Publications by authors named "Philip J Horner"

72 Publications

Neural Stimulation and Molecular Mechanisms of Plasticity and Regeneration: A Review.

Front Cell Neurosci 2020 14;14:271. Epub 2020 Oct 14.

Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Research Institute, Houston Methodist Hospital, Houston, TX, United States.

Neural stimulation modulates the depolarization of neurons, thereby triggering activity-associated mechanisms of neuronal plasticity. Activity-associated mechanisms in turn play a major role in post-mitotic structure and function of adult neurons. Our understanding of the interactions between neuronal behavior, patterns of neural activity, and the surrounding environment is evolving at a rapid pace. Brain derived neurotrophic factor is a critical mediator of activity-associated plasticity, while multiple immediate early genes mediate plasticity of neurons following bouts of neural activity. New research has uncovered genetic mechanisms that govern the expression of DNA following changes in neural activity patterns, including RNAPII pause-release and activity-associated double stranded breaks. Discovery of novel mechanisms governing activity-associated plasticity of neurons hints at a layered and complex molecular control of neuronal response to depolarization. Importantly, patterns of depolarization in neurons are shown to be important mediators of genetic expression patterns and molecular responses. More research is needed to fully uncover the molecular response of different types of neurons-to-activity patterns; however, known responses might be leveraged to facilitate recovery after neural damage. Physical rehabilitation through passive or active exercise modulates neurotrophic factor expression in the brain and spinal cord and can initiate cortical plasticity commensurate with functional recovery. Rehabilitation likely relies on activity-associated mechanisms; however, it may be limited in its application. Electrical and magnetic stimulation direct specific activity patterns not accessible through passive or active exercise and work synergistically to improve standing, walking, and forelimb use after injury. Here, we review emerging concepts in the molecular mechanisms of activity-derived plasticity in order to highlight opportunities that could add value to therapeutic protocols for promoting recovery of function after trauma, disease, or age-related functional decline.
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http://dx.doi.org/10.3389/fncel.2020.00271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591397PMC
October 2020

Quantification of Myelinated Nerve Fraction and Degeneration in Spinal Cord Neuropil by SHIFT MRI.

J Magn Reson Imaging 2021 04 24;53(4):1162-1174. Epub 2020 Oct 24.

Center for Neuroregneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston Methodist Hospital, Houston, Texas, USA.

Background: Neurodegeneration is a complex cellular process linked to prompt changes in myelin integrity and gradual neuron loss. Current imaging techniques offer estimations of myelin volumes in lesions/remyelinated areas but are limited to detect subtle injury.

Purpose: To investigate whether measurements detected by a signal hierarchically isolated as a function of time-to-echo (SHIFT) MRI technique can determine changes in myelin integrity and fiber axolemma.

Study Type: Prospective animal model.

Animal Model: Surgically demyelinated spinal cord (SC) injury model in rodents (n = 6).

Field Strength/sequence: Gradient-echo spin-echo at 3T.

Assessment: Multicompartment T relaxations were computed by SHIFT MRI in 75-microns-resolution images of the SC injury penumbra region 2 weeks post-trauma. G-ratio and axolemma delamination were assessed by transmission electron microscopy (TEM) in intact and injured samples. SC myelinated nerve fraction was computed by SHIFT MRI prospectively and assessed histologically.

Statistical Tests: Relations between SHIFT-isolated T -components and TEM measurements were studied using linear regression and t-tests. Pearson's correlation and significance were computed to determine the SHIFT's sensitivity to detect myelinated fibers ratio in gray matter. Regularized least-squares-based ranking analysis was employed to determine SHIFT MRI's ability to discern intact and injured myelinated nerves.

Results: Biexponential signals isolated by SHIFT MRI for intact vs. lesion penumbra exhibited changes in T , shifting from intermediate components (25 ± 2 msec) to long (43 ± 11 msec) in white matter, and similarly in gray matter regions-of-interest (31 ± 2 to 46 ± 16 msec). These changes correlated highly with TEM g-ratio and axon delamination measurements (P < 0.05). Changes in short T components were observed but not statistically significant (8.5 ± 0.5 to 7 ± 3 msec, P = 0.445, and 4.0 ± 0.9 to 7 ± 3 msec, P = 0.075, respectively). SHIFT MRI's ability to detect myelinated fibers within gray matter was confirmed (P < 0.001).

Data Conclusion: Changes detected by SHIFT MRI are associated with abnormal intermembrane spaces formed upon mild injury, directly correlated with early neuro integrity loss. Level of Evidence 1 Technical Efficacy Stage 2.
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http://dx.doi.org/10.1002/jmri.27397DOI Listing
April 2021

Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.

