Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) an... more Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) and cell discharge (population spike) were analyzed in the dentate gyrus of the rat following repetitive low-frequency stimulation of the medial and lateral entorhinal cortex. Stimulation of either subdivision of the entorhinal projection system resulted in comparable habituation-like response decrements with similar stimulation regimens. However, habituating stimulation of one subdivision did not result in decreased responsiveness to stimulation of the other. Repetitive lowfrequency stimulation or even a single pulse delivered to either subdivision did, however, result in a potentiation of granule cell discharge in response to stimulation of the other subdivision (a form of heterosynaptic potentiation). This heterosynaptic potentiation of granule cell discharge was not accompanied by any increase in the extracellular EPSP. Comparisons of the relationship between the population EPSP and population spike before and during habituating stimulation revealed changes in cell discharge in response to the habituating stimulus which could not be accounted for by changes in synaptic activation alone. The results suggest that repetitive activation of the temporodentate pathway alters granule cell output as a result of two processes, a habituation-like decrement in synaptic activation, and a potentiation of granule cell discharge as a consequence of prior activation.
Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylatio... more Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/ 236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.
ABSTRACTInjury responses require communication between different cell types in the skin. Sensory ... more ABSTRACTInjury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of privileged translation in vitro and in vivo. Inflammatory cues promote local translation of Arc in the skin. Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found...
While interest toward caloric restriction (CR) in various models of brain injury has increased in... more While interest toward caloric restriction (CR) in various models of brain injury has increased in recent decades, studies have predominantly focused on the benefits of chronic or intermittent CR. The effects of ultra-short, including overnight, CR on acute ischemic brain injury, however, are not well studied. Here, we show that overnight caloric restriction (75% over 14 h) prior to asphyxial cardiac arrest and resuscitation (CA) improves survival and neurological recovery, including complete prevention of neurodegeneration in multiple regions of the brain. We also show that overnight CR normalizes stress-induced hyperglycemia, while significantly decreasing insulin and glucagon production and increasing corticosterone and ketone body production. The benefits seen with ultra-short CR appear independent of Sirtuin 1 (SIRT-1) and brain-derived neurotrophic factor (BDNF) expression, which have been strongly linked to neuroprotective benefits seen in chronic CR. Further exploration into ...
Cover: A hippocampal neuron grown in culture. The neuron is immunostained with an antibody to the... more Cover: A hippocampal neuron grown in culture. The neuron is immunostained with an antibody to the microtubule associated protein MAP2. Courtesy G.A. Banker. Library of Congress Cataloging-in-Publication Data Steward, Oswald. Principles of cellular. molecular, and developmental neuroscience/ Oswald Steward. p. cm. Includes bibliographies and index.
Demyelination contributes to loss of function after spinal cord injury, and thus a potential ther... more Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.
Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part ... more Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part of chromosome 18 (chr18) is a major determinant of vulnerability to hippocampal neurodegeneration following kainic acid (KA)-induced seizures in inbred mouse strains. We assessed excitotoxic vulnerability in two congenic, "genome tagged" mouse strains carrying segments of either distal or proximal/medial chr18 from vulnerable DBA/2J mice on a resistant C57BL/6 background. Systemic KA injections triggered brain-wide neurodegeneration in the distal chr18 congenic strain, and specifically in the hilus of the dentate gyrus, but not in CA3. In contrast, the proximal/medial chr18 congenic strain exhibited enhanced degeneration in CA1 and CA3, but little neurodegeneration elsewhere. Both strains exhibited low levels of QUIN-induced striatal neurodegeneration comparable to what is seen in C57BL/6 mice. These results suggest that gene(s) on distal chr18 are important determinants of vulnerability to KA-induced hippocampal neurodegeneration, but not QUIN-induced striatal neurodegeneration.
Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of in... more Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. Adult rats received unilateral intra-cortical injections of AAV expressing shRNA against PTEN and were allowed to survive for 2 months. Immunostaining for c-fos under resting conditions (home cage, HC) and after 1hr of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphosphorylation of ERK1/2.
et al., Regulatory T cells promote remyelination in the murine experimental autoimmune encephalom... more et al., Regulatory T cells promote remyelination in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis following human neural stem cell transplant, Neurobiology of Disease (2019),
In mice, the genetic background determines susceptibility to hippocampal neurodegeneration induce... more In mice, the genetic background determines susceptibility to hippocampal neurodegeneration induced by the excitotoxin kainic acid (KA). If genetic background plays as significant a role in the striatum, the area most affected in Huntington's disease (HD), it is important to characterize intrinsic differences in neuronal susceptibility in mouse strains used in HD models. This study was performed to investigate whether strain differences of different HD mouse models are determinants of striatal resistance to excitotoxicity. We conducted a survey of the susceptibility of striatal neurons to neurodegeneration induced by quinolinic acid and KA in six inbred, two outbred and two F1 hybrid (resistant*vulnerable) strains. These are the same strains in which we have assessed vulnerability to KA-induced hippocampal neurodegeneration. We found significant strain differences in response to both excitotoxins and, for the most part, the strain-dependent patterns of susceptibility to quinolinic acid and KA were similar and comparable to those previously found with KA-induced hippocampal neurodegeneration. There were some incongruities, suggesting that the genetic determinants may be different for the two forms of excitotoxicity or that there are important interacting factors. For example, the F1 hybrid strains exhibited neurodegeneration similar to their vulnerable parent, indicating that the vulnerable phenotype is dominant. This is in contrast to KA-induced hippocampal neurodegeneration, where F1 hybrids exhibit the resistant phenotype. These results are also of significance with regard to the issue of region-specific vulnerability in the context of different diseases in which genetic modifiers affect age of onset and ⁄ or disease progression.
Over the past century, the question of whether the cells of origin of the corticospinal tract (CS... more Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent study reported an approximately 20% loss of retrogradely labeled cortical motoneurons following damage to their axons resulting from SCI at T9 (Hains et al. [2003] J. Comp. Neurol. 462:328-341). In follow-up studies, however, we failed to find any evidence of loss of CST axons in the medullary pyramid, which must occur if CST neurons die. Here, we seek to resolve the discrepancy by re-evaluating possible loss of CST neurons using the same techniques as Hains et al. (quantitative analysis of retrograde labeling and staining for cell death markers including TUNEL and Hoechst labeling of the nuclei). Following either dorsal funiculus lesions at thoracic level 9 (T9) or lateral hemisection at cervical level 5 (C5), our results reveal no evidence for a loss of retrogradely labeled neurons and no evidence for TUNEL staining of axotomized cortical motoneurons. These results indicate that CST cell bodies do not undergo retrograde cell death following SCI, and therefore targeting such cell death is not a valid therapeutic target.
Previous studies in mice have demonstrated that forepaw gripping ability, as measured by a grip s... more Previous studies in mice have demonstrated that forepaw gripping ability, as measured by a grip strength meter (GSM), is dependent on the contralateral sensorimotor cortex, but this dependency changes after hemisection injury at cervical level 4 (C4). Initially, the mouse fails to grip with the forepaw ipsilateral to the hemisection but gripping recovers. Additionally, a mouse's gripping by the contralateral paw becomes independent of the sensorimotor cortex, indicating a reorganization of cortical control of gripping function (Blanco et al, 2007). Here we explore whether a similar reorganization occurs after cervical hemisection injuries in rats. We show that as in mice, unilateral lesions of the sensorimotor cortex impair rats' griping by the contralateral paw. We also confirm from previous studies that cervical hemisections in the impair rats' griping by the ipsilateral paw. In contrast to mice, however there is minimal recovery of gripping after complete lateral hemisections and secondary lesions of the sensorimotor cortex continue to impair rats' gripping by the contralateral paw. Thus, forelimb gripping ability as measured by the GSM is dependent on the contralateral sensorimotor cortex in rats even after a cervical hemisection.
