This comparison revealed that KLS-13019 was 31-fold more potent than CBD in preventing neuronal toxicity from the combined toxin treatment, while both compounds exhibited complete protective efficacy back to control values. In addition, treatment with KLS-13019 alone was 5-fold less toxic (TC50) than CBD. Previous studies suggested that CBD targeted the Na+-Ca2+ exchanger in mitochondria (mNCX) to regulate intracellular calcium levels, an important determinant of neuronal survival. After treatment with an inhibitor of mNCX (CGP-37157), no detectable neuroprotection from ethanol toxicity was observed for either CBD or KLS-13019. Furthermore, AM630 (CB2 antagonist) significantly attenuated CBD-mediated neuroprotection, while having no detectable effect on neuroprotection from KLS-13019. Our studies indicated KLS-13019 was more potent and less toxic than CBD. Both compounds can act through mNCX. KLS-13019 may provide an alternative to CBD as a therapeutic candidate to treat diseases associated with oxidative stress.
Assuntos
Canabidiol/análogos & derivados , Canabidiol/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Acetatos/toxicidade , Animais , Células Cultivadas , Etanol/toxicidade , Hipocampo/citologia , Neurônios/metabolismo , Ratos , Trocador de Sódio e Cálcio/metabolismoRESUMO
There is confusion in the literature on the measurement of the drug activity onset time (AOT) for both clinical and non-clinical studies of antidepressant and antimanic drugs. The questions asked are: How often and at which time points should drug effects be measured? At what level of a drug effect should AOT be determined? Is the placebo (control) effect important for consideration of drug AOT? This paper reviews approaches taken to answer these questions and to assess drug therapeutic AOT. The first part of the paper is devoted to a review of methods used in clinical trials with depression as an indication. The second part is focused on approaches taken in animal models of depression and how they could help in assessing drug AOT. Finally, a summary of pharmacological values on which the AOT depends is presented and a new statistical approach to data analysis method proposed. The allied experimental design for pre-clinical and clinical studies may help to characterize and differentiate AOT for available and new generation of antidepressants and antimanic drugs.
Assuntos
Antidepressivos/farmacologia , Antimaníacos/farmacologia , Comportamento Animal/efeitos dos fármacos , Dominação-Subordinação , Tempo de Reação/efeitos dos fármacos , Animais , Transtorno Bipolar/tratamento farmacológico , Transtorno Depressivo/tratamento farmacológico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fatores de TempoRESUMO
A new aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline ring was identified by high-throughput screening for the inhibition of BACE-1. X-ray crystallography revealed that the exocyclic amino group participated in a hydrogen bonding array with the two catalytic aspartic acids of BACE-1 (Asp(32), Asp(228)). BACE-1 inhibitory potency was increased (0.9 microM to 11 nM K(i)) by substitution into the unoccupied S(1)' pocket.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Modelos Moleculares , Quinazolinas/síntese química , Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/química , Células CHO , Células CACO-2 , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Conformação Molecular , Mutação , Oligopeptídeos/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Quinazolinas/química , Quinazolinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.
Assuntos
Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Quimiocinas/metabolismo , Humanos , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Technical variants of mania and depression models that were based on dominant-submissive relationships (DSR) have been analyzed and compared in the present paper. In these paradigms, one animal of a pair developed the behavioral trait of dominance while the other submissiveness in a food competition test after repeated interactions in a specially designed apparatus. Data collection methods and timelines have been compared in variants of the DSR-based models. In addition, different selection criteria to assign dominant or submissive status to animals and two different scoring systems were evaluated. The importance of the selection criteria for DSR stability has been emphasized. Our data showed that (1) only animals selected with the strict criteria form clear dominant and submissive relationships that hold throughout the study period, (2) submissive animals were influenced by fluoxetine and dominant animals were influenced by sodium valproate similarly in pairs scored by human observer and by a video-tracking system. These studies indicate that the model variant using stringent selection criteria and automatic scoring was the most reliable for use in depression-related studies.