Sci Rep 2020 07 31;10(1):12939. Epub 2020 Jul 31.

Nanobioscience Constellation, Colleges of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, NanoFab East, 257 Fuller Road, Albany, NY, 12203, USA.

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.
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http://dx.doi.org/10.1038/s41598-020-69274-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395100PMC
July 2020

Formulation of thrombin-inhibiting hydrogels via self-assembly of ionic peptides with peptide-modified polymers.

Soft Matter 2020 Apr;16(15):3762-3768

Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. and Molecular Engineering and Sciences, University of Washington, Seattle, WA 98195, USA.

Cell therapy for spinal cord injuries offers the possibility of replacing lost cells after trauma to the central nervous system (CNS). In preclinical studies, synthetic hydrogels are often co-delivered to the injury site to support survival and integration of the transplanted cells. These hydrogels ideally mimic the mechanical and biochemical features of a healthy CNS extracellular matrix while also providing the possibility of localized drug delivery to promote healing. In this work, we synthesize peptide-functionalized polymers that contain both a peptide sequence for incorporation into self-assembled peptide hydrogels along with bioactive peptides that inhibit scar formation. We demonstrate that peptide hydrogels formulated with the peptide-functionalized polymers possess similar mechanical properties (soft and shear-thinning) as peptide-only hydrogels. Small angle neutron scattering analysis reveals that polymer-containing hydrogels possess larger inhomogeneous domains but small-scale features such as mesh size remain the same as peptide-only hydrogels. We further confirm that the integrated hydrogels containing bioactive peptides exhibit thrombin inhibition activity, which has previously shown to reduce scar formation in vivo. Finally, while the survival of encapsulated cells was poor, cells cultured on the hydrogels exhibited good viability. Overall, the described composite hydrogels formed from self-assembling peptides and peptide-modified polymers are promising, user-friendly materials for CNS applications in regeneration.
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http://dx.doi.org/10.1039/d0sm00209gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204513PMC
April 2020

Polyplex transfection from intracerebroventricular delivery is not significantly affected by traumatic brain injury.

J Control Release 2020 06 18;322:149-156. Epub 2020 Mar 18.

Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, United States. Electronic address:

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.
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http://dx.doi.org/10.1016/j.jconrel.2020.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250725PMC
June 2020

pH-sensitive polymer micelles provide selective and potentiated lytic capacity to venom peptides for effective intracellular delivery.

Biomaterials 2019 02 9;192:235-244. Epub 2018 Nov 9.

Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, 98195, United States. Electronic address:

Endocytosed biomacromolecule delivery systems must escape the endosomal trafficking pathway in order for their cargo to exert effects in other cellular compartments. Although endosomal release is well-recognized as one of the greatest barriers to efficacy of biologic drugs with intracellular targets, most drug carriers have relied on cationic materials that passively induce endosomal swelling and membrane rupture with low efficiency. To address the endosome release challenge, our lab has developed a diblock copolymer system for nucleic acid delivery that selectively displays a potent membrane-lytic peptide (melittin) in response to the pH drop during the endosomal maturation. To further optimize this system, we evaluated a panel of peptides with reported lytic activity in comparison to melittin. Nineteen different lytic peptides were synthesized and their membrane-lytic properties at both neutral and acidic pH characterized using a red blood cell hemolysis assay. The top five performing peptides were then conjugated to our pH-sensitive diblock copolymer via disulfide linkers and used to deliver a variety of nucleic acids to cultured mammalian cells as well as in vivo to the mouse brain. We demonstrate that the sharp pH-transition of VIPER compensates for potential advantages from pH-sensitive peptides, such that polymer-peptide conjugates with poorly selective but highly lytic peptides achieve safe and effective transfection both in vitro and in vivo. In addition, peptides that require release from polymer backbones for lysis were less effective in the VIPER system, likely due to limited endosomal reducing power of target cells. Finally, we show that certain peptides are potentiated in lytic ability by polymer conjugation and that these peptide-polymer constructs are most effective in vivo.
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http://dx.doi.org/10.1016/j.biomaterials.2018.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331286PMC
February 2019

Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes.

Stem Cell Reports 2017 12 30;9(6):1745-1753. Epub 2017 Nov 30.

Department of Ophthalmology, University of California, San Francisco, CA 94143, USA; Department of Physiology, University of California, San Francisco, CA 94143, USA.

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785708PMC
December 2017

Myelin plasticity, neural activity, and traumatic neural injury.

Dev Neurobiol 2018 02 4;78(2):108-122. Epub 2017 Oct 4.

