Nerve Wrap for Local Delivery of FK506/Tacrolimus Accelerates Nerve Regeneration.

Peripheral nerve injuries (PNIs) occur frequently and can lead to devastating and permanent sensory and motor function disabilities. Systemic tacrolimus (FK506) administration has been shown to hasten recovery and improve functional outcomes after PNI repair. Unfortunately, high systemic levels of FK506 can result in adverse side effects. The localized administration of FK506 could provide the neuroregenerative benefits of FK506 while avoiding systemic, off-target side effects. This study investigates the utility of a novel FK506-impregnated polyester urethane urea (PEUU) nerve wrap to treat PNI in a previously validated rat infraorbital nerve (ION) transection and repair model. ION function was assessed by microelectrode recordings of trigeminal ganglion cells responding to controlled vibrissae deflections in ION-transected and -repaired animals, with and without the nerve wrap. Peristimulus time histograms (PSTHs) having 1 ms bins were constructed from spike times of individual single units. Responses to stimulus onsets (ON responses) were calculated during a 20 ms period beginning 1 ms after deflection onset; this epoch captures the initial, transient phase of the whisker-evoked response. Compared to no-wrap controls, rats with PEUU-FK506 wraps functionally recovered earlier, displaying larger response magnitudes. With nerve wrap treatment, FK506 blood levels up to six weeks were measured nearly at the limit of quantification (LOQ ≥ 2.0 ng/mL); whereas the drug concentrations within the ION and muscle were much higher, demonstrating the local delivery of FK506 to treat PNI. An immunohistological assessment of ION showed increased myelin expression for animals assigned to neurorrhaphy with PEUU-FK506 treatment compared to untreated or systemic-FK506-treated animals, suggesting that improved PNI outcomes using PEUU-FK506 is mediated by the modulation of Schwann cell activity.

Author Correction: Matrix-bound nanovesicles prevent ischemia-induced retinal ganglion cell axon degeneration and death and preserve visual function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transforming the Early Drug Development Paradigm at the National Cancer Institute: The Formation of NCI's Experimental Therapeutics Clinical Trials Network (ETCTN).

Early drug development for cancer requires broad collaboration and skilled clinical investigators to enable enrollment of patients whose tumors have defined molecular profiles. To respond to these challenges, the National Cancer Institute (NCI) transformed its 60-year-old early-phase drug development program in 2014 into the Experimental Therapeutics Clinical Trials Network (ETCTN). The ETCTN is a consolidated, national network of 40+ academic institutions responsible for conducting more than 100 early-phase clinical trials. It promotes team science coordinated among basic, translational, and clinical investigators, emphasizing the inclusion of early career trialists. This perspective provides a brief overview of the ETCTN, summarizes its successes and challenges over its first grant funding cycle, and discusses the program's future directions. Measures indicated strong connectivity across the institutions, significant increases in investigator approval of the ETCTN scientific portfolio from years 1 to 4, and substantial research activity over 5 years, with 334 letters of intent submitted, 102 trials activated, and 3,570 patients accrued. The ETCTN's successful adoption relied heavily on the inclusion of senior investigators who have long-standing interactions with the NCI and a willingness to participate in a team science approach and to mentor early career investigators. In addition, NCI invested substantial resources in a centralized infrastructure to conduct trials and to support the inclusion of biomarkers in its studies. The ETCTN provides evidence that a collaborative national clinical trial network for early drug development is feasible and can address the demands of precision medicine approaches to oncologic clinical trials.

Matrix-bound nanovesicles prevent ischemia-induced retinal ganglion cell axon degeneration and death and preserve visual function.

