Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.

While major inroads have been made in identifying the genetic causes of rare Mendelian disorders, little progress has been made in the discovery of common gene variations that predispose to complex diseases. The single gene variants that have been shown to associate reproducibly with complex diseases typically have small effect sizes or attributable risks. However, the joint actions of common gene variants within pathways may play a major role in predisposing to complex diseases (the paradigm of complex genetics). The goal of this study was to determine whether polymorphism in a candidate pathway (axon guidance) predisposed to a complex disease (Parkinson disease [PD]). We mined a whole-genome association dataset and identified single nucleotide polymorphisms (SNPs) that were within axon-guidance pathway genes. We then constructed models of axon-guidance pathway SNPs that predicted three outcomes: PD susceptibility (odds ratio = 90.8, p = 4.64 x 10(-38)), survival free of PD (hazards ratio = 19.0, p = 5.43 x 10(-48)), and PD age at onset (R(2) = 0.68, p = 1.68 x 10(-51)). By contrast, models constructed from thousands of random selections of genomic SNPs predicted the three PD outcomes poorly. Mining of a second whole-genome association dataset and mining of an expression profiling dataset also supported a role for many axon-guidance pathway genes in PD. These findings could have important implications regarding the pathogenesis of PD. This genomic pathway approach may also offer insights into other complex diseases such as Alzheimer disease, diabetes mellitus, nicotine and alcohol dependence, and several cancers.

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, vertebrate fish spinal cord tissue regenerates significantly to restore function. Cord transection in zebrafish (Danio rerio) initially causes paralysis and neural cell death. Subsequently, ependymal glia proliferate, bipolar glia extend across the lesion, and new neurons are born; axons from spared and nascent neurons extend along trans-lesional glial bridges to restore functional connectivity. Here we report that glucocorticoids, used in the clinical management of spinal cord injury, directly inhibit neural repair by targeting ependymal glia independently of hematogenous cells and microglia. After transecting injury, the glucocorticoid receptor in ependymal glia is regulated differentially in zebrafish (becoming inactive) vs. the rat (becoming active). Glucocorticoid blockade of neural regeneration via a direct effect on ependymal glia has important therapeutic implications for the putative benefit of corticosteroids in early management of spinal cord injury.

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

We have previously demonstrated that high concentrations of nerve growth factor suppress neurite outgrowth from sensory neurons. Inhibition could be mediated by either the p75NTR or TrkA receptor. We used a functional block of p75NTR by REX antibody in rat dorsal root ganglion neurons and dorsal root ganglion cultures from p75NTR knockout mice. In both systems, high-dose NGF inhibited neurite outgrowth, implying that p75NTR is not involved in suppression of neurite outgrowth. Confocal images of dissociated dorsal root ganglion neurons exposed to fluorescence-tagged NGF showed ligand internalization. Radioligand binding indicated disappearance of high-affinity binding sites from the surface of dorsal root ganglia after treatment with 200 ng/ml NGF for 1 h. Downstream signaling showed sustained hyperphosphorylation of MAPK (Erk(1-2)) but not of SNT or Akt. High-dose NGF may induce cytoplasmic relocation of the receptor TrkA and axonal growth arrest independently of p75NTR.

Cisplatin is primarily used for treatment of ovarian and testicular cancer. Oxaliplatin is the only effective treatment for metastatic colorectal cancer. Both are known to cause dose related, cumulative toxic effects on the peripheral nervous system and thirty to forty percent of cancer patients receiving these agents experience painful peripheral neuropathy. The mechanisms underlying painful platinum-induced neuropathy remain poorly understood. Previous studies have demonstrated important roles for TRPV1, TRPM8, and TRPA1 in inflammation and nerve injury induced pain.

The spinal cord has a poor ability to regenerate after an injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treating a spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface on the other side. When the cells are cultured on OPF via chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to that of the multichannel scaffold control, which is likely due to the greater number of axons growing across it. The immune cell number (microglia or hemopoietic cells: 50-120 cells/mm2 in all conditions), scarring (5-10% in all conditions), and ECM deposits (Laminin or Fibronectin: approximately 10-20% in all conditions) were equal in all conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery. This study provides a hydrogel scaffold construct that can be used in vitro for cell characterization or in vivo for future neuroprosthetics, devices, or cell and ECM delivery.