Center for Neuroregeneration, Houston Methodist Research Institute, 6670 Bertner Avenue, MSR10-112, Houston, Texas.

The possibility that adult organisms exhibit myelin plasticity has recently become a topic of great interest. Many researchers are exploring the role of myelin growth and adaptation in daily functions such as memory and motor learning. Here we consider evidence for three different potential categories of myelin plasticity: the myelination of previously bare axons, remodeling of existing sheaths, and the removal of a sheath with replacement by a new internode. We also review evidence that points to the importance of neural activity as a mechanism by which oligodendrocyte precursor cells (OPCs) are cued to differentiate into myelinating oligodendrocytes, which may potentially be an important component of myelin plasticity. Finally, we discuss demyelination in the context of traumatic neural injury and present an argument for altering neural activity as a potential therapeutic target for remyelination following injury. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 108-122, 2018.
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http://dx.doi.org/10.1002/dneu.22540DOI Listing
February 2018

Tunable, Injectable Hydrogels Based on Peptide-Cross-Linked, Cyclized Polymer Nanoparticles for Neural Progenitor Cell Delivery.

Biomacromolecules 2017 Sep 25;18(9):2723-2731. Epub 2017 Aug 25.

Department of Bioengineering, University of Washington , Seattle, Washington 98195, United States.

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.
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http://dx.doi.org/10.1021/acs.biomac.7b00510DOI Listing
September 2017

Age-related accumulation of phosphorylated mitofusin 2 protein in retinal ganglion cells correlates with glaucoma progression.

Exp Neurol 2017 10 3;296:49-61. Epub 2017 Jul 3.

Department of Neurological Surgery, University of Washington, Seattle, WA, USA. Electronic address:

Dysregulation of axonal bioenergetics is likely a key mechanism in the initiation and progression of age-related neurodegenerative diseases. Glaucoma is a quintessential neurodegenerative disorder characterized by progressive deterioration of the optic nerve (ON) and eventual death of retinal ganglion cells (RGCs). Age and elevation of intraocular pressure are key risk factors in glaucoma, but the common early hallmarks of decreased axonal transport and increased bioenergetic vulnerability likely underlie disease initiation. We examined the correlation between bioenergetics and axonal transport with mitochondrial mutation frequency and post-translational modifications of mitofusin 2 (Mfn2) in RGCs during glaucoma progression. No increase in the frequency of mtDNA mutations was detected, but we observed significant shifts in mitochondrial protein species. Mfn2 is a fusion protein that functions in mitochondrial biogenesis, maintenance, and mitochondrial transport. We demonstrate that Mfn2 accumulates selectively in RGCs during glaucomatous degeneration, that two novel states of Mfn2 exist in retina and ON, and identify a phosphorylated form that selectively accumulates in RGCs, but is absent in ON. Phosphorylation of Mfn2 is correlated with higher ubiquitination, and failure of the protein to reach the ON. Together, these data suggest that post-translational modification of Mfn2 is associated with its dysregulation during a window of metabolic vulnerability that precedes glaucomatous degeneration. Future work to either manipulate expression of Mfn2 or to prevent its degradation could have therapeutic value in the treatment of neurodegenerative diseases where long-tract axons are vulnerable.
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http://dx.doi.org/10.1016/j.expneurol.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021128PMC
October 2017

Fabrication of multipoint side-firing optical fiber by laser micro-ablation.

Opt Lett 2017 May;42(9):1808-1811

A multipoint, side-firing design enables an optical fiber to output light at multiple desired locations along the fiber body. This provides advantages over traditional end-to-end fibers, especially in applications requiring fiber bundles such as brain stimulation or remote sensing. This Letter demonstrates that continuous wave (CW) laser micro-ablation can controllably create conical-shaped cavities, or side windows, for outputting light. The dimensions of these cavities determine the amount of firing light and their firing angle. Experimental data show that a single side window on a 730 μm fiber can deliver more than 8% of the input light. This can be increased to more than 19% on a 65 μm fiber with side windows created using femtosecond laser ablation and chemical etching. Fine control of light distribution along an optical fiber is critical for various biomedical applications such as light-activated drug-release and optogenetics studies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769456PMC
http://dx.doi.org/10.1364/OL.42.001808DOI Listing
May 2017

Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers.

Biomaterials 2017 05 1;127:89-96. Epub 2017 Mar 1.

