Elena S. Pak

Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in various animal models of neurodegenerative disease; however, few studies have examined the ability of ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to examine the ability of pre-differentiated murine ES cells (neuronal and glial precursors) to improve functional outcome. Rats were prepared with a unilateral controlled cortical impact injury or sham and then transplanted 7 days later with 100K ES cells (WW6G) (~30% neurons) or media. Two days following transplantation rats were tested on a battery of behavioral tests. It was found that transplantation of ES cells improved behavioral outcome by reducing the initial magnitude of the deficit on the bilateral tactile removal and locomotor placing tests. ES cells also induced almost complete recovery on the vibrissae --> forelimb placing test, whereas, media-transplanted rats failed to show recovery. Acquisition of a reference memory task in the Morris water maze was not improved by transplantation of ES cells. Histological analysis revealed a large number of surviving ES cells in the lesion cavity and showed migration of ES cells into subcortical structures. It was found that transplantation of ES cells prevented the occurrence of multiple small necrotic cavities that were seen in the cortex adjacent to the lesion cavity in media transplanted rats. Additionally, ES cells transplants also significantly reduced lesion size. Results of this study suggest that ES cells that have been pre-differentiated into neuronal precursors prior to transplantation have therapeutic potential.

RNAi pathway is functional in peripheral nerve axons

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase—components of the RNA‐induced silencing complex (RISC). Application of short‐interfering RNAs against neuronal β‐tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal β‐tubulin III mRNA and corresponding protein, as well as a signifi‐cant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is func‐tional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.—Murashov, A. K., Chintalgattu, V., Islamov, R. R., Lever, T. E., Pak, E. S., Sierpinski, P. L., Katwa, L. C., Van Scott, M. R. RNAi pathway is functional in peripheral nerve axons. FASEB J. 21, 656–670 (2007)

Directed differentiation of embryonic stem cells into dorsal interneurons

During neural development caudalization and dorsoventral patterning of the neural tube is directed by several inductive factors including retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt signaling. The purpose of the current study was to investigate whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals. Here we show that specific combination of developmental signaling molecules including all trans‐retinoic acid, Shh, bone morphogenetic protein 2 (BMP2), and Wnt3A can direct differentiation of ES cells into dorsal interneurons possessing appropriate neuronal markers, synaptic proteins and functional neurotransmitter machineries. We introduce a concept that Wnt3A morphogenic action relies on crosstalk with both Shh and BMP2 signaling pathways.

17β‐Estradiol enhances neuronal differentiation of mouse embryonic stem cells

Existing protocols show a variety in the percentage of neurons that can be generated from mouse embryonic stem (ES) cells. In the current study, we compared effects of various differentiating conditions, including gelatin and poly‐l‐ornithine/fibronectin coatings, and NGF and 17β‐estradiol treatments on the total yield of neurons, as well as, neurite growth and branching. Here, we show that combination of fibronectin coating with 17β‐estradiol increased number of generated neurons over 50%. Poly‐l‐ornithine/fibronectin increased the percent of neurons in all cultures, suggesting its direct influence on neurogenesis. Addition of 17β‐estradiol reduced mean neurite length in culture, but significantly increased branching. Our results indicate a substrate‐dependent regulation of estrogen‐induced ES cells differentiation into neuronal cells.

Effect of 17β-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-alpha and estrogen receptor-beta localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-alpha and estrogen receptor-beta mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-alpha and -beta mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i). axotomized motor neurons increase expression of estrogen receptors-alpha and -beta mRNA, (ii). estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii). estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in many animal models of injury and disease. However, few studies have examined the ability of pre-differentiated ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to compare the effect of murine ES cells that were pre-differentiated into GABAergic neurons or astrocytes on functional recovery following TBI. Neural and astrocyte induction was achieved by co-culturing ES cells on a bone marrow stromal fibroblast (M2-10B4) feeder layer and incubating them with various mitogenic factors. Rats were initially prepared with a unilateral controlled cortical contusion injury of the sensorimotor cortex or sham procedure. Rats were transplanted 7 days following injury with approximately 100K GABAergic neurons, astrocytes, fibroblasts, or media. Animals were assessed on a battery of sensorimotor tasks following transplantation. The stromal fibroblast cells (M2-10B4), as a control cell line, did not differ significantly from media infusions. Transplantation of GABAergic neurons facilitated complete and total recovery on the vibrissae-forelimb placing test as opposed to all other groups, which failed to show any recovery. It was also found that GABAergic neurons reduced the magnitude of the initial impairment on the limb use test. Histological analysis revealed infiltration of host brain with transplanted neurons and astrocytes. The results of the present study suggest that transplantation of pre-differentiated GABAergic neurons significantly induces recovery of sensorimotor function; whereas, astrocytes do not.

