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The Effect of Nanoparticle Diameter on TAT-mediated Delivery to the CNS In Vivo

Description

Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method

Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.

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2016-05

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Antibody based diagnostic and therapeutic approach for Alzheimer's disease

Description

Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among

Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among top 10 death causes that cannot be prevented, cured or slowed. An estimated 5.4 million Americans live with AD, and this number is expected to triple by year 2050 as the baby boomers age. The cost of care for AD in the US is about $200 billion each year. Unfortunately, in addition to the lack of an effective treatment or AD, there is also a lack of an effective diagnosis, particularly an early diagnosis which would enable treatment to begin before significant neuronal damage has occurred.

Increasing evidence implicates soluble oligomeric forms of beta-amyloid and tau in the onset and progression of AD. While many studies have focused on beta-amyloid, soluble oligomeric tau species may also play an important role in AD pathogenesis. Antibodies that selectively identify and target specific oligomeric tau variants would be valuable tools for both diagnostic and therapeutic applications and also to study the etiology of AD and other neurodegenerative diseases.

Recombinant human tau (rhTau) in monomeric, dimeric, trimeric and fibrillar forms were synthesized and purified to perform LDH assay on human neuroblastoma cells, so that trimeric but not monomeric or dimeric rhTau was identified as extracellularly neurotoxic to neuronal cells. A novel biopanning protocol was designed based on phage display technique and atomic force microscopy (AFM), and used to isolate single chain antibody variable domain fragments (scFvs) that selectively recognize the toxic tau oligomers. These scFvs selectively bind tau variants in brain tissue of human AD patients and AD-related tau transgenic rodent models and have potential value as early diagnostic biomarkers for AD and as potential therapeutics to selectively target toxic tau aggregates.

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Date Created
2014

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Antibody-based Diagnostics and Therapeutics for Alzheimer's disease and Frontotemporal Dementia

Description

Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD) are the leading causes of early onset dementia. There are currently no ways to slow down progression, to prevent or cure AD and FTD. Both AD and FTD share a lot of the

Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD) are the leading causes of early onset dementia. There are currently no ways to slow down progression, to prevent or cure AD and FTD. Both AD and FTD share a lot of the symptoms and pathology. Initial symptoms such as confusion, memory loss, mood swings and behavioral changes are common in both these dementia subtypes. Neurofibrillary tau tangles and intraneuronal aggregates of TAR DNA Binding Protein 43 (TDP-43) are also observed in both AD and FTD. Hence, FTD cases are often misdiagnosed as AD due to a lack of accurate diagnostics. Prior to the formation of tau tangles and TDP-43 aggregates, tau and TDP-43 exist as intermediate protein variants which correlate with cognitive decline and progression of these neurodegenerative diseases. Effective diagnostic and therapeutic agents would selectively recognize these toxic, disease-specific variants. Antibodies or antibody fragments such as single chain antibody variable domain fragments (scFvs), with their diverse binding capabilities, can aid in developing reagents that can selectively bind these protein variants. A combination of phage display library and Atomic Force Microscopy (AFM)-based panning was employed to identify antibody fragments against immunoprecipitated tau and immunoprecipitated TDP-43 from human postmortem AD and FTD brain tissue respectively. Five anti-TDP scFvs and five anti-tau scFvs were selected for characterization by Enzyme Linked Immunosorbent Assays (ELISAs) and Immunohistochemistry (IHC). The panel of scFvs, together, were able to identify distinct protein variants present in AD but not in FTD, and vice versa. Generating protein variant profiles for individuals, using the panel of scFvs, aids in developing targeted diagnostic and therapeutic plans, gearing towards personalized medicine.

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Date Created
2018

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An Integrated Biomanufacturing Platform for the Large-Scale Expansion and Differentiation of Neural Progenitor Cells

Description

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications.

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Date Created
2018

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Using bioengineering approaches to generate a three-dimensional (3D) human pluripotent stem cell (hPSC)-based model for neurodegenerative diseases

Description

The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human

The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment.

I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.

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Date Created
2016

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The effect of Rho kinase inhibitors on Alzheimer's disease

Description

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects 5.4 million Americans. AD leads to memory loss, changes in behavior, and death. The key hallmarks of the disease are amyloid plaques and tau tangles, consisting of amyloid-β oligomers and

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects 5.4 million Americans. AD leads to memory loss, changes in behavior, and death. The key hallmarks of the disease are amyloid plaques and tau tangles, consisting of amyloid-β oligomers and hyperphosphorylated tau, respectively.

Rho-associated, coiled-coil-containing protein kinase (ROCK) is an enzyme that plays important roles in neuronal cells including mediating actin organization and dendritic spine morphogenesis. The ROCK inhibitor Fasudil has been shown to increase learning and working memory in aged rats, but another ROCK inhibitor, Y27632, was shown to impair learning and memory. I am interested in exploring how these, and other ROCK inhibitors, may be acting mechanistically to result in very different outcomes in treated animals.

Preliminary research on thirteen different ROCK inhibitors provides evidence that while Fasudil and a novel ROCK inhibitor, T343, decrease tau phosphorylation in vitro, Y27632 increases tau phosphorylation at a low dose and decreases at a high dose. Meanwhile, novel ROCK inhibitor T299 increases tau phosphorylation at a high dosage.

Further, an in vivo study using triple transgenic AD mice provides evidence that Fasudil improves reference memory and fear memory in both transgenic and wild-type mice, while Y27632 impairs reference memory in transgenic mice. Fasudil also decreases tau phosphorylation and Aβ in vivo, while Y27632 significantly increases the p-tau to total tau ratio.

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Date Created
2017

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Generation of a Human Induced Pluripotent Stem Cell Based Model of Progerin Induced Aging

Description

An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease

An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells.

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2017