Department of Bioengineering and Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA 98195, United States. Electronic address:

Cationic polymer gene delivery vehicles that effectively resist premature serum degradation often have difficulty releasing their nucleic acid cargoes. In this work, we report a pH-sensitive polymer (SP), poly(oligo(ethylene glycol) monomethyl ether methacrylate)-co-poly(2-(dimethylamino)ethyl methacrylate)-block- poly(propargyl methacrylate-graft-propyl-(4-methoxy-benzylidene)-amine) (p(PMA-PMBA)-b-(p(OEGMA-DMAEMA)), for successful in vitro and in vivo gene transfer. In the physiological condition, the hydrophobization of p(OEGMA-DMAEMA) polycations by p(PMA-PMBA) significantly enhanced the stability of its polyplexes counterpart. In endosomes, the polymer undergoes an acid-triggered hydrophilic transition through the cleavage of benzoic imines, thus allowing the vector to quickly release nucleic acid cargo due to the loss of hydrophobic functionalization. Compared to a pH-insensitive polymer (IP), SP exhibited more significant luciferase plasmid delivery efficiency with HeLa cells in vitro and with in vivo intraventricular brain injections. Therefore, the polymer designed here is a good solution to address the dilemma of stability and cargo release in gene delivery, and may have broad potential applications in therapeutic agent delivery.
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http://dx.doi.org/10.1016/j.biomaterials.2017.02.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382035PMC
May 2017

Non-Viral Nucleic Acid Delivery Strategies to the Central Nervous System.

Front Mol Neurosci 2016 1;9:108. Epub 2016 Nov 1.

Center for Neuroregenerative Medicine, Houston Methodist Research Institute Houston, TX, USA.

With an increased prevalence and understanding of central nervous system (CNS) injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the challenges hindering non-viral nucleic acid delivery to the CNS and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated gene transfer to the CNS are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for CNS applications and will ultimately bring non-viral vectors closer to clinical application.
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http://dx.doi.org/10.3389/fnmol.2016.00108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088201PMC
November 2016

Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

Small 2016 May 7;12(20):2750-8. Epub 2016 Apr 7.

Department of Bioengineering and Molecular, Engineering and Sciences Institute, University of Washington, Seattle, WA, 98195, USA.

The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052141PMC
http://dx.doi.org/10.1002/smll.201502930DOI Listing
May 2016

Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration.

Proc Natl Acad Sci U S A 2016 Mar 17;113(9):2514-9. Epub 2016 Feb 17.

Department of Bioengineering, University of Washington, Seattle, WA 98195; Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.
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http://dx.doi.org/10.1073/pnas.1515526113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780603PMC
March 2016

Microbubbles and ultrasound increase intraventricular polyplex gene transfer to the brain.

J Control Release 2016 06 6;231:86-93. Epub 2016 Feb 6.

Department of Bioengineering, University of Washington, Seattle, WA 98195, USA; Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, USA. Electronic address:

Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits further research.
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http://dx.doi.org/10.1016/j.jconrel.2016.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944389PMC
June 2016

Early astrocyte redistribution in the optic nerve precedes axonopathy in the DBA/2J mouse model of glaucoma.

Exp Eye Res 2016 09 2;150:22-33. Epub 2015 Dec 2.

Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN 37205, United States. Electronic address:

Glaucoma challenges the survival of retinal ganglion cell axons in the optic nerve through processes dependent on both aging and ocular pressure. Relevant stressors likely include complex interplay between axons and astrocytes, both in the retina and optic nerve. In the DBA/2J mouse model of pigmentary glaucoma, early progression involves axonopathy characterized by loss of functional transport prior to outright degeneration. Here we describe novel features of early pathogenesis in the DBA/2J nerve. With age the cross-sectional area of the nerve increases; this is associated generally with diminished axon packing density and survival and increased glial coverage of the nerve. However, for nerves with the highest axon density, as the nerve expands mean cross-sectional axon area enlarges as well. This early expansion was marked by disorganized axoplasm and accumulation of hyperphosphorylated neurofilamants indicative of axonopathy. Axon expansion occurs without loss up to a critical threshold for size (about 0.45-0.50 μm(2)), above which additional expansion tightly correlates with frank loss of axons. As well, early axon expansion prior to degeneration is concurrent with decreased astrocyte ramification with redistribution of processes towards the nerve edge. As axons expand beyond the critical threshold for loss, glial area resumes an even distribution from the center to edge of the nerve. We also found that early axon expansion is accompanied by reduced numbers of mitochondria per unit area in the nerve. Finally, our data indicate that both IOP and nerve expansion are associated with axon enlargement and reduced axon density for aged nerves. Collectively, our data support the hypothesis that diminished bioenergetic resources in conjunction with early nerve and glial remodeling could be a primary inducer of progression of axon pathology in glaucoma.
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http://dx.doi.org/10.1016/j.exer.2015.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889569PMC
September 2016

A Cervical Hemi-Contusion Spinal Cord Injury Model for the Investigation of Novel Therapeutics Targeting Proximal and Distal Forelimb Functional Recovery.

