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Licochalcone-A (Lico-A) PLGA NPs functionalized with cell penetrating peptides B6 and Tet-1 are proposed for the treatment of ocular anti-inflammatory diseases. In this work, we report the in vitro biocompatibility of cell penetrating peptides-functionalized Lico-A-loaded PLGA NPs in Caco-2 cell lines revealing a non-cytotoxic profile, and their anti-inflammatory activity against RAW 264.7 cell lines. Given the risk of hydrolysis of the liquid suspensions, freeze-drying was carried out testing different cryoprotectants (e.g., disaccharides, alcohols, and oligosaccharide-derived sugar alcohol) to prevent particle aggregation and mitigate physical stress. As the purpose is the topical eye instillation of the nanoparticles, to reduce precorneal wash-out, increase residence time and thus Lico-A bioavailability, an in-situ forming gel based on poloxamer 407 containing Lico-A loaded PLGA nanoparticles functionalized with B6 and Tet-1 for ocular administration has been developed. Developed formulations remain in a flowing semi-liquid state under non-physiological conditions and transformed into a semi-solid state under ocular temperature conditions (35 °C), which is beneficial for ocular administration. The pH, viscosity, texture parameters and gelation temperature results met the requirements for ophthalmic formulations. The gel has characteristics of viscoelasticity, suitable mechanical and mucoadhesive performance which facilitate its uniform distribution over the conjunctiva surface. In conclusion, we anticipate the potential clinical significance of our developed product provided that a synergistic effect is achieved by combining the high anti-inflammatory activity of Lico-A delivered by PLGA NPs with B6 and Tet-1 for site-specific targeting in the eye, using an in-situ forming gel., R. M. Galindo-Camacho acknowledges the financial support of the Generalitat de Catalunya for the PhD scholarship FI-DGR EMC/2199/2017 (DOGC-7459-2017) and for the scholarship Santander Research/Convocatòria d’estades 2022 per a doctorands de la UB. Author are also thankful to the Spanish Ministry of Economy, Industry and Competitiveness, and the European Regional Development Fund (grants RTI2018-094120-B-I00 and PID2021-122216OB-I00). E. B. Souto wishes to acknowledge the national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB

Protein tyrosine phosphatase 1B (PTP1B) is a typical member of the PTP family, considered a direct negative regulator of several receptor and receptor-associated tyrosine kinases. This widely localized enzyme has been involved in the pathophysiology of several diseases. More recently, PTP1B has attracted attention in the field of neuroscience, since its activation in brain cells can lead to schizophrenia-like behaviour deficits, anxiety-like effects, neurodegeneration, neuroinflammation and depression. Conversely, PTP1B inhibition has been shown to prevent microglial activation, thus exerting a potent anti-inflammatory effect and has also shown potential to increase the cognitive process through the stimulation of hippocampal insulin, leptin and BDNF/TrkB receptors. Notwithstanding, most research on the clinical efficacy of targeting PTP1B has been developed in the field of obesity and type 2 diabetes mellitus (TD2M). However, despite the link existing between these metabolic alterations and neurodegeneration, no clinical trials assessing the neurological advantages of PTP1B inhibition have been performed yet. Preclinical studies, though, have provided strong evidence that targeting PTP1B could allow to reach different pathophysiological mechanisms at once. herefore, specific interventions or trials should be designed to modulate PTP1B activity in brain, since it is a promising strategy to decelerate or prevent neurodegeneration in aged individuals, among other neurological diseases. The present paper fails to include all neurological conditions in which PTP1B could have a role; instead, it focuses on those which have been related to metabolic alterations and neurodegenerative processes. Moreover, only preclinical data is discussed, since clinical studies on the potential of PTP1B inhibition for treating neurological diseases are still required., This work was supported by funds from the Spanish Ministerio de Ciencia e Innovación (SAF2017-84283‑R and PID2021-123462OB-I00 to AC and CA), the Generalitat de Catalunya (2014SGR‑525 to CA), CIBERNED (Grant CB06/05/2004 to AC) and AARG-NTF-22-924702, (MB). Jordi Olloquequi and Miren Ettcheto are Serra Húnter fellows.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases whose activity depends on association with a noncatalytic regulatory subunit called cyclin. The genes that encode CDKs were initially identified in screens for conditional mutants of Saccharomyces cerevisiae , which reversibly arrested at characteristic points in the cell cycle upon transfer to the restrictive conditions. Cyclins were initially named for their periodic accumulation and degradation (cycling) through the early cell cycles of fertilized sea urchin eggs. Their activation markedly changes during cell cycle progression. The fusion of these two lines of research identified a ubiquitous cell cycle engine responsible for coordinating cell growth, DNA replication, and mitosis in an orderly fashion required to ensure the viability of progeny cells. On the basis of their functions, CDKs can be classified into two groups: those involved in the control of the cell cycle and others responsible for transcriptional regulation.

et al., The objective of the EU funded integrated project “ACuteTox” is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABAA receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro–in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity., The project was financed by the EU-FP6 Grant (FP6-LIFESCIHEALTH-2004-512051, the Swedish Animal Welfare Agency, the Swedish Fund for Research without Animal Experiments, Spain’s Ministerio de Educación y Ciencia (SAF2005-01604, SAF2006-13092) and the Spanish PI061212 project from Ministry of Health.

