Inducible Knockout of 14-3-3ß Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG.

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3ß, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3ß has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3ß promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3ß, a CRISPR/Cas9 knockout model of 14-3-3ß has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3ß results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3ß. Using this model, we show that loss of 14-3-3ß significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Curcumin Loaded Dendrimers Specifically Reduce Viability of Glioblastoma Cell Lines.

Glioblastoma (GB) is a deadly and aggressive cancer of the CNS. Even with extensive resection and chemoradiotherapy, patient survival is still only 15 months. To maintain growth and proliferation, cancer cells require a high oxidative state. Curcumin, a well-known anti-inflammatory antioxidant, is a potential candidate for treatment of GB. To facilitate efficient delivery of therapeutic doses of curcumin into cells, we encapsulated the drug in surface-modified polyamidoamine (PAMAM) dendrimers. We studied the in vitro effectiveness of a traditional PAMAM dendrimer (100% amine surface, G4 NH2), surface-modified dendrimer (10% amine and 90% hydroxyl-G4 90/10-Cys), and curcumin (Cur)-encapsulated dendrimer (G4 90/10-Cys-Cur) on three species of glioblastoma cell lines: mouse-GL261, rat-F98, and human-U87. Using an MTT assay for cell viability, we found that G4 90/10-Cys-Cur reduced viability of all three glioblastoma cell lines compared to non-cancerous control cells. Under similar conditions, unencapsulated curcumin was not effective, while the non-modified dendrimer (G4 NH2) caused significant death of both cancerous and normal cells. By harnessing and optimizing the components of PAMAM dendrimers, we are providing a promising new route for delivering cancer therapeutics. Our results with curcumin suggest that antioxidants are good candidates for treating glioblastoma.

Solid Lipid Curcumin Particles Protect Medium Spiny Neuronal Morphology, and Reduce Learning and Memory Deficits in the YAC128 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by massive neuronal degeneration in the striatum. In this study, we utilized solid lipid curcumin particles (SLCPs) and solid lipid particles (SLPs) to test their efficacy in reducing deficits in YAC128 HD mice. Eleven-month-old YAC128 male and female mice were treated orally with SLCPs (100 mg/kg) or equivalent volumes of SLPs or vehicle (phosphate-buffered saline) every other day for eight weeks. Learning and memory performance was assessed using an active-avoidance task on week eight. The mice were euthanized, and their brains were processed using Golgi-Cox staining to study the morphology of medium spiny neurons (MSNs) and Western blots to quantify amounts of DARPP-32, brain-derived neurotrophic factor (BDNF), TrkB, synaptophysin, and PSD-95. We found that both SLCPs and SLPs improved learning and memory in HD mice, as measured by the active avoidance task. We also found that SLCP and SLP treatments preserved MSNs arborization and spinal density and modulated synaptic proteins. Our study shows that SLCPs, as well as the lipid particles, can have therapeutic effects in old YAC128 HD mice in terms of recovering from HD brain pathology and cognitive deficits.

A Mixed-Surface Polyamidoamine Dendrimer for In Vitro and In Vivo Delivery of Large Plasmids.

Drug delivery to the brain is highly hindered by the presence of the blood-brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.

PAMAM Dendrimer Nanomolecules Utilized as Drug Delivery Systems for Potential Treatment of Glioblastoma: A Systematic Review.

Glioblastoma (GB) is a grade IV astrocytoma that maintains a poor prognosis with respect to current treatment options. Despite major advancements in the fields of surgery and chemoradiotherapy over the last few decades, the life expectancy for someone with glioblastoma remains virtually unchanged and warrants a new approach for treatment. Poly(amidoamine) (PAMAM) dendrimers are a type of nanomolecule that ranges in size (between 1 and 100 nm) and shape and can offer a new viable solution for the treatment of intracranial tumors, including glioblastoma. Their ability to deliver a variety of therapeutic cargo and penetrate the blood-brain barrier (BBB), while preserving low cytotoxicity, make them a favorable candidate for further investigation into the treatment of glioblastoma. Here, we present a systematic review of the current advancements in PAMAM dendrimer technology, including the wide spectrum of dendrimer generations formulated, surface modifications, core modifications, and conjugations developed thus far to enhance tumor specificity and tumor penetration for treatment of glioblastoma. Furthermore, we highlight the extensive variety of therapeutics capable of delivery by PAMAM dendrimers for the treatment of glioblastoma, including cytokines, peptides, drugs, siRNAs, miRNAs, and organic polyphenols. While there have been prolific results stemming from aggressive research into the field of dendrimer technology, there remains a nearly inexhaustible amount of questions that remain unanswered. Nevertheless, this technology is rapidly developing and is nearing the cusp of use for aggressive tumor treatment. To that end, we further highlight future prospects in focus as researchers continue developing more optimal vehicles for the delivery of therapeutic cargo.