J Neurotrauma 2015 Dec 29;32(24):1994-2007. Epub 2015 Sep 29.

3 Department of Neurological Surgery, University of Washington , Seattle, Washington.

Cervical spinal cord contusion is the most common human spinal cord injury, yet few rodent models replicate the pathophysiological and functional sequela of this injury. Here, we modified an electromechanical injury device and characterized the behavioral and histological changes occurring in response to a lateralized C4 contusion injury in rats. A key feature of the model includes a non-injurious touch phase where the spinal cord surface is dimpled with a consistent starting force. Animals were either left intact as a control, received a non-injury-producing touch on the surface of the cord ("sham"), or received a 0.6 mm or a 0.8 mm displacement injury. Rats were then tested on the forelimb asymmetry use test, CatWalk, and the Irvine, Beatties, and Bresnahan (IBB) cereal manipulation task to assess proximal and distal upper limb function for 12 weeks. Injuries of moderate (0.6 mm) and large (0.8 mm) displacement showed consistent differences in forelimb asymmetry, metrics of the CatWalk, and sub-scores of the IBB. Overall findings indicated long lasting proximal and distal upper limb deficits following 0.8 mm injury but transient proximal with prolonged distal limb deficits following 0.6 mm injury. Significant differences in loss of ipsilateral unmyelinated and myelinated white matter was detected between injury severities. Demyelination was primarily localized to the dorsolateral region of the hemicord and extended further rostral following 0.8 mm injury. These findings establish the C4 hemi-contusion injury as a consistent, graded model for testing novel treatments targeting forelimb functional recovery.
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http://dx.doi.org/10.1089/neu.2014.3792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4677514PMC
December 2015

Neural Stem Cells in the Adult Subventricular Zone Oxidize Fatty Acids to Produce Energy and Support Neurogenic Activity.

Stem Cells 2015 Jul 4;33(7):2306-19. Epub 2015 Jun 4.

Centre for Brain Ageing and Vitality, Newcastle University, Newcastle upon Tyne, United Kingdom.

Neural activity is tightly coupled to energy consumption, particularly sugars such as glucose. However, we find that, unlike mature neurons and astrocytes, neural stem/progenitor cells (NSPCs) do not require glucose to sustain aerobic respiration. NSPCs within the adult subventricular zone (SVZ) express enzymes required for fatty acid oxidation and show sustained increases in oxygen consumption upon treatment with a polyunsaturated fatty acid. NSPCs also demonstrate sustained decreases in oxygen consumption upon treatment with etomoxir, an inhibitor of fatty acid oxidation. In addition, etomoxir decreases the proliferation of SVZ NSPCs without affecting cellular survival. Finally, higher levels of neurogenesis can be achieved in aged mice by ectopically expressing proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), a factor that increases cellular aerobic capacity by promoting mitochondrial biogenesis and metabolic gene transcription. Regulation of metabolic fuel availability could prove a powerful tool in promoting or limiting cellular proliferation in the central nervous system. Stem Cells 2015;33:2306-2319.
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http://dx.doi.org/10.1002/stem.2042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478223PMC
July 2015

Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency.

Biomaterials 2015 Jun 30;54:87-96. Epub 2015 Mar 30.

Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, USA. Electronic address:

There is currently no cure for neuron loss in the brain, which can occur due to traumatic injury or neurodegenerative disease. One proposed method to enhance brain neurogenesis is gene transfer to neural progenitor cells. In this work, a guanidine-based copolymer was synthesized and compared to an amine-based copolymer analog previously shown to effectively deliver genes in the murine brain. The guanidine-based copolymer was more efficient at gene transfer to immortalized, cultured cell lines; however, the amine-based copolymer was more effective at gene transfer in the brain. DNA condensation studies revealed that the nucleic acid complexes formed with the guanidine-based copolymer were more susceptible to unpackaging in the presence of anionic proteoglycans compared to complexes formed with the amine-based copolymer. Therefore, polyplexes formed from the amine-based copolymer may be more resistant to destabilization by the heparan sulfate proteoglycans present in the stem cell niches of the brain.
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http://dx.doi.org/10.1016/j.biomaterials.2015.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409668PMC
June 2015

A functionalized, injectable hydrogel for localized drug delivery with tunable thermosensitivity: synthesis and characterization of physical and toxicological properties.

J Control Release 2015 Jun 4;208:76-84. Epub 2015 Mar 4.

Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, United States. Electronic address:

Thermosensitive injectable hydrogels have been used for the delivery of pharmacological and cellular therapies in a variety of soft tissue applications. A promising class of synthetic, injectable hydrogels based upon oligo(ethylene glycol) methacrylate (OEGMA) monomers has been previously reported, but these polymers lack reactive groups for covalent attachment of therapeutic molecules. In this work, thermosensitive, amine-reactive and amine-functionalized polymers were developed by incorporation of methacrylic acid N-hydroxysuccinimide ester or 2-aminoethyl methacrylate into OEGMA-based polymers. A model therapeutic peptide, bivalirudin, was conjugated to the amine-reactive hydrogel to investigate effects on the polymer thermosensitivity and gelation properties. The ability to tune the thermosensitivity of the polymer in order to compensate for peptide hydrophilicity and maintain gelation capability below physiological temperature was demonstrated. Cell encapsulation studies using an H9 T-cell line (CD4+) were conducted to evaluate feasibility of the hydrogel as a carrier for cellular therapies. Although this class of polymers is generally considered to be non-toxic, it was found that concentrations required for gelation were incompatible with cell survival. Investigation into the cause of cytotoxicity revealed that a hydrolysis byproduct, diethylene glycol monomethyl ether, is likely a contributing factor. While modifications to structure or composition will be required to enable viable cell encapsulation, the functionalized injectable hydrogel has the potential for controlled delivery of a wide range of drugs.
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http://dx.doi.org/10.1016/j.jconrel.2015.03.003DOI Listing
June 2015

Mitochondrial morphology differences and mitophagy deficit in murine glaucomatous optic nerve.

Invest Ophthalmol Vis Sci 2015 Feb 5;56(3):1437-46. Epub 2015 Feb 5.

Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, Ohio, United States.

Purpose: Decreased ATP correlates with intraocular pressure exposure in the optic nerves of mice with glaucoma. To understand what underlies this energy deficit, we examined mitochondria in the myelinated optic nerve axons of the DBA/2J mouse, a model of glaucoma secondary to iris pigment disease, and the DBA/2(wt-gpnmb) control strain.

Methods: Mitochondrial length, width, surface area, and health status were measured in 30 electron microscopic fields within the myelinated portion of optic nerves from DBA/2J and DBA/2(wt-gpnmb) mice at 3, 6, and 10 months of age. Protein was isolated from optic nerve for analysis of PINK1, Parkin, LC3-I and -II, and lysosome-associated membrane protein 1 (LAMP1) by Western blot.

Results: The number of mitochondria in DBA/2J optic nerve was increased, and they had significantly smaller surface area. Mitochondria in DBA/2J were closer to the axolemma, more spatially isolated, and their cristae were more disrupted at every age group as compared to DBA/2(wt-gpnmb). Autophagosomes were significantly increased in DBA/2J optic nerve at all ages. Protein analysis showed higher LC3-II to LC3-I ratio in aged DBA/2J optic nerve than in DBA/2(wt-gpnmb). PINK1 and Parkin levels were not statistically different across age groups. LAMP1 was significantly decreased in the aged DBA/2J optic nerve.

Conclusions: Decreased surface area, combined with reduced oxidative capacity in mitochondria from the aged DBA/2J axon, indicate that mitochondrial pathology may contribute to the energy deficit in glaucomatous optic nerve. Though autophagosomes were increased in DBA/2J optic nerve, the increased mitochondria and decreased LAMP1 suggest deteriorating mitochondria are not being efficiently recycled by mitophagy.
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http://dx.doi.org/10.1167/iovs.14-16126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347310PMC
February 2015

Poly(lactic-co-glycolic) acid microspheres encapsulated in Pluronic F-127 prolong hirudin delivery and improve functional recovery from a demyelination lesion.

Biomaterials 2014 Oct 23;35(31):8895-8902. Epub 2014 Jul 23.

Department of Neurological Surgery, University of Washington, Seattle, WA 98104, USA; Neurobiology and Behavior Program, Center on Human Development and Disability, University of Washington, Seattle, Washington, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.

Components of the blood have been proposed as potential therapeutic targets for improving cellular regeneration after injury and neurodegenerative disease. In this work, thrombin is shown to increase endogenous neural progenitor proliferation in the intact murine spinal cord. A local injection of heparin before a spinal cord injury reduces cell proliferation and astrogliogenesis associated with scarring. We sought to create depot-formulations of PLGA microsphere and Pluronic F-127 for sustained local delivery of two thrombin inhibitors, heparin and hirudin. Each hydrogel depot-formulation showed delayed drug release compared to microspheres or hydrogel alone. Animals with a lateral demyelination lesion showed a reduction in CD68+ macrophages when treated with hirudin-loaded PLGA/F-127 gels compared to control and heparin-treated animals. Moreover, hirudin-loaded materials showed an accelerated recovery in coordinated stepping and increased oligodendrocyte densities. Together, these data demonstrate that controlled delivery of hirudin accelerates functional recovery from a demyelination lesion in the spinal cord.
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http://dx.doi.org/10.1016/j.biomaterials.2014.06.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136545PMC
October 2014

Therapeutic intraspinal stimulation to generate activity and promote long-term recovery.