Senescence-accelerated mice (SAM) are used as a model of aging and age-associated diseases. SAMP8 are prone strains that show shortened life span and deficits in learning and memory processes, while SAMR1 are strains of accelerated senescence-resistant, long-lived mice. Due to their abnormal APP (amyloid precursor protein) metabolism in brain, SAMP8 may be an Alzheimer-type model. Adenosine receptors are G-protein coupled receptors which are altered in brain from Alzheimer disease (AD) cases. The analysis of adenosine receptors in brain from young (21 days old) and middle-aged (180 days old) SAMP8 as compared with SAMR1 mice revealed differences between these strains associated with age. The age-related increase in mRNA coding A1 and A2B receptors observed in SAMR1 was absent in SAMP8. A1 receptors were significantly decreased with age in SAMR1, while no differences were observed in SAMP8. However, the levels of A1 receptors in young SAMP8 were even lower than those obtained in middle-aged SAMR1. In addition, A2A receptors were significantly increased only in aged SAMR1. A similar age-related decrease in A1 receptors level was also observed in brain from male Wistar rats. These results suggest different age-related effects on adenosine receptors in SAMR1 and SAMP8 strains. Since A1 receptors are mainly neuroprotective, their important loss in very young SAMP8 strain suggests the involvement of these receptors in the pathogenesis of neurodegenerative diseases associated with aging., This work was supported in part by the European Union through the Marie-Curie Research Training Network PRAIRIES (Contract MRTN-CT-2006-035810), by the Consejería de Educación y Ciencia (PCI08-0125), the Consejería de Sanidad (PI-2007/50 and G-2007-C/13) of the Junta de Comunidades de Castilla-La Mancha, the Ministerio de Ciencia e Innovación (BFU2008-00138 and SAF2006-13092) and the Instituto de Salud Carlos III (PI080400). We are grateful to the Autonomous Government of Catalonia for supporting research groups (2005/SGR00893) and to the TV3 Marathon(063230).

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3β (GSK-3β) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3β is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3β activity after 2 h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3β and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3β inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3β inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3β with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3β inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li+ and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3β. Therefore, there is a connection between cell cycle and GSK-3β activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal. © 2007 Elsevier Ltd. All rights reserved., This study was supported by grants SAF2005-01604, SAF-2005-05179 and SAF-2006-13092 from Ministerio de Educación y Ciencia, PI 041300, PI040376 (RT) and Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) from Instituto de Salud Carlos III. We also thanks to Generalitat de Catalunya 2005/SGR00893.

The biochemical pathways involved in neuronal cell death in Parkinson's disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 μM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinson's disease. © 2007 IBRO., This study was supported by grants SAF2005-01604, SAF2005-05179 and SAF2006-13092 from Ministerio de Educación y Ciencia, La Marató de TV3, PI 041300, PI040376 (R.T.) and Centros de Investigación Biomédica en Red (CIBER) from Instituto de Salud Carlos III. We thank the Generalitat de Catalunya 2005/SGR00893.

Publisher Summary The method used to measure changes in cytoplasmic oxidative stress and in mitochondrial membrane potential (MMP) in viable freshly dissociated cerebellar granule cells obtained from 9- to 11-day-old rat pups or in granule cerebellar cells from in vitro cultures is described. The same methodology (with little modification) is used to study other processes, such as changes in cytoplasmic calcium, cytoplasmic membrane potential, and apoptosis. Changes in oxidative stress and MMP can be detected using other fluorometric techniques, such as spectrofluorimetry or fluorescence microscopy. Similar to these methods, flow cytometry is specific and has a wide noise-to-signal ratio, but it also has other advantages: it is fast, sensitive, and can separate a particular cell type for further examination or purification. Cell suspensions can be obtained either from freshly dissected tissue or from cell cultures. Flow cytometry is a technique that has been used mainly in hematology and immunology. From the study of neuronal or glial markers to the purification and separation of particular cell types, flow cytometry has demonstrated its usefulness to neuroscience.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that was histopathologically characterized in the brain by the presence of extracellular senile plaques made of amyloid β peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated Tau protein. Over the years, AD has been classified in two subgroups: early onset or familial AD and late onset or sporadic AD. On the one hand, familial AD has been described to be the result of genetic mutations that cause, in some cases, for the overproduction of amyloid β. On the other, the cause of late onset or sporadic AD is still unclear even though several hypotheses have been proposed to explain the process of severe and progressive memory and cognitive loss. In the present review, some of the current hypotheses that try to explain the origin of late onset or sporadic AD have been summarized. Also, their potential implication in the development of new drugs for the presymptomatic treatment of late onset or sporadic AD has been considered.