Possible roles of epigenetics in stem cell therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease with loss of dopaminergic neurons. PD has genetic and epigenetic influences that determine specific changes in the brain. Epigenetic changes result in defective methylation of genes leading to differential gene-expression causing PD. This review provides an overview of stem cell transplantations as potential therapies for PD, with a focus on the epigenetic changes, prior or following transplantation. To date, no reports have addressed epigenetic alterations following stem cell transplantation into the PD brain. Given the potential for affecting the efficacy of stem cell therapy, increased attention needs to be given to the epigenetic processes that occur during stem cell culture and transplantation to maximize the therapeutic potential of stem cells to PD.

Prion-like mechanisms in neurodegenerative disease: Implications for Huntington's disease therapy.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansions in the huntingtin gene resulting in the synthesis of a misfolded form of the huntingtin protein (mHTT) which is toxic. The current treatments for HD are only palliative. Some of the potential therapies for HD include gene therapy (using antisense oligonucleotides and clustered regularly interspaced short palindromic repeats-Cas9 system) and stem-cell-based therapies. Various types of stem cell transplants, such as mesenchymal stem cells, neural stem cells, and reprogrammed stem cells, have the potential to either replace the lost neurons or support the existing neurons by releasing trophic factors. Most of the transplants are xenografts and allografts; however, recent reports on HD patients who received grafts suggest that the mHTT aggregates are transferred from the host neurons to the grafted cells as well as to the surrounding areas of the graft by a "prion-like" mechanism. This observation seems to be true for autotransplantation paradigms, as well. This article reviews the different types of stem cells that have been transplanted into HD patients and their therapeutic efficacy, focusing on the transfer of mHTT from the host cells to the graft. Autotransplants of reprogramed stem cells in HD patients are a promising therapeutic option. However, this needs further attention to ensure a better understanding of the transfer of mHTT aggregates following transplantation of the gene-corrected cells back into the patient.

Surface-Modified G4 PAMAM Dendrimers Cross the Blood-Brain Barrier Following Multiple Tail-Vein Injections in C57BL/6J Mice.

Intracranial injections are currently used to deliver drugs into the brain, as most drugs cannot cross the blood-brain barrier (BBB) following systemic injections. Moreover, multiple dosing is difficult with invasive techniques. Therefore, viable systemic techniques are necessary to facilitate treatment paradigms that require multiple dosing of therapeutics across the BBB. In this study, we show that mixed-surface fourth-generation poly(amidoamine) (PAMAM) dendrimers containing predominantly biocompatible hydroxyl groups and a few amine groups are taken up by cultured primary cortical neurons derived from mouse embryo. We also show that these dendrimers cross the BBB following their administration to healthy mice in multiple doses via tail-vein injections and are taken up by neurons and the glial cells as evidenced by appropriate staining methods. Besides the brain, the dendrimers were found mostly in the kidneys compared to other peripheral organs, such as liver, lungs, and spleen, implying that they may be readily excreted, thereby preventing potential toxic accumulation in the body. Our findings provide a proof-of-concept that appropriate surface modifications of dendrimers provide safe, biocompatible nanomaterial with the potential to deliver therapeutic cargo across the BBB into the brain via multiple tail-vein injections.

A novel approach to label bone marrow-derived mesenchymal stem cells with mixed-surface PAMAM dendrimers.