Front Neurosci 2014 27;8:21. Epub 2014 Feb 27.

Department of Rehabilitation Medicine, University of Washington Seattle, WA, USA ; Center for Sensorimotor Neural Engineering, University of Washington Seattle, WA, USA ; Graduate Program in Neurobiology and Behavior, University of Washington Seattle, WA, USA ; Department of Physiology and Biophysics, University of Washington Seattle, WA, USA.

Neuroprosthetic approaches have tremendous potential for the treatment of injuries to the brain and spinal cord by inducing appropriate neural activity in otherwise disordered circuits. Substantial work has demonstrated that stimulation applied to both the central and peripheral nervous system leads to immediate and in some cases sustained benefits after injury. Here we focus on cervical intraspinal microstimulation (ISMS) as a promising method of activating the spinal cord distal to an injury site, either to directly produce movements or more intriguingly to improve subsequent volitional control of the paretic extremities. Incomplete injuries to the spinal cord are the most commonly observed in human patients, and these injuries spare neural tissue bypassing the lesion that could be influenced by neural devices to promote recovery of function. In fact, recent results have demonstrated that therapeutic ISMS leads to modest but sustained improvements in forelimb function after an incomplete spinal cord injury (SCI). This therapeutic spinal stimulation may promote long-term recovery of function by providing the necessary electrical activity needed for neuron survival, axon growth, and synaptic stability.
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http://dx.doi.org/10.3389/fnins.2014.00021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936503PMC
February 2014

Differential progression of structural and functional alterations in distinct retinal ganglion cell types in a mouse model of glaucoma.

J Neurosci 2013 Oct;33(44):17444-57

Department of Biological Structure, University of Washington, Seattle, Washington 98195, and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109.

Intraocular pressure (IOP) elevation is a principal risk factor for glaucoma. Using a microbead injection technique to chronically raise IOP for 15 or 30 d in mice, we identified the early changes in visual response properties of different types of retinal ganglion cells (RGCs) and correlated these changes with neuronal morphology before cell death. Microbead-injected eyes showed reduced optokinetic tracking as well as cell death. In such eyes, multielectrode array recordings revealed that four RGC types show diverse alterations in their light responses upon IOP elevation. OFF-transient RGCs exhibited a more rapid decline in both structural and functional organizations compared with other RGCs. In contrast, although the light-evoked responses of OFF-sustained RGCs were perturbed, the dendritic arbor of this cell type remained intact. ON-transient and ON-sustained RGCs had normal functional receptive field sizes but their spontaneous and light-evoked firing rates were reduced. ON- and OFF-sustained RGCs lost excitatory synapses across an otherwise structurally normal dendritic arbor. Together, our observations indicate that there are changes in spontaneous activity and light-evoked responses in RGCs before detectable dendritic loss. However, when dendrites retract, we found corresponding changes in receptive field center size. Importantly, the effects of IOP elevation are not uniformly manifested in the structure and function of diverse RGC populations, nor are distinct RGC types perturbed within the same time-frame by such a challenge.
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http://dx.doi.org/10.1523/JNEUROSCI.5461-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812509PMC
October 2013

Caudalized human iPSC-derived neural progenitor cells produce neurons and glia but fail to restore function in an early chronic spinal cord injury model.

Exp Neurol 2013 Oct 25;248:491-503. Epub 2013 Jul 25.

Department of Neurological Surgery, University of Washington, Seattle, WA 98104, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.