Injury to retinal ganglion cells (RGC), central nervous system neurons that relay visual information to the brain, often leads to RGC axon degeneration and permanently lost visual function. Herein this study shows matrix-bound nanovesicles (MBV), a distinct class of extracellular nanovesicle localized specifically to the extracellular matrix (ECM) of healthy tissues, can neuroprotect RGCs and preserve visual function after severe, intraocular pressure (IOP) induced ischemia in rat. Intravitreal MBV injections attenuated IOP-induced RGC axon degeneration and death, protected RGC axon connectivity to visual nuclei in the brain, and prevented loss in retinal function as shown by histology, anterograde axon tracing, manganese-enhanced magnetic resonance imaging, and electroretinography. In the optic nerve, MBV also prevented IOP-induced decreases in growth associated protein-43 and IOP-induced increases in glial fibrillary acidic protein. In vitro studies showed MBV suppressed pro-inflammatory signaling by activated microglia and astrocytes, stimulated RGC neurite growth, and neuroprotected RGCs from neurotoxic media conditioned by pro-inflammatory astrocytes. Thus, MBV can positively modulate distinct signaling pathways (e.g., inflammation, cell death, and axon growth) in diverse cell types. Since MBV are naturally derived, bioactive factors present in numerous FDA approved devices, MBV may be readily useful, not only experimentally, but also clinically as immunomodulatory, neuroprotective factors for treating trauma or disease in the retina as well as other CNS tissues.

Fetal extracellular matrix nerve wraps locally improve peripheral nerve remodeling after complete transection and direct repair in rat.

In peripheral nerve (PN) injuries requiring surgical repair, as in PN transection, cellular and ECM remodeling at PN epineurial repair sites is hypothesized to reduce PN functional outcomes by slowing, misdirecting, or preventing axons from regrowing appropriately across the repair site. Herein this study reports on deriving and analyzing fetal porcine urinary bladder extracellular matrix (fUB-ECM) by vacuum assisted decellularization, fabricating fUBM-ECM nerve wraps, and testing fUB-ECM nerve wrap biocompatibility and bioactivity in a trigeminal, infraorbital nerve (ION) branch transection and direct end-to-end repair model in rat. FUB-ECM nerve wraps significantly improved epi- and endoneurial organization and increased both neovascularization and growth associated protein-43 (GAP-43) expression at PN repair sites, 28-days post surgery. However, the number of neurofilament positive axons, remyelination, and whisker-evoked response properties of ION axons were unaltered, indicating improved tissue remodeling per se does not predict axon regrowth, remyelination, and the return of mechanoreceptor cortical signaling. This study shows fUB-ECM nerve wraps are biocompatible, bioactive, and good experimental and potentially clinical devices for treating epineurial repairs. Moreover, this study highlights the value provided by precise, analytic models, like the ION repair model, in understanding how PN tissue remodeling relates to axonal regrowth, remyelination, and axonal response properties.

Correction to: Extracellular Vesicles: Biomarkers, Therapeutics, and Vehicles in the Visual System.

[This corrects the article DOI: 10.1007/s40135-017-0153-0.].

An Elastomeric Polymer Matrix, PEUU-Tac, Delivers Bioactive Tacrolimus Transdurally to the CNS in Rat.

Central nervous system (CNS) neurons fail to regrow injured axons, often resulting in permanently lost neurologic function. Tacrolimus is an FDA-approved immunosuppressive drug with known neuroprotective and neuroregenerative properties in the CNS. However, tacrolimus is typically administered systemically and blood levels required to effectively treat CNS injuries can lead to lethal, off-target organ toxicity. Thus, delivering tacrolimus locally to CNS tissues may provide therapeutic control over tacrolimus levels in CNS tissues while minimizing off-target toxicity. Herein we show an electrospun poly(ester urethane) urea and tacrolimus elastomeric matrix (PEUU-Tac) can deliver tacrolimus trans-durally to CNS tissues. In an acute CNS ischemia model in rat, the optic nerve (ON) was clamped for 10s and then PEUU-Tac was used as an ON wrap and sutured around the injury site. Tacrolimus was detected in PEUU-Tac wrapped ONs at 24h and 14days, without significant increases in tacrolimus blood levels. Similar to systemically administered tacrolimus, PEUU-Tac locally decreased glial fibrillary acidic protein (GFAP) at the injury site and increased growth associated protein-43 (GAP-43) expression in ischemic ONs from the globe to the chiasm, consistent with decreased astrogliosis and increased retinal ganglion cell (RGC) axon growth signaling pathways. These initial results suggest PEUU-Tac is a biocompatible elastic matrix that delivers bioactive tacrolimus trans-durally to CNS tissues without significantly increasing tacrolimus blood levels and off-target toxicity.

Urinary bladder extracellular matrix hydrogels and matrix-bound vesicles differentially regulate central nervous system neuron viability and axon growth and branching.

