A Reevaluation of Chitosan-Decorated Nanoparticles to Cross the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a sophisticated and very selective dynamic interface composed of endothelial cells expressing enzymes, transport systems, and receptors that regulate the passage of nutrients, ions, oxygen, and other essential molecules to the brain, regulating its homeostasis. Moreover, the BBB performs a vital function in protecting the brain from pathogens and other dangerous agents in the blood circulation. Despite its crucial role, this barrier represents a difficult obstacle for the treatment of brain diseases because many therapeutic agents cannot cross it. Thus, different strategies based on nanoparticles have been explored in recent years. Concerning this, chitosan-decorated nanoparticles have demonstrated enormous potential for drug delivery across the BBB and treatment of Alzheimer's disease, Parkinson's disease, gliomas, cerebral ischemia, and schizophrenia. Our main objective was to highlight the high potential of chitosan adsorption to improve the penetrability through the BBB of nanoformulations for diseases of CNS. Therefore, we describe the BBB structure and function, as well as the routes of chitosan for crossing it. Moreover, we define the methods of decoration of nanoparticles with chitosan and provide numerous examples of their potential utilization in a variety of brain diseases. Lastly, we discuss future directions, mentioning the need for extensive characterization of proposed nanoformulations and clinical trials for evaluation of their efficacy.

Coexistence of D3 R typical and atypical signaling in striatonigral neurons during dopaminergic denervation. Correlation with D3 nf expression changes.

Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals.

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

D2 autoreceptor switches CB2 receptor effects on [3 H]-dopamine release in the striatum.

CB2 receptors (CB2 R) are expressed in midbrain neurons. To evidence the control of dopamine release in dorsal striatum by CB2 R, we performed experiments of [3 H]-dopamine release in dorsal striatal slices. We found a paradoxical increase in K+ -induced [3 H]-dopamine release by CB2 R activation with GW 833972A and JWH 133 two selective agonist. To understand the mechanism involved, we tested for a role of the D2 autoreceptor in this effect; because in pallidal structures, the inhibitory effect of CB1 receptors (CB1 R) on GABA release is switched to a stimulatory effect by D2 receptors (D2 R). We found that the blockade of D2 autoreceptors with sulpiride prevented the stimulatory effect of CB2 R activation; in fact, under this condition, CB2 R decreased dopamine release, indicating the role of the D2 autoreceptor in the paradoxical increase. We also found that the effect occurs in nigrostriatal terminals, since lesions with 6-OH dopamine in the middle forebrain bundle prevented CB2 R effects on release. In addition, D2 -CB2 R interaction promoted cAMP accumulation, and the increase in [3 H]-dopamine release was prevented by PKA blockade. D2 -CB2 R coprecipitation and proximity ligation assay studies indicated a close interaction of receptors that could participate in the observed effects. Finally, intrastriatal injection of CB2 R agonist induced contralateral turning in amphetamine-treated rats, which was prevented by sulpiride, indicating the role of the interaction in motor behavior. Thus, these data indicate that the D2 autoreceptor switches, from inhibitory to stimulatory, the CB2 R effects on dopamine release, involving the cAMP â†’ PKA pathway in nigrostriatal terminals.

Nanotechnology As Potential Tool for siRNA Delivery in Parkinson's Disease.

BACKGROUND: The lack of an outright treatment for Parkinson's disease (PD) is a pivotal concern in medicine and has driven the search for novel alternatives for treating the disease. Among the proposed approaches, small interfering RNA (siRNA)-based therapy is attracting significant attention as a potential method for the treatment of PD; however, siRNAs delivery possesses potential drawbacks, such as reduced stability in blood circulation and low capacity for reaching the target site. OBJECTIVE: This review aims to explore siRNA-based approaches to PD and the latest advances for designing nanoparticles that effectively target siRNAs to the action site and that protect these against degradation in blood circulation. RESULTS: siRNA-based approaches provide an interesting option for designing new strategies for treating PD through the silencing of genes, whose abnormal expressions contribute to the pathophysiology of the disease; however, siRNA delivery to the brain is a key issue that remains unsolved to date. Current research efforts are focused on designing vectors that effectively transport and protect siRNAs. In this regard, nanoparticles are being developed as carriers for siRNAs with controlled delivery efficiency and low toxicity profiles, and these represent an alternative to common vectors. CONCLUSION: Identification of putative gene targets for siRNA therapy of PD has set the pace for researching non-viral vectors; however, the technological aspects for tackling the challenge that siRNAs targeting to the brain represents are essentials. In this respect, the formulation of siRNAs in nanoparticles would avoid harmful side effects, such as immunogenic and oncogenic drawbacks.

Eag1 channels as potential early-stage biomarkers of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide. HCC is usually asymptomatic at potential curative stages, and it has very poor prognosis if detected later. Thus, the identification of early biomarkers and novel therapies is essential to improve HCC patient survival. Ion channels have been proposed as potential tumor markers and therapeutic targets for several cancers including HCC. Especially, the ether à-go-go-1 (Eag1) voltage-gated potassium channel has been suggested as an early marker for HCC. Eag1 is overexpressed during HCC development from the cirrhotic and the preneoplastic lesions preceding HCC in a rat model. The channel is also overexpressed in human HCC. Astemizole has gained great interest as a potential anticancer drug because it targets several proteins involved in cancer including Eag1. Actually, in vivo studies have shown that astemizole may have clinical utility for HCC prevention and treatment. Here, we will review first some general aspects of HCC including the current biomarkers and therapies, and then we will focus on Eag1 channels as promising tools in the early diagnosis of HCC.