Cisplatin has been in use for 40 years for treatment of germ line and other forms of cancer. Oxaliplatin is approved for treatment of metastatic colorectal cancer. Thirty to forty percent of cancer patients receiving these agents develop pain and sensory loss. Oxaliplatin induces distinctive cold-associated dysesthesias in up to 80% of patients.

Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases.

Drosophila melanogaster has recently been developed as a simple, in vivo, genetic model of chemotherapy-induced peripheral neuropathy. Flies treated with the chemotherapy agent cisplatin display both a neurodegenerative phenotype and cell death in rapidly dividing follicles, mimicking the cell specific responses seen in humans. Cisplatin induces climbing deficiencies and loss of fertility in a dose dependent manner. Drosophila sensitivity to cisplatin in both cell types is affected by genetic background. We show that mutation or RNAi-based knockdown of genes known to be associated with CIPN incidence in humans affect sensitivity of flies to CIPN. Drosophila is a promising model with which to study the effect of genetics on sensitivity to CIPN.

To determine the safety and efficacy of mesenchymal stem cell (MSC)-neurotrophic factor (NTF) cells (NurOwn®, autologous bone marrow-derived MSCs, induced to secrete NTFs) delivered by combined intrathecal and intramuscular administration to participants with amyotrophic lateral sclerosis (ALS) in a phase 2 randomized controlled trial.

Mechano growth factor (MGF) is a splice variant of IGF-1 first described in skeletal muscle. MGF induces muscle cell proliferation in response to muscle stress and injury. In control mice we found endogenous expression of MGF in neurogenic areas of the brain and these levels declined with age. To better understand the role of MGF in the brain, we used transgenic mice that constitutively overexpressed MGF from birth. MGF overexpression significantly increased the number of BrdU+ proliferative cells in the dentate gyrus (DG) of the hippocampus and subventricular zone (SVG). Although MGF overexpression increased the overall rate of adult hippocampal neurogenesis at the proliferation stage it did not alter the distribution of neurons at post-mitotic maturation stages. We then used the lac-operon system to conditionally overexpress MGF in the mouse brain beginning at 1, 3 and 12 months with histological and behavioral observation at 24 months of age. With conditional overexpression there was an increase of BrdU+ proliferating cells and BrdU+ differentiated mature neurons in the olfactory bulbs at 24 months when overexpression was induced from 1 and 3 months of age but not when started at 12 months. This was associated with preserved olfactory function. In vitro, MGF increased the size and number of neurospheres harvested from SVZ-derived neural stem cells (NSCs). These findings indicate that MGF overexpression increases the number of neural progenitor cells and promotes neurogenesis but does not alter the distribution of adult newborn neurons at post-mitotic stages. Maintaining youthful levels of MGF may be important in reversing age-related neuronal loss and brain dysfunction.

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1) s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1) growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+)-imaging revealed local elevation of cytoplasmic Ca(2+) concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+) signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.