Paternal long-term exercise programs offspring for low energy expenditure and increased risk for obesity in mice

Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin‐resistant phenotype. Although diet‐induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long‐term voluntary wheel‐running in C57BL/6J male mice on their offsprings predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel‐running for 12 wk produced offspring that were more susceptible to the adverse effects of a high‐fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long‐term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro‐RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA‐induced modification of sperm.—Murashov, A. K., Pak, E. S., Koury, M., Ajmera, A., Jeyakumar, M., Parker, M., Williams, O., Ding, J., Walters, D., Neufer, P. D. Paternal long‐term exercise programs offspring for low energy expenditure and increased risk for obesity in mice. FASEB J. 30, 775–784 (2016). www.fasebj.org

Multi-walled carbon nanotubes inhibit regenerative axon growth of dorsal root ganglia neurons of mice

Recent observations have demonstrated that nanomaterials may be toxic to human tissue. While the ability of nano-scaled particulate matter is known to cause a range of problems in respiratory system, recent observations suggest that the nervous system may be vulnerable as well. In the current paper we asked whether exposure of primary neuronal cell cultures to nanoparticles might compromise regenerative axon growth. Regenerative response was triggered by performing a conditioning lesion of sciatic nerve five days prior to collection of dorsal root ganglia (DRG). DRG neurons were plated at a low density and incubated with multi-walled carbon nanotubes (MWCNTs) (0.1-10 μg/ml in 10% of surfactant in saline) overnight. The experiments showed that exposure of DRG cultures to MWCNT significantly impaired regenerative axonogenesis without concomitant cell death. These results indicate that MWNCTs may have detrimental effect on nerve regeneration and may potentially trigger axonal pathology.

Differential expression of endothelin receptors in regenerating spinal motor neurons in mice.

On day 4 after sciatic nerve crush injury, expression and localization of endothelin receptors ET(A) and ET(B) in the lumbar spinal cord were examined. Immunohistochemical staining with antibodies to ET(A) and ET(B) receptors showed cytoplasmic distribution of ET(A) receptors in motor neurons, whereas ET(B) receptors were localized in the perinuclear region. On the injured side of the lumbar spinal cord, when compared to contralateral, results demonstrated an up-regulation of ET(B) and a down-regulation of ET(A) receptors expression at the level of both mRNA and protein. These results suggest that ET(B) receptors may play a role in the regeneration of axotomized motor neurons.

miRNA-431 Prevents Amyloid-β-Induced Synapse Loss in Neuronal Cell Culture Model of Alzheimer's Disease by Silencing Kremen1

Synapse loss is well regarded as the underlying cause for the progressive decline of memory function over the course of Alzheimers disease (AD) development. Recent observations suggest that the accumulation of the Wnt antagonist Dickkopf-1 (Dkk1) in the AD brain plays a critical role in triggering synaptic degeneration. Mechanistically, Dkk1 cooperates with Kremen1 (Krm1), its transmembrane receptor, to block the Wnt/β-catenin signaling pathway. Here, we show that silencing Krm1 with miR-431 prevents amyloid-β-mediated synapse loss in cortico-hippocampal cultures isolated from triple transgenic 3xTg-AD mice. Exposure to AβDDL (an amyloid-β derived diffusive ligand) or Dkk1 reduced the number of pre- and post-synaptic puncta in primary neuronal cultures, while treatment with miR-431 prevented synapse loss. In addition, treatment with miR-431 also prevented neurite degeneration. Our findings demonstrate that miR-431 protects synapses and neurites from Aβ-toxicity in an AD cell culture model and may be a promising therapeutic target.