Drug-loaded polymer-coated silver nanoparticles for lung cancer theranostics.

Lung cancer is the leading cause of death in cancer across the globe. To minimize these deaths, the replacement of traditional chemotherapy with novel strategies is significant. We have developed a nanotheranostic approach using silver nanoparticles for imaging and treatment. Silver nanoparticles (AgNPs) are fabricated by chemical reduction method. The formulation of AgNPs was confirmed by different characterization techniques like stability test, UV-Visible spectroscopy, Confocal Raman Spectroscopy, and Energy-Dispersive X-ray analysis. Further, AgNPs are coated with poly lactic-co-glycolic acid (PLGA) and then loaded with paclitaxel (Pac). Then the drug-loaded PLGA-coated AgNPs were characterized for size and zeta potential measurement by zetasizer, surface morphology study by atomic force microscopy, Fourier transform infrared spectroscopy, and release kinetics study. The imaging and anticancer properties of these nanoformulations are investigated using lung cancer cell lines. The results proved that the particles are in the nanometer range with smooth surface morphology. Moreover, the drug-loaded NPs showed a sustained release of the drug for a longer period of time. Further the formulations showed imaging property with greater anticancer efficacy. Thus, the results suggest the effective use of these nanoformulation in both lung cancer imaging and treatment using a simple and efficient approach.

Chrysin-Loaded Chitosan Nanoparticle-Mediated Neuroprotection in Aß1-42-Induced Neurodegenerative Conditions in Zebrafish.

Amyloid ß plaques and neurofibrillary tangles are the characteristic features of Alzheimer's disease (AD). Plaques of amyloid ß play a pivotal role in affecting cognitive functions and memory. Alzheimer's disease is a progressive neurodegenerative disease and is one of the leading causes of dementia worldwide. Several treatment strategies focusing on the amyloid cascade have been implemented to treat AD. The blood-brain barrier (BBB) poses the main obstructive barrier by refraining drugs from penetrating the brain. Nanotechnology is a promising research field for brain drug delivery using nanosized particles. Zebrafish is emerging as a model of interest to elaborate on brain targeting and nanotechnology-based therapeutics for neurodegenerative diseases. In the current study, we have synthesized and characterized chrysin-loaded chitosan nanoparticles (Chr-Chi NPs) and evaluated them for neuroprotection against amyloid-ß-induced toxicity. We find that treatment with Chr-Chi NPs helps to retain memory, cognition, and synaptic connections, which are otherwise compromised due to Aß1-42 toxicity. The NPs further help in reducing aggregates of amyloid ß, thus decreasing neuronal death and generation of reactive oxygen species (ROS). Taken together, our study brings to light a novel strategy for treating AD by a combined action on the neurons and amyloid aggregates mediated by chrysin and chitosan, respectively. Chr-Chi NPs, therefore, have the potential to provide a beneficial combinatorial treatment strategy for AD.

BH3-only proteins Puma and Beclin1 regulate autophagic death in neurons in response to Amyloid-ß.

Alzheimer's disease (AD) is characterized by accumulation of senile amyloid-ß (Aß) plaques and hyperphosphorylated tau tangles causing progressive loss of synapse and neuronal death. Out of the various neuron death modalities, autophagy and apoptosis are reported to be the major death paradigms in AD. However, how these two processes lead to neuronal loss is still inconspicuous. Here we report that under Aß toxicity, aberrant autophagy is induced with inefficient autophagic flux in neurons. Simultaneous activation of both autophagy and apoptosis are seen in primary cortical neurons as well as in transgenic mice brains. We found that induction of autophagy by rapamycin is detrimental for neurons; whereas downregulation of Beclin1, an important autophagy inducing protein, provides significant protection in Aß treated neuronal cells by blocking cytochrome-c release from the mitochondria. We further report that downregulation of Puma, a BH3-only pro-apoptotic protein, inhibits the induction of aberrant autophagy and also ameliorates the autophagy flux under the influence of Aß. Notably, stereotactic administration of shRNAs against Puma and Beclin1 in adult Aß-infused rat brains inhibits both apoptotic and autophagic pathways. The regulation of both of the death processes is brought about by the direct interaction between Puma and Beclin1 upon Aß treatment. We conclude that both Beclin1 and Puma play essential roles in the neuronal death caused by the induction of aberrant autophagy in AD and targeting their interaction could be vital to understand the crosstalk of autophagy and apoptosis as well as to develop a potential therapeutic strategy in AD.

Zebrafish: A Promising Real-Time Model System for Nanotechnology-Mediated Neurospecific Drug Delivery.