Assuntos
Antidepressivos/farmacologia , Antimaníacos/farmacologia , Comportamento Animal/efeitos dos fármacos , Transtorno Bipolar/tratamento farmacológico , Transtorno Depressivo/tratamento farmacológico , Dominação-Subordinação , Animais , Comportamento Animal/fisiologia , Transtorno Bipolar/fisiopatologia , Transtorno Bipolar/psicologia , Interpretação Estatística de Dados , Transtorno Depressivo/fisiopatologia , Transtorno Depressivo/psicologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Comportamento Alimentar/fisiologia , Fluoxetina/farmacologia , Testes Neuropsicológicos/normas , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Comportamento Social , Ácido Valproico/farmacologia , Gravação de Videoteipe/métodosRESUMO
Juvenile Batten disease (JBD) is an inherited disorder that is characterized by the development of blindness, seizures, and progressive motor, psychiatric, and cognitive impairment. A model of JBD expressing the predominant human mutation (Cln3 ∆ex7/8 ) has been explored. Dissociated brain cultures from Cln3 ∆ex7/8 knock-in mice were compared to wild type (WT) for effects on granules of ceroid lipofuscin (CL) and neuronal structure. Utilizing high content image analysis of CL granules identified with antibodies to mitochondrial ATP synthase subunit c or tripeptidyl peptidase-1, significant increases in the areas for both immunoreactive granules were observed in Cln3 ∆ex7/8 cultures in comparison to WT. CL granules also exhibit autofluorescence at 488 and 560 nm, and the areas of these autofluorescent spots were found to be significantly increased in Cln3 ∆ex7/8 cultures in comparison to WT. Progressive increases in CL granule area in Cln3 ∆ex7/8 cultures were observed during culture development. Because current therapies for JBD provide only symptomatic support, a therapeutic strategy has been explored based on the observations that JBD-related tissues are deficient in ß-galactosyl ceramide. Treatment of cultures for 40 h with a potent analog of ß-galactosyl ceramide (SNB-4050) produced significant decreases in CL granule area in the Cln3 ∆ex7/8 cultures; whereas identical studies on WT cultures produced no detectible changes. Significant decreases in average neurite length and neurite branch point number were also observed in the Cln3 ∆ex7/8 cultures that were attenuated by treatment with 1 nM SNB-4050. These studies indicate Cln3 ∆ex7/8 brain cultures may be useful to screen therapeutic agents for treatment of JBD.
Assuntos
Encéfalo/citologia , Galactosilceramidas/farmacologia , Lipofuscina/metabolismo , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/efeitos dos fármacos , Complexos de ATP Sintetase/metabolismo , Aminopeptidases/metabolismo , Animais , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Serina Proteases/metabolismo , Tripeptidil-Peptidase 1RESUMO
The Ts65Dn segmental mouse model of Down syndrome (DS) possesses a triplication of the section of chromosome 16 that is most homologous to the human chromosome 21 that is trisomic in DS. This model exhibits many of the characteristics of DS including small size, developmental delays, and a decline of cholinergic systems and cognitive function with age. Recent studies have shown that vasoactive intestinal peptide (VIP) systems are upregulated in aged Ts65Dn mice and that VIP dysregulation during embryogenesis is followed by the hypotonia and developmental delays as seen in both DS and in Ts65Dn mice. Additionally, astrocytes from aged Ts65Dn brains do not respond to VIP stimulation to release survival-promoting substances. To determine if VIP dysregulation is age-related in Ts65Dn mice, the current study examined VIP and VIP receptors (VPAC-1 and VPAC-2) in postnatal day 8 Ts65Dn mice. VIP and VPAC-1 expression was significantly increased in the brains of trisomic mice compared with wild-type mice. VIP-binding sites were also significantly increased in several brain areas of young Ts65Dn mice, especially in the cortex, caudate/putamen, and hippocampus. Further, in vitro treatment of normal neurons with conditioned medium from VIP-stimulated Ts65Dn astrocytes from neonatal mice did not enhance neuronal survival. This study indicates that VIP anomalies are present in neonatal Ts65Dn mice, a defect occurs in the signal transduction mechanism of the VPAC-1 VIP receptor, cortical astrocytes from neonatal brains are dysfunctional, and further, that VIP dysregulation may play a significant role in DS.
Assuntos
Astrócitos/fisiologia , Síndrome de Down/fisiopatologia , Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Cromossomos Humanos Par 21 , Primers do DNA , Diploide , Modelos Animais de Doenças , Síndrome de Down/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Reação em Cadeia da Polimerase , Receptores de Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/genéticaRESUMO
Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for alpha-, beta-, and gamma-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex = hippocampus > striatum > cerebellum for alpha-synuclein; cortex > hippocampus = cerebellum > striatum for beta-synuclein; and hippocampus = striatum > cortex = cerebellum for gamma-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for alpha-synuclein were significantly reduced with age in all structures except hippocampus. For beta- and gamma-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, gamma-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , RNA Mensageiro/biossíntese , Ratos/metabolismo , alfa-Sinucleína/genética , beta-Sinucleína/genética , gama-Sinucleína/genética , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Feminino , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Masculino , Especificidade de Órgãos , Ratos/genética , Ratos/crescimento & desenvolvimento , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Sinucleína/biossíntese , beta-Sinucleína/biossíntese , gama-Sinucleína/biossínteseRESUMO
OBJECTIVE: Previous work has demonstrated that two synthetic peptides can prevent prenatal alcohol-induced damage as assessed by prevention of learning abnormalities in adult offspring as well as improve outcome from traumatic brain damage. The current studies were undertaken to evaluate whether these peptides could enhance performance in a learning and memory paradigm when administered either prenatally or to aged mice. STUDY DESIGN: For prenatal treatment, C57Bl6/J mice were treated on gestational day 8 with 1 oral administration of D-NAP or D-SAL alone or D-NAP+D-SAL or a double dose of D-SAL. Control groups were same-regimen treated with vehicle alone. Learning was assessed in adult male offspring (35-50 days) by using the Morris water maze. To evaluate aged animals, 12-month-old mice were treated with D-NAP and D-SAL or vehicle alone daily and tested on the Morris water maze. RESULTS: Offspring exposed prenatally to D-NAP+D-SAL learned significantly faster than controls, with an earlier onset of learning and an overall decreased latency to find the hidden platform (P < .05). Animals exposed prenatally to either D-NAP or D-SAL alone learned similar to control, with a trend toward faster latencies. Aged animals who received D-NAP+D-SAL learned significantly faster than age-matched controls, with an earlier onset of learning (P < .05). CONCLUSION: Combined D-NAP+D-SAL enhanced learning in healthy young mice and aged mice. These findings suggest potential therapeutic interventions not only during a critical developmental period, but also in aged animals.