BACKGROUND: Transplantation of mesenchymal stem cells has created enormous opportunities as a potential treatment for various diseases including neurodegenerative diseases. Given current techniques, such as Hoechst labeling, have safety and leakage issues, our study focused, as a proof-of-concept, on a new dendrimer-based technique for labeling these stem cells to ensure their efficacy and safety following transplantation into the brain of a healthy mice. METHODS AND RESULTS: The bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled using polyaminoamine (PAMAM) dendrimers following which their stemness based on their proliferation and differentiation ability were analyzed by gold standard methods. These labeled BM-MSCs were transplanted into the striatum of C57BL/6J mice and were tracked using in vivo imaging system (IVIS) and analyzed using tissue imaging, 2 weeks after transplantation. Our results showed that the dendrimer-labeled BM-MSCs were able to successfully maintain their stemness and were tracked in vivo following transplantation. Unlike Hoechst, we did not find the dendrimers to be leaking out of the cells and were very specific to the cells that up took the dendrimers. Moreover, no adverse events were found in the transplanted animals proving that this is a safer method. CONCLUSIONS: Labeling BM-MSCs using fluorescently tagged PAMAM dendrimers can be used as a potentially safe and efficient method for labeling cells, particularly stem cells, in vitro and in vivo following transplantation in rodents.

Current Therapeutic Strategies for Glioblastoma.

Glioblastomas (GB) are grade 4 brain tumors, one of the most aggressive forms of brain cancer found in humans. Although current treatments for GB are largely ineffective, new alternate approaches, beyond standard chemo- or radiation therapies have shown promising results in both pre-clinical and clinical settings. Some of these approaches include stem cell therapy, new pharmaceuticals and nutraceuticals, and the use of tumor specific peptides or GB-targeted antibodies and immune-based therapies. A common limitation in the efficacy of these treatments is the inability of these therapeutic agents to readily cross the blood-brain barrier (BBB) to reach the tumor site. Therefore, many strategies are being developed to achieve targeted delivery of drugs across the BBB so that they can kill the cancer cells, while sparing healthy tissue. One of the most promising new approaches involves the use of nanoparticles that can carry therapeutic drugs and genes across the BBB and home in on the GB tumor site.

Use of Polyamidoamine Dendrimers in Brain Diseases.

Polyamidoamine (PAMAM) dendrimers are one of the smallest and most precise nanomolecules available today, which have promising applications for the treatment of brain diseases. Each aspect of the dendrimer (core, size or generation, size of cavities, and surface functional groups) can be precisely modulated to yield a variety of nanocarriers for delivery of drugs and genes to brain cells in vitro or in vivo. Two of the most important criteria to consider when using PAMAM dendrimers for neuroscience applications is their safety profile and their potential to be prepared in a reproducible manner. Based on these criteria, features of PAMAM dendrimers are described to help the neuroscience researcher to judiciously choose the right type of dendrimer and the appropriate method for loading the drug to form a safe and effective delivery system to the brain.

Zika virus induces astrocyte differentiation in neural stem cells.

Zika virus (ZIKV) is a rapidly emerging flavivirus that has been associated with a number of congenital neurological manifestations. Here, we show that ZIKV replicated efficiently in mouse neural stem cells (mNSCs). ZIKV infection caused a cytopathic effect without affecting cell viability, yet led to a significant decrease in the number of proteins secreted into mNSC supernatants. A gene expression array of neural stem cell progenitor and differentiation markers suggested that infection reduced the number of neuronal and oligodendrocyte progenitors while increasing the number of astrocyte progenitors. Infection in astrocytes increased transcription of key genes involved in the antiviral response. These data provide molecular and cellular evidence that ZIKV significantly alters neural development in the vertebrate host and that astrocyte differentiation may be a protective response that limits neuropathogenesis.