Dopaminergic denervation switches dopamine D3 receptor signaling and disrupts its Ca(2+) dependent modulation by CaMKII and calmodulin in striatonigral projections of the rat.

In striatonigral projections activation of dopamine D3 receptors (D3Rs) potentiates the stimulation of GABA release and cAMP production caused by activation of dopamine D1 receptors (D1Rs). Cytoplasmic [Ca(2+)] in the terminals controls this response by modulating CaMKII, an enzyme that depresses D3R action. To examine the effects of dopamine deprivation on D3R signaling we investigated their function in striatonigral terminals of hemiparkinsonian rats. Denervation switched the signaling cascade initiated by D3R activation. In the non-lesioned side activation of D3R potentiated the stimulatory effects of D1R activation on cAMP production and K(+)-depolarization induced [(3)H] GABA release. In contrast, in the denervated side the stimulatory effects of both D1R activation and forskolin administration were blocked by D3R activation. In non-lesioned slices, D3R responses were inhibited by the activation of CaMKII produced by K(+)-depolarization (via increased Ca(2+) entry). The CaMKII-induced inhibition was blocked by the selective inhibitor KN-62. In denervated tissues the response to D3R stimulation was not modified either by K(+) depolarization or by blocking CaMKII with KN-62. Immunoblotting studies showed that depolarization-induced CaMKII binding to the D3 receptor and CaMKII phosphorylation were suppressed in denervated tissues. We also determined calmodulin expression with PCR and immunoblot techniques. Both techniques showed that calmodulin expression was depressed in the lesioned side. In sum, our studies show that dopaminergic denervation switches the D3R signaling cascade and depresses CaMKII signaling through a process that appears to involve reduced calmodulin levels. Since calmodulin is a major cytoplasmic Ca(2+) buffer our findings suggest that abnormal Ca(2+) buffering may be an important component of the abnormalities observed during dopaminergic denervation.

Presynaptic CaMKIIα modulates dopamine D3 receptor activation in striatonigral terminals of the rat brain in a Ca²âº dependent manner.

CaMKIIα is expressed at high density in the nucleus accumbens where it binds to postsynaptic D3 receptors inhibiting their effects. In striatonigral projections, activation of presynaptic D3 receptors potentiates D1 receptor-induced stimulation of cAMP production and GABA release. In this study we examined whether the presynaptic effects of D3 receptor stimulation in the substantia nigra reticulata (SNr) are modulated by Ca²âº activation of CaMKIIα. In SNr synaptosomes two procedures that increase cytoplasmic Ca²âº, ionomycin and K⁺-depolarization, blocked the additional stimulation of cAMP accumulation produced by coactivating D3 and D1 dopamine receptors. The selective CaMKIIα inhibitor KN-62 reversed the blockade produced by ionomycin and K⁺-depolarization. Incubation in either Ca²-free solutions or with the selective Ca²âº blocker nifedipine, also reversed the blocking effects of K⁺-depolarization. Immunoblot studies showed that K⁺-depolarization increased CaMKIIα phosphorylation in a KN-62 sensitive manner and promoted CaMKIIα binding to D3 receptors. In K⁺-depolarized tissues, D3 receptors potentiated D1 receptor-induced stimulation of [³H]GABA release only when CaMKIIα was blocked with KN-62. In the presence of this inhibitor, the selective D3 agonist PD 128,907 reduced the ED50 for the D1 agonist SKF 38393 from 56 to 4 nM. KN-62 also enhanced the effects of dopamine on depolarization induced [³H]GABA release. KN-62 changed ED50 for dopamine from 584 to 56 nM. KN-62 did not affect D1 and D4 receptor responses. These experiments show that in striatonigral projections, CaMKIIα inhibits the action of D3 receptors in a Ca²âº dependent manner blocking their modulatory effects on GABA release. These findings suggest a mechanism through which the frequency of action potential discharge in presynaptic terminals regulates dopamine effects.

D3 dopamine receptors interact with dopamine D1 but not D4 receptors in the GABAergic terminals of the SNr of the rat.

The firing rate of substantia nigra reticulata (SNr) neurons is modulated by GABA release from striatonigral and pallidonigral projections. This release is, in turn, modulated by dopamine acting on dopamine D1 receptors at striatonigral terminals and D4 receptors at pallidonigral terminals. In addition, striatal neurons that express D1 receptors also express D3 receptors. In this study we analyzed the possible significance of D3 and D1 receptor colocalization in striatonigral projections. We found that these receptors coprecipitate in SNr synaptosomes suggesting their close association in this structure. D1 agonist SKF 38393 administered alone increased mIPSC frequency in SNr slices and cAMP production in SNr synaptosomes, however, the selective D3 agonist PD 128,907 increased mIPSC frequency and cAMP production only when D1 receptors were concurrently stimulated. The D1 antagonist SCH 23390 blocked completely the effects of the concurrent administration of these agonists while the selective D3 antagonist GR 103691 blocked only the potentiating effects of PD 128,907. These findings further indicate that D1 and D3 receptors are localized in the same structure. The D4 agonist PD 168,077 decreased mIPSCs frequency without changing amplitude, an effect that was blocked by the selective D4 antagonist L 745,870. The effects of D4 receptor stimulation disappeared after lesioning the globus pallidus. D3 agonist PD 128,907 did not reduce mIPSC frequency even in neurons that responded to D4 agonist. In sum, activation of D3 receptors in SNr potentiates the stimulation of transmitter release and cAMP production caused by D1 receptor activation of striatonigral projections while it is without effects in terminals, probably of pallidal origin, that are inhibited by activation of D4 receptors.