Neural progenitor cells (NPCs) have shown modest potential and some side effects (e.g. allodynia) for treatment of spinal cord injury (SCI). In only a few cases, however, have NPCs shown promise at the chronic stage. Given the 1.275 million people living with chronic paralysis, there is a significant need to rigorously evaluate the cell types and methods for safe and efficacious treatment of this devastating condition. For the first time, we examined the pre-clinical potential of NPCs derived from human induced pluripotent stem cells (hiPSCs) to repair chronic SCI. hiPSCs were differentiated into region-specific (i.e. caudal) NPCs, then transplanted into a new, clinically relevant model of early chronic cervical SCI. We established the conditions for successful transplantation of caudalized hiPSC-NPCs and demonstrate their remarkable ability to integrate and produce multiple neural lineages in the early chronic injury environment. In contrast to prior reports in acute and sub-acute injury models, survival and integration of hiPSC-derived neural cells in the early chronic cervical model did not lead to significant improvement in forelimb function or induce allodynia. These data indicate that while hiPSCs show promise, future work needs to focus on the specific hiPSC-derivatives or co-therapies that will restore function in the early chronic injury setting.
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http://dx.doi.org/10.1016/j.expneurol.2013.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109283PMC
October 2013

α-Lipoic acid antioxidant treatment limits glaucoma-related retinal ganglion cell death and dysfunction.

PLoS One 2013 5;8(6):e65389. Epub 2013 Jun 5.

Department of Neurological Surgery, University of Washington, Seattle, Washington, United States of America.

Oxidative stress has been implicated in neurodegenerative diseases, including glaucoma. However, due to the lack of clinically relevant models and expense of long-term testing, few studies have modeled antioxidant therapy for prevention of neurodegeneration. We investigated the contribution of oxidative stress to the pathogenesis of glaucoma in the DBA/2J mouse model of glaucoma. Similar to other neurodegenerative diseases, we observed lipid peroxidation and upregulation of oxidative stress-related mRNA and protein in DBA/2J retina. To test the role of oxidative stress in disease progression, we chose to deliver the naturally occurring, antioxidant α-lipoic acid (ALA) to DBA/2J mice in their diet. We used two paradigms for ALA delivery: an intervention paradigm in which DBA/2J mice at 6 months of age received ALA in order to intervene in glaucoma development, and a prevention paradigm in which DBA/2J mice were raised on a diet supplemented with ALA, with the goal of preventing glaucoma development. At 10 and 12 months of age (after 4 and 11 months of dietary ALA respectively), we measured changes in genes and proteins related to oxidative stress, retinal ganglion cell (RGC) number, axon transport, and axon number and integrity. Both ALA treatment paradigms showed increased antioxidant gene and protein expression, increased protection of RGCs and improved retrograde transport compared to control. Measures of lipid peroxidation, protein nitrosylation, and DNA oxidation in retina verified decreased oxidative stress in the prevention and intervention paradigms. These data demonstrate the utility of dietary therapy for reducing oxidative stress and improving RGC survival in glaucoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065389PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673940PMC
January 2014

The impact of age on oncogenic potential: tumor-initiating cells and the brain microenvironment.

Aging Cell 2013 Oct 28;12(5):733-41. Epub 2013 Jun 28.

Institute for Aging and Health, Newcastle University, Newcastle upon Tyne, UK.

Paradoxically, aging leads to both decreased regenerative capacity in the brain and an increased risk of tumorigenesis, particularly the most common adult-onset brain tumor, glioma. A shared factor contributing to both phenomena is thought to be age-related alterations in neural progenitor cells (NPCs), which function normally to produce new neurons and glia, but are also considered likely cells of origin for malignant glioma. Upon oncogenic transformation, cells acquire characteristics known as the hallmarks of cancer, including unlimited replication, altered responses to growth and anti-growth factors, increased capacity for angiogenesis, potential for invasion, genetic instability, apoptotic evasion, escape from immune surveillance, and an adaptive metabolic phenotype. The precise molecular pathogenesis and temporal acquisition of these malignant characteristics is largely a mystery. Recent studies characterizing NPCs during normal aging, however, have begun to elucidate mechanisms underlying the age-associated increase in their malignant potential. Aging cells are dependent upon multiple compensatory pathways to maintain cell cycle control, normal niche interactions, genetic stability, programmed cell death, and oxidative metabolism. A few multi-functional proteins act as 'critical nodes' in the coordination of these various cellular activities, although both intracellular signaling and elements within the brain environment are critical to maintaining a balance between senescence and tumorigenesis. Here, we provide an overview of recent progress in our understanding of how mechanisms underlying cellular aging inform on glioma pathogenesis and malignancy.
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http://dx.doi.org/10.1111/acel.12104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199664PMC
October 2013

Dual responsive, stabilized nanoparticles for efficient in vivo plasmid delivery.

Angew Chem Int Ed Engl 2013 May 16;52(20):5377-81. Epub 2013 Apr 16.

Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, USA.

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http://dx.doi.org/10.1002/anie.201301896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738056PMC
May 2013

FMR1 transcript isoforms: association with polyribosomes; regional and developmental expression in mouse brain.

PLoS One 2013 7;8(3):e58296. Epub 2013 Mar 7.

Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1-3, 6-9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591412PMC
September 2013