Spinal cord injury (SCI) results in cell death, demyelination, and axonal loss. The spinal cord has a limited ability to regenerate, and current clinical therapies for SCI are not effective in helping promote neurologic recovery. We have developed a novel scaffold biomaterial that is fabricated from the biodegradable hydrogel oligo(poly(ethylene glycol)fumarate) (OPF). We have previously shown that positively charged OPF scaffolds (OPF+) in an open spaced, multichannel design can be loaded with Schwann cells to support axonal generation and functional recovery following SCI. We have now developed a hybrid OPF+ biomaterial that increases the surface area available for cell attachment and that contains an aligned microarchitecture and extracellular matrix (ECM) proteins to better support axonal regeneration. OPF+ was fabricated as 0.08 mm thick sheets containing 100 μm high polymer ridges that self-assemble into a spiral shape when hydrated. Laminin, fibronectin, or collagen I coating promoted neuron attachment and axonal outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and neurites aligned in the direction of the ridge. Schwann cells seeded on laminin coated OPF+ sheets aligned along the ridges over a 6-day period and could myelinate dorsal root ganglion neurons over 4 weeks. This novel scaffold design, with closer spaced ridges and Schwann cells, is a novel biomaterial construct to promote regeneration after SCI.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a median lifespan of 2-3 years after diagnosis. There are few meaningful treatments that alter progression in this disease. Preclinical and clinical studies have demonstrated that neuroinflammation may play a key role in the progression rate of ALS. Despite this, there are no validated biomarkers of neuroinflammation for use in clinical practice or clinical trials. Biomarkers of neuroinflammation could improve patient management, provide new therapeutic targets, and possibly help stratify clinical trial selection and monitoring. However, attempts to identify a singular cause of neuroinflammation have not been successful. Here, we performed multi-parameter flow cytometry to comprehensively assess 116 leukocyte populations and phenotypes from lymphocytes, monocytes, and granulocytes in a cohort of 80 ALS patients. We identified 32 leukocyte phenotypes that were altered in ALS patients compared to age and gender matched healthy volunteers (HV) that included phenotypes of both inflammation and immune suppression. Unsupervised hierarchical clustering and principle component analysis of ALS and HV immunophenotypes revealed two distinct immune profiles of ALS patients. ALS patients were clustered into a profile distinct from HVs primarily due to differences in a multiple T cell phenotypes, CD3+CD56+ T cells and HLA-DR on monocytes. Patients clustered into an abnormal immune profile were younger, more likely to have a familial form of the disease, and survived longer than those patients who clustered similarly with healthy volunteers (344 weeks versus 184 weeks; p = 0.012). The data set generated from this study establishes an extensive accounting of immunophenotypic changes readily suitable for biomarker validation studies. The extensive immune system changes measured in this study indicate that normal immune homeostatic mechanisms are disrupted in ALS patients, and that multiple immune states likely exist within a population of patients with ALS.

Glioblastoma multiforme (GBM) is the most common primary brain tumor, with a median survival of only 15 months. A subpopulation of cells, the brain tumor stem cells (BTSCs), may be responsible for the malignancy of this disease. Xenografts have proven to be a robust model of human BTSCs, but the effects of long-term passaging have yet to be determined. Here we present a study detailing changes in BTSC multipotency, invasive migration, and proliferation after serial passaging of human GBM xenografts. Immunocytochemistry and tumorsphere formation assays demonstrated the presence of BTSCs in both early generation (EG-BTSCs; less than 15 passages) and late generation (LG-BTSCs; more than 24 passages) xenografts. The EG-BTSCs upregulated expression of lineage markers for neurons and oligodendrocytes upon differentiation, indicating multipotency. In contrast, the LG-BTSCs were restricted to an astrocytic differentiation. Quantitative migration and proliferation assays showed that EG-BTSCs are more migratory and proliferative than LG-BTSCs. However, both populations respond similarly to the chemokine SDF-1 by increasing invasive migration. These differences between the EG- and LG-BTSCs were correlated with a significant decrease in nanog expression as determined by qRT-PCR. Mice implanted intracranially with EG-BTSCs showed shorter survival when compared to LG-BTSCs. Moreover, differentiation prior to implantation of EG-BTSCs, but not LG-BTSCs, led to increased survival. Thus, nanog may identify multipotent BTSCs. Furthermore, limited passaging of xenografts preserves these multipotent BTSCs, which may be an essential underlying feature of GBM lethality.

The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.

Here, we report the effect of newly regenerated axons via scaffolds on reorganization of spinal circuitry and restoration of motor functions with epidural electrical stimulation (EES). Motor recovery was evaluated for 7 weeks after spinal transection and following implantation with scaffolds seeded with neurotrophin producing Schwann cell and with rapamycin microspheres. Combined treatment with scaffolds and EES-enabled stepping led to functional improvement compared to groups with scaffold or EES, although, the number of axons across scaffolds was not different between groups. Re-transection through the scaffold at week 6 reduced EES-enabled stepping, still demonstrating better performance compared to the other groups. Greater synaptic reorganization in the presence of regenerated axons was found in group with combined therapy. These findings suggest that newly regenerated axons through cell-containing scaffolds with EES-enabled motor training reorganize the sub-lesional circuitry improving motor recovery, demonstrating that neuroregenerative and neuromodulatory therapies cumulatively enhancing motor function after complete SCI.