Assuntos
Aprendizagem em Labirinto/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/farmacologia , Neuropeptídeos/fisiologia , Oligopeptídeos/fisiologia , GravidezRESUMO
Cannabidiol is the nonpsychoactive natural component of C. sativa that has been shown to be neuroprotective in multiple animal models. Our interest is to advance a therapeutic candidate for the orphan indication hepatic encephalopathy (HE). HE is a serious neurological disorder that occurs in patients with cirrhosis or liver failure. Although cannabidiol is effective in models of HE, it has limitations in terms of safety and oral bioavailability. Herein, we describe a series of side chain modified resorcinols that were designed for greater hydrophilicity and "drug likeness", while varying hydrogen bond donors, acceptors, architecture, basicity, neutrality, acidity, and polar surface area within the pendent group. Our primary screen evaluated the ability of the test agents to prevent damage to hippocampal neurons induced by ammonium acetate and ethanol at clinically relevant concentrations. Notably, KLS-13019 was 50-fold more potent and >400-fold safer than cannabidiol and exhibited an in vitro profile consistent with improved oral bioavailability.
RESUMO
Activity-dependent neurotrophic factor (ADNF) is a novel, femtomolar-acting, glial-derived polypeptide (14 kDa) known to protect neurons from a variety of toxic insults. The active site for ADNF function is localized to a 9-amino-acid stretch (SALLRSIPA; ADNF-9). A few years later, a novel ADNF-9-like active peptide (NAPVSIPQ or NAP) was identified and shown to be expressed in the CNS and exhibit an activity profile similar to ADNF-9. Such studies suggest that ADNF-9 and NAP might function like other known neurotrophins and play a role in neural development and maintenance. The purpose of the present studies was to determine if ADNF-9 or NAP affects neurite outgrowth and synaptogenesis in rat hippocampal and cortical cultures. Using MAP2-FITC immunofluorescent labeling, we found that ADNF-9 and NAP promoted neurite outgrowth in a concentration-dependent manner, with maximal activity observed at femtomolar concentrations. Both peptides stimulated robust outgrowth in hippocampal cells (approximately 150% of control; p < 0.01) with a modest effect on cortical cells (approximately 20% of control; p < 0.05) similar to other known growth factors. However, the outgrowth-promoting effect was abolished in the absence of serum, suggesting that soluble factors might be necessary for the neurotrophic activity. Finally, we found that ADNF-9 and NAP increased synaptophysin expression in both rat hippocampal and cortical cultures. These results suggest that ADNF-9 and NAP might contribute to neuronal plasticity associated with development and repair after injury.
Assuntos
Proteínas do Tecido Nervoso/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/fisiologia , Oligopeptídeos/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Hipocampo/citologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Gravidez , Ratos , Sinaptofisina/metabolismoRESUMO
NAP is a short octapeptide sequence (single letter code, NAPVSIPQ) that protects neurons against a wide variety of insults. The NAP sequence was identified by peptide structure/function scanning of activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation. To further evaluate the in vivo efficacy of NAP neuroprotection we used a mouse model of head trauma; a condition that presents a risk factor for the development of Alzheimer's disease in injured patients. In the mouse model, NAP treatment (prophylactic or curative) indicated improvement in longitudinal clinical, biochemical and anatomical outcomes. Furthermore, closed head injury was associated with a delayed increase in the expression of the immune cell surface glycoprotein Mac-1 (CD11B antigen) at the injury site that was decreased in NAP-treated mice. Additional experiments with Mac-1-deficient mice suggested partial protection against death related to severe head injury. NAP protection in Mac-1-deficient mice against adverse clinical outcome was concomitant with the time period when increases in Mac-1 transcripts were observed in the Mac-1 expressing mice ( approximately four weeks after the injury). The expression of ADNP (the NAP parent protein) was also increased at the injured brain site four weeks after the traumatic event, only in Mac-1 expressing mice. Here, using immunocytochemistry, we localized the increase in ADNP to microglia and astrocyte-like cells. The increase in ADNP in injured brains is now suggested to be a part of an endogenous compensatory mechanism and NAP treatment provides an additional protection. Toxicology studies suggest NAP as safe for further clinical development.