Induced Pluripotent Stem Cell-Derived Neural Stem Cell Transplantations Reduced Behavioral Deficits and Ameliorated Neuropathological Changes in YAC128 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by neuronal loss and motor dysfunction. Although there is no effective treatment, stem cell transplantation offers a promising therapeutic strategy, but the safety and efficacy of this approach needs to be optimized. The purpose of this study was to test the potential of intra-striatal transplantation of induced pluripotent stem cell-derived neural stem cells (iPS-NSCs) for treating HD. For this purpose, we developed mouse adenovirus-generated iPSCs, differentiated them into neural stem cells in vitro, labeled them with Hoechst, and transplanted them bilaterally into striata of 10-month old wild type (WT) and HD YAC128 mice. We assessed the efficiency of these transplanted iPS-NSCs to reduce motor deficits in YAC128 mice by testing them on an accelerating rotarod task at 1 day prior to transplantation, and then weekly for 10 weeks. Our results showed an amelioration of locomotor deficits in YAC128 mice that received iPS-NSC transplantations. Following testing, the mice were sacrificed, and their brains were analyzed using immunohistochemistry and Western blot (WB). The results from our histological examinations revealed no signs of tumors and evidence that many iPS-NSCs survived and differentiated into region-specific neurons (medium spiny neurons) in both WT and HD mice, as confirmed by co-labeling of Hoechst-labeled transplanted cells with NeuN and DARPP-32. Also, counts of Hoechst-labeled cells revealed that a higher proportion were co-labeled with DARPP-32 and NeuN in HD-, compared to WT- mice, suggesting a dissimilar differentiation pattern in HD mice. Whereas significant decreases were found in counts of NeuN- and DARPP-32-labeled cells, and for neuronal density measures in striata of HD vehicle controls, such decrements were not observed in the iPS-NSCs-transplanted-HD mice. WB analysis showed increase of BDNF and TrkB levels in striata of transplanted HD mice compared to HD vehicle controls. Collectively, our data suggest that iPS-NSCs may provide an effective option for neuronal replacement therapy in HD.

PAMAM Dendrimers Cross the Blood-Brain Barrier When Administered through the Carotid Artery in C57BL/6J Mice.

Drug delivery into the central nervous system (CNS) is challenging due to the blood-brain barrier (BBB) and drug delivery into the brain overcoming the BBB can be achieved using nanoparticles such as dendrimers. The conventional cationic dendrimers used are highly toxic. Therefore, the present study investigates the role of novel mixed surface dendrimers, which have potentially less toxicity and can cross the BBB when administered through the carotid artery in mice. In vitro experiments investigated the uptake of amine dendrimers (G1-NH2 and G4-NH2) and novel dendrimers (G1-90/10 and G4-90/10) by primary cortical cultures. In vivo experiments involved transplantation of G4-90/10 into mice through (1) invasive intracranial injections into the striatum; and (2) less invasive carotid injections. The animals were sacrificed 24-h and 1-week post-transplantations and their brains were analyzed. In vivo experiments proved that the G4-90/10 can cross the BBB when injected through the carotid artery and localize within neurons and glial cells. The dendrimers were found to migrate through the corpus callosum 1-week post intracranial injection. Immunohistochemistry showed that the migrating cells are the dendrimer-infected glial cells. Overall, our results suggest that poly-amidoamine (PAMAM) dendrimers may be used as a minimally invasive means to deliver biomolecules for treating neurological diseases or disorders.

Role of Epigenetics in Stem Cell Proliferation and Differentiation: Implications for Treating Neurodegenerative Diseases.

The main objectives of this review are to survey the current literature on the role of epigenetics in determining the fate of stem cells and to assess how this information can be used to enhance the treatment strategies for some neurodegenerative disorders, like Huntington's disease, Parkinson's disease and Alzheimer's disease. Some of these epigenetic mechanisms include DNA methylation and histone modifications, which have a direct impact on the way that genes are expressed in stem cells and how they drive these cells into a mature lineage. Understanding how the stem cells are behaving and giving rise to mature cells can be used to inform researchers on effective ways to design stem cell-based treatments. In this review article, the way in which the basic understanding of how manipulating this process can be utilized to treat certain neurological diseases will be presented. Different genetic factors and their epigenetic changes during reprogramming of stem cells into induced pluripotent stem cells (iPSCs) have significant potential for enhancing the efficacy of cell replacement therapies.