Central nervous system neurons often degenerate after trauma due to the inflammatory innate immune response to injury, which can lead to neuronal cell death, scarring, and permanently lost neurologic function. Extracellular matrix bioscaffolds, derived by decellularizing healthy tissues, have been widely used in both preclinical and clinical studies to promote positive tissue remodeling, including neurogenesis, in numerous tissues, with extracellular matrix from homologous tissues often inducing more positive responses. Extracellular matrix hydrogels are liquid at room temperature and enable minimally invasive extracellular matrix injections into central nervous system tissues, before gelation at 37℃. However, few studies have analyzed how extracellular matrix hydrogels influence primary central nervous system neuron survival and growth, and whether central nervous system and non-central nervous system extracellular matrix specificity is critical to neuronal responses. Urinary bladder extracellular matrix hydrogels increase both primary hippocampal neuron survival and neurite growth to similar or even greater extents, suggesting extracellular matrix from non-homologous tissue sources, such as urinary bladder matrix-extracellular matrix, may be a more economical and safer alternative to developing central nervous system extracellular matrices for central nervous system applications. Additionally, we show matrix-bound vesicles derived from urinary bladder extracellular matrix are endocytosed by hippocampal neurons and positively regulate primary hippocampal neuron neurite growth. Matrix-bound vesicles carry protein and RNA cargos, including noncoding RNAs and miRNAs that map to the human genome and are known to regulate cellular processes. Thus, urinary bladder matrix-bound vesicles provide natural and transfectable cargoes which offer new experimental tools and therapeutic applications to study and treat central nervous system neuron injury.

Extracellular Vesicles: Biomarkers, Therapeutics, and Vehicles in the Visual System.

PURPOSE: We discuss recent advances in extracellular vesicle (EV) technology as biomarkers, therapeutics, and drug delivery vehicles in the visual system with an emphasis on the retina. RECENT FINDINGS: Retinal cell-type specific EVs can be detected in the blood and in the aqueous humor and EV miRNA cargoes can be used diagnostically to predict retinal disease progression. Studies have now shown EVs can deliver bioactive miRNA and AAV cargoes to the inner retinal cell layers and, in some models, improve retinal ganglion cell (RGC) survival and axon regeneration. SUMMARY: EV molecular profiles and cargoes are attractive biomarkers for retinal and optic nerve disease and trauma and EVs offer a safe and tunable platform for delivering therapies to ocular tissues. However, EVs are heterogeneous by nature with variable lipid membranes, cargoes, and biologic effects, warranting stringent characterization to understand how heterogeneous EV populations modulate positive tissue remodeling.

Developing Extracellular Matrix Technology to Treat Retinal or Optic Nerve Injury(1,2,3).

Adult mammalian CNS neurons often degenerate after injury, leading to lost neurologic functions. In the visual system, retinal or optic nerve injury often leads to retinal ganglion cell axon degeneration and irreversible vision loss. CNS axon degeneration is increasingly linked to the innate immune response to injury, which leads to tissue-destructive inflammation and scarring. Extracellular matrix (ECM) technology can reduce inflammation, while increasing functional tissue remodeling, over scarring, in various tissues and organs, including the peripheral nervous system. However, applying ECM technology to CNS injuries has been limited and virtually unstudied in the visual system. Here we discuss advances in deriving fetal CNS-specific ECMs, like fetal porcine brain, retina, and optic nerve, and fetal non-CNS-specific ECMs, like fetal urinary bladder, and the potential for using tissue-specific ECMs to treat retinal or optic nerve injuries in two platforms. The first platform is an ECM hydrogel that can be administered as a retrobulbar, periocular, or even intraocular injection. The second platform is an ECM hydrogel and polymer "biohybrid" sheet that can be readily shaped and wrapped around a nerve. Both platforms can be tuned mechanically and biochemically to deliver factors like neurotrophins, immunotherapeutics, or stem cells. Since clinical CNS therapies often use general anti-inflammatory agents, which can reduce tissue-destructive inflammation but also suppress tissue-reparative immune system functions, tissue-specific, ECM-based devices may fill an important need by providing naturally derived, biocompatible, and highly translatable platforms that can modulate the innate immune response to promote a positive functional outcome.

Promoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles.

Filopodia are 5-10 µm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration. From the clinical editor: Mechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.

Regulation of intrinsic axon growth ability at retinal ganglion cell growth cones.

PURPOSE: Mammalian central nervous system neurons fail to regenerate after injury or disease, in part due to a progressive loss in intrinsic axon growth ability after birth. Whether lost axon growth ability is due to limited growth resources or to changes in the axonal growth cone is unknown. METHODS: Static and time-lapse images of purified retinal ganglion cells (RGCs) were analyzed for axon growth rate and growth cone morphology and dynamics without treatment and after manipulating Kruppel-like transcription factor (KLF) expression or applying mechanical tension. RESULTS: Retinal ganglion cells undergo a developmental switch in growth cone dynamics that mirrors the decline in postnatal axon growth rates, with increased filopodial adhesion and decreased lamellar protrusion area in postnatal axonal growth cones. Moreover, expressing growth-suppressive KLF4 or growth-enhancing KLF6 transcription factors elicits similar changes in postnatal growth cones that correlate with axon growth rates. Postnatal RGC axon growth rate is not limited by an inability to achieve axon growth rates similar to embryonic RGCs; indeed, postnatal axons support elongation rates up to 100-fold faster than postnatal axonal growth rates. Rather, the intrinsic capacity for rapid axon growth is due to both growth cone pausing and retraction, as well as to a slightly decreased ability to achieve rapid instantaneous rates of forward progression. Finally, we observed that RGC axon and dendrite growth are regulated independently in vitro. CONCLUSIONS: Together, these data support the hypothesis that intrinsic axon growth rate is regulated by an axon-specific growth program that differentially regulates growth cone motility.

Mitochondrial Dynamics in Retinal Ganglion Cell Axon Regeneration and Growth Cone Guidance.

Failed axon regeneration and retinal ganglion cell (RGC) death after trauma or disease, including glaucomatous and mitochondrial optic neuropathies, are increasingly linked to mitochondrial dysfunction. Mitochondria are highly dynamic organelles whose size, organization, and function are regulated by a balance between mitochondrial fission and fusion. Mitochondria are ubiquitous in axonal growth cones both in vitro and in vivo and during development and regeneration. However, the roles that mitochondrial fission and fusion dynamics play in the growth cone during axon regeneration are largely unstudied. Here we discuss recent data suggesting mitochondria in the distal axon and growth cone play a central role in axon growth by integrating intrinsic axon growth states with signaling from extrinsic cues. Mitochondrial fission and fusion are intrinsically regulated in the distal axon in the growth cones of acutely purified embryonic and postnatal RGCs with differing intrinsic axon growth potentials. These differences in fission and fusion correlate with differences in mitochondrial bioenergetics; embryonic RGCs with high intrinsic axon growth potential rely more on glycolysis whereas RGCs with low intrinsic axon growth potential rely more on oxidative phosphorylation. Mitochondrial fission and fusion are also differentially modulated by KLFs that either promote or suppress intrinsic axon growth, and altering the balance between mitochondrial fission and fusion can differentially regulate axon growth rate and growth cone guidance responses to both inhibitory and permissive guidance cues.

Signaling endosomes and growth cone motility in axon regeneration.

During development and regeneration, growth cones guide neurites to their targets by altering their motility in response to extracellular guidance cues. One class of cues critical to nervous system development is the neurotrophins. Neurotrophin binding to their cognate receptors stimulates their endocytosis into signaling endosomes. Current data indicate that the spatiotemporal localization of signaling endosomes can direct diverse processes regulating cell motility, including membrane trafficking, cytoskeletal remodeling, adhesion dynamics, and local translation. Recent experiments manipulating signaling endosome localization in neuronal growth cones support these views and place the neurotrophin signaling endosome in a central role regulating growth cone motility during axon growth and regeneration.

Mitochondrial dynamics regulate growth cone motility, guidance, and neurite growth rate in perinatal retinal ganglion cells in vitro.