In situ hybridization was used to assess the subcellular distribution of rRNA and poly(A) RNA in ... more In situ hybridization was used to assess the subcellular distribution of rRNA and poly(A) RNA in hippocampal neurons maintained in culture. Labeling produced with 35S-labeled probes to either rRNA or poly(A) was heaviest over the cell body with lighter, patchy labeling of proximal dendrites. In contrast, 3H-labeled probes labeled dendrites throughout their length, and the ratio of dendritic to cell body labeling was higher with 3H-labeled probes. There was no detectable labeling of axons of mature neurons with either probe. The pattern of hybridization produced by 35S-labeled oligonucleotide probes to rRNA varied depending on the concentration of the oligonucleotide. These studies provide the first detailed study of the subcellular distribution of rRNA and poly(A) RNA in neurons, and highlight technical issues to consider when evaluating results of hybridizations carried out with 35S- and 3H-labeled probes on cells in culture.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1993
This study evaluates whether protein synthesis plays a role in targeting RNA molecules to differe... more This study evaluates whether protein synthesis plays a role in targeting RNA molecules to different subcellular domains within neurons. Transport of newly synthesized RNA (labeled with [3HJuridine) was emined in the presence of the protein synthesis inhibitors puromycin and cycloheximide. In situ hybridization was used to determine whether inhibition of protein synthesis altered the subcellular distribution ofmRNAs. Transport ofrecently synthesized RNA was not disrupted after prolonged exposure to either inhibitor. However, inhibition ofprotein synthesis caused several mRNAs that are normally confined to the cell body to appear in dendrites. The distribution of mRNAs that are normally present in dendrites was unaffected. These findings suggest that protein synthesis is not required to translocate RNA into the dendrites but may play a role in restricting particular mRNAs to the neuronal cell body. Abbreviations: DMSO, dimethyl sulfoxide; MAP2, microtubuleassociated protein 2; GAP-43, growth-associated protein 43; cRNA, complementary RNA.
Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervou... more Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system (CNS) and for which there is no cure. Current treatments focus on suppression of the immune system but fail to repair the resulting damage to the CNS. Neural stem cell (NSC) transplantation is a promising therapeutic strategy for treating neurodegenerative diseases through cell replacement and repair however it is unclear how these cells would mediate repair in MS. We report that human NSCs promote CNS specific T regulatory cells (Tregs) which activate endogenous repair pathways and promote remyelination in a murine model of MS. We observed remyelination, decreased inflammation and an increase in (CNS)-infiltrating CD4+CD25+FoxP3+ Tregs in EAE mice receiving an intra-spinal transplant of hNSCs. Recovery was not a result of cell replacement, as hNSCs underwent xenograft rejection, and was Treg dependent, as ablation of Tregs abrogated histopathological improvement. Treg expansion is antigen driven as hNSCs expanded CD25+FoxP3+ Tregs in-vitro when cultured with neural antigen restricted RAG2−/−2D2+ (R2D2) splenocytes but not RAG2−/−OT-II+ splenocytes. When co-cultured with B6 splenocytes, hNSCs drove the expansion of unique TCRs when compared to controls. Additionally, hNSC-Tregs also appear to derive from the exTreg pool suggesting both antigen specific expansion and antigen dependent maintenance of FOXP3 in CNS-specific Tregs. hNSC Tregs also have a unique expression profile and express transglutimase-2 which is implicated in oligodendrocyte dependent repair in the CNS.
It has been recognized since the time of Cajal that different parts of the neuron are specialized... more It has been recognized since the time of Cajal that different parts of the neuron are specialized for different functions; indeed, this is the basis for what Cajal called the “dynamic polarization of the neuron.” Axons are specialized for transmitting information over great distances, dendrites and cell bodies for receiving and integrating inputs from other cells, and synapses for communicating information from one neuron to the next. Each of these functional attributes depends upon two features: (1) the “molecular anatomy” of the neuron, which includes the composition and spatial distribution of receptors, channels, etc. in the neuronal plasma membrane and the intracellular distribution of organelles, second messenger systems, and enzyme systems; and (2) neuronal microstructure, i. e., the shape and spatial configuration of axons, dendrites, and the synaptic connections between them.
This study evaluates the temperature sensitivity of transport of recently synthesized RNA from th... more This study evaluates the temperature sensitivity of transport of recently synthesized RNA from the nucleus to the cytoplasm (nucleocytoplasmic transport) in CNS neurons. Rat hippocampal slices were incubated with [3H]uridine for 1 h to label recently synthesized RNA. Slices were then fixed immediately or maintained at 27°C or 37°C for chase intervals of 3, 4.5, and 6 h to allow for nucleocytoplasmic transport of recently synthesized RNA. The time-dependent translocation of recently synthesized RNA was evaluated autoradiographically. At the end of the i h pulse at either 27°C or 37°C, the label was localized exclusively over nuclei. In slices maintained at 37°C, labeling expanded to cover the cell body and proximal dendrites. However, in slices that were labeled and maintained at room temperature, labeling remained confined to the nucleus. In slices that were pulse-labeled at room temperature, and then transferred to 37°C medium, cytoplasmic labeling