Delivering drugs to the brain has always remained a challenge for the research community and physicians. The blood-brain barrier (BBB) acts as a major hurdle for delivering drugs to specific parts of the brain and the central nervous system. It is physiologically comprised of complex network of capillaries to protect the brain from any invasive agents or foreign particles. Therefore, there is an absolute need for understanding of the BBB for successful therapeutic interventions. Recent research indicates the strong emergence of zebrafish as a model for assessing the permeability of the BBB, which is highly conserved in its structure and function between the zebrafish and mammals. The zebrafish model system offers a plethora of advantages including easy maintenance, high fecundity and transparency of embryos and larvae. Therefore, it has the potential to be developed as a model for analysing and elucidating the permeability of BBB to novel permeation technologies with neurospecificity. Nanotechnology has now become a focus area within the industrial and research community for delivering drugs to the brain. Nanoparticles are being developed with increased efficiency and accuracy for overcoming the BBB and delivering neurospecific drugs to the brain. The zebrafish stands as an excellent model system to assess nanoparticle biocompatibility and toxicity. Hence, the zebrafish model is indispensable for the discovery or development of novel technologies for neurospecific drug delivery and potential therapies for brain diseases.

Apoptosis, Autophagy, Necrosis and Their Multi Galore Crosstalk in Neurodegeneration.

The progression of neurodegenerative disorders is mainly characterized by immense neuron loss and death of glial cells. The mechanisms which are active and regulate neuronal cell death are namely necrosis, necroptosis, autophagy and apoptosis. These death paradigms are governed by a set of molecular determinants that are pivotal in their performance and also exhibit remarkable overlapping functional pathways. A large number of such molecules have been demonstrated to be involved in the switching of death paradigms in various neurodegenerative diseases. In this review, we discuss various molecules and the concurrent crosstalk mediated by them. According to our present knowledge and research in neurodegeneration, molecules like Atg1, Beclin1, LC3, p53, TRB3, RIPK1 play switching roles toggling from one death mechanism to another. In addition, the review also focuses on the exorbitant number of newer molecules with the potential to cross communicate between death pathways and create a complex cell death scenario. This review highlights recent studies on the inter-dependent regulation of cell death paradigms in neurodegeneration, mediated by cross-communication between pathways. This will help in identifying potential targets for therapeutic intervention in neurodegenerative diseases.

Human placental laminin: Role in neuronal differentiation, cell adhesion and proliferation.

Human placental extract has wound healing potential. Immuno-blots revealed presence of laminin in placental extract (70 +/- 0.257 µg/ml; n=3). It was purified using immuno-affinity chromatography. SDS-PAGE and SEHPLC indicated a188 kDa protein with some small peptides. Since placental laminin existed in its truncated form, its roles in cellular migration, differentiation and wound healing were verified. Induction of cellular migration and motility in rat fibroblasts were enhanced by placental laminin as observed from scratch wound assay. Promotion of neuronal differentiation of PC12 cells by placental laminin was observed by phase contrast microscopy. Confocal images showed presence of laminin on the cell surface and along the axonal processes. Significant interaction between integrin receptors and laminin responsible for cellular differentiation was demonstrated from co-localization experiments. Union between integrin receptor and its synthetic antagonist revealed retarded pattern of neurite outgrowth in laminin treated cells. Animal model studies revealed faster wound healing in the presence of placental laminin. Induction of re-epithelialization and angiogenesis in wound area by cellular proliferation and adhesion were observed. The cytokine levels showed an initial rise and gradual fall over the duration of wound healing on application of the fragmented laminin. Thus, roles of placental laminin in neuronal differentiation and wound healing were indicated.

The energetic brain - A review from students to students.

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.

Exogenous human beta amyloid peptide interferes osteogenesis through Sox9a in embryonic zebrafish.

The two major hallmarks of Alzheimer's disease (AD) are beta-amyloid plaques and neurofibrillary tangles. Amyloid peptide aggregations in the brain cause loss of synaptic connections and subsequent neurotoxicity leading to neurodegeneration and memory deficits. However, the physiological effects of beta-amyloid on early embryonic development still remain unclear. Administration of human beta-amyloid peptide (1-42) through cerebrospinal ventricular injection was carried out at 24 hpf (hours post fertilization) and it was uptaken into the cellular layers of the early ventricular development without any plaque aggregation. Whole-mount Immunostaining of zebrafish embryos injected with the beta-amyloid at 60 hpf revealed the delay in Sox9a expression. Decreased level of cartilage to bone transformation rate in 15 dpf (days post fertilization) zebrafish was observed by differential staining. These results suggest the possible existence of a genetic relationship between extrinsic amyloid peptide and Sox9a expression. Thus, our results demonstrated that the human beta-amyloid influences bone development through Sox9a expression during osteogenesis in zebrafish.

Multifunctional transcriptional coactivator PC4 is a global co-regulator of p53-dependent stress response and gene regulation.

Human positive coactivator 4 (PC4), a multifunctional chromatin-associated protein, is known to directly interact with p53 and modulate expressions of a few p53-dependent genes. However, the role of PC4 in p53's myriad of other regulatory functions is not known. The p53-PC4 interaction was selectively perturbed by a small peptide which led to abrogation of genotoxic stress-induced up-regulation of many p53-dependent genes and reduction of apoptosis in A549 cells. Over-expression of a PC4 point mutant, incapable of binding p53, recapitulated many of the effects of the peptide. Global gene expression profiling in A549 cells, upon peptide treatment, revealed PC4's involvement in the regulation of many p53-dependent pathways, including the Hippo pathway. Introduction of the peptide in neuronal cells significantly reduced its amyloid-ß-induced death. Thus, PC4 emerges as a global co-regulator of p53 and a therapeutic target against pathogeneses where the p53-dependent cell death process plays a crucial role.

Zebrafish: an emerging real-time model system to study Alzheimer's disease and neurospecific drug discovery.

Zebrafish (Danio rerio) is emerging as an increasingly successful model for translational research on human neurological disorders. In this review, we appraise the high degree of neurological and behavioural resemblance of zebrafish with humans. It is highly validated as a powerful vertebrate model for investigating human neurodegenerative diseases. The neuroanatomic and neurochemical pathways of zebrafish brain exhibit a profound resemblance with the human brain. Physiological, emotional and social behavioural pattern similarities between them have also been well established. Interestingly, zebrafish models have been used successfully to simulate the pathology of Alzheimer's disease (AD) as well as Tauopathy. Their relatively simple nervous system and the optical transparency of the embryos permit real-time neurological imaging. Here, we further elaborate on the use of recent real-time imaging techniques to obtain vital insights into the neurodegeneration that occurs in AD. Zebrafish is adeptly suitable for Ca2+ imaging, which provides a better understanding of neuronal activity and axonal dystrophy in a non-invasive manner. Three-dimensional imaging in zebrafish is a rapidly evolving technique, which allows the visualisation of the whole organism for an elaborate in vivo functional and neurophysiological analysis in disease condition. Suitability to high-throughput screening and similarity with humans makes zebrafish an excellent model for screening neurospecific compounds. Thus, the zebrafish model can be pivotal in bridging the gap from the bench to the bedside. This fish is becoming an increasingly successful model to understand AD with further scope for investigation in neurodevelopment and neurodegeneration, which promises exciting research opportunities in the future.

The pro-apoptotic protein Bmf co-operates with Bim and Puma in neuron death induced by ß-amyloid or NGF deprivation.

The pro-apoptotic Bcl-2 homology 3 domain only (BH3-only) proteins are central regulators of cell death in various physiological and pathological conditions, including Alzheimer's disease (AD). Bcl-2 modifying factor (Bmf) is one such BH3-only protein that is implicated in various death paradigms such as anoikis, seizures, cancer and autoimmunity. It also co-operates with other BH3-only proteins such as Bim in various death paradigms. However, its role in neurodegeneration is under-investigated. Here, we report for the first time the essential role of Bmf and its co-operativity with direct activator BH3-only proteins Bim and Puma in neuron death induced by beta-amyloid (Aß) toxicity or NGF deprivation. Oligomeric Aß is main pathologic species in AD and NGF deprivation is relevant for both developmental as well as pathologic neuron death. We find that Bmf over-expression causes cell death and Bmf knockdown protects neurons against death evoked by Aß or NGF deprivation. We also find that Bmf co-operates with other important BH3-only proteins such as Bim and Puma in neuron death induced by Aß or NGF deprivation. Simultaneous knocking down of these molecules by their respective shRNAs provide enhanced protection against Aß. Taken together, our results elucidate the essential role of Bmf and its co-operative effects with already known neuron death inducers, Bim and Puma, in neuron death evoked by Aß treatment or NGF deprivation.

Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-ß-induced Neuronal Death.

Amyloid-ß (Aß)-induced neuron death is considered central to the pathogenesis of Alzheimer's disease (AD). Among several death modalities, autophagy and apoptosis play important roles in Aß-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon Aß treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro Increased Trib3 levels inhibited the activity of the kinase Akt by interacting with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to Aß insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. We found that Trib3 indirectly activates unc-51-like autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from Aß insult. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt, and FoxO1 in Aß-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD.

Novel hexapeptide interacts with tubulin and microtubules, inhibits Aß fibrillation, and shows significant neuroprotection.

Herein, we report a novel hexapeptide, derived from activity dependent neuroprotective protein (ADNP), that spontaneously self-assembles to form antiparallel ß-sheet structure and produces nanovesicles under physiological conditions. This peptide not only strongly binds with ß-tubulin in the taxol binding site but also binds with the microtubule lattice in vitro as well as in intracellular microtubule networks. Interestingly, it shows inhibition of amyloid fibril formation upon co-incubation with Aß peptide following an interesting mechanistic pathway and excellent neuroprotection in PC12 cells treated with anti-nerve growth factor (NGF). The potential of this hexapeptide opens up a new paradigm in design and development of novel therapeutics for AD.