PURPOSE: Retinal ganglion cell (RGC) death and failed axonal regeneration after trauma or disease, including glaucomatous and mitochondrial optic neuropathies, are linked increasingly to dysfunctional mitochondrial dynamics. However, how mitochondrial dynamics influence axon growth largely is unstudied. We examined intrinsic mitochondrial organization in embryonic and postnatal RGCs and the roles that mitochondrial dynamics have in regulating neurite growth and guidance. METHODS: RGCs were isolated from embryonic day 20 (E20) or postnatal days 5 to 7 (P5-7) Sprague-Dawley rats by anti-Thy1 immunopanning. After JC-1 loading, mitochondria were analyzed in acutely purified RGCs by flow cytometry and in RGC neurites by fluorescence microscopy. Intrinsic axon growth was modulated by overexpressing Krüppel-like family (KLF) transcription factors, or mitochondrial dynamics were altered by inhibiting dynamin related protein-1 (DRP-1) pharmacologically or by overexpressing mitofusin-2 (Mfn-2). Mitochondrial organization, neurite growth, and growth cone motility and guidance were analyzed. RESULTS: Mitochondrial dynamics and function are regulated developmentally in acutely purified RGCs and in nascent RGC neurites. Mitochondrial dynamics are modulated differentially by KLFs that promote or suppress growth. Acutely inhibiting mitochondrial fission reversibly suppressed axon growth and lamellar extension. Inhibiting DRP-1 or overexpressing Mfn-2 altered growth cone responses to chondroitin sulfate proteoglycan, netrin-1, and fibronectin. CONCLUSIONS: These results support the hypothesis that mitochondria locally modulate signaling in the distal neurite and growth cone to affect the direction and the rate of neurite growth.

Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth.

Understanding neurite growth regulation remains a seminal problem in neurobiology. During development and regeneration, neurite growth is modulated by neurotrophin-activated signaling endosomes that transmit regulatory signals between soma and growth cones. After injury, delivering neurotrophic therapeutics to injured neurons is limited by our understanding of how signaling endosome localization in the growth cone affects neurite growth. Nanobiotechnology is providing new tools to answer previously inaccessible questions. Here, we show superparamagnetic nanoparticles (MNPs) functionalized with TrkB agonist antibodies are endocytosed into signaling endosomes by primary neurons that activate TrkB-dependent signaling, gene expression and promote neurite growth. These MNP signaling endosomes are trafficked into nascent and existing neurites and transported between somas and growth cones in vitro and in vivo. Manipulating MNP-signaling endosomes by a focal magnetic field alters growth cone motility and halts neurite growth in both peripheral and central nervous system neurons, demonstrating signaling endosome localization in the growth cone regulates motility and neurite growth. These data suggest functionalized MNPs may be used as a platform to study subcellular organelle localization and to deliver nanotherapeutics to treat injury or disease in the central nervous system.

Three functionally distinct adhesions in filopodia: shaft adhesions control lamellar extension.

In this study, adhesions on individual filopodial shafts were shown to control veil (lamellar) advance and to be modulated by guidance cues. Adhesions were detected in individual filopodia of sensory growth cones using optical recordings, adhesion markers, and electron microscopy. Veils readily advanced along filopodia lacking shaft adhesions but rarely advanced along filopodia displaying shaft adhesions. Experiments altering adhesion showed that this relationship is not caused by veils removing adhesions as they advanced. Reducing adhesion with antibodies decreased the proportion of filopodia with shaft adhesions and coordinately increased veil advance. Moreover, the inhibitory relationship was maintained: veils still failed to advance on individual filopodia that retained shaft adhesions. These results support the idea that shaft adhesions inhibit veil advance. Of particular interest, guidance cues can act by altering shaft adhesions. When a cellular cue was contacted by a filopodial tip, veil extension and shaft adhesions altered in concert. Contact with a Schwann cell induced veil advance and inhibited shaft adhesions. In contrast, contact with a posterior sclerotome cell prohibited veil advance and promoted shaft adhesions. These results show that veil advance is controlled by shaft adhesions and that guidance signal cascades can alter veil advance by altering these adhesions. Shaft adhesions thus differ functionally from two other adhesions identified on individual filopodia. Tip adhesions suffice to signal. Basal adhesions do not influence veil advance but are critical to filopodial initiation and dynamics. Individual growth cone filopodia thus develop three functionally distinct adhesions that are vital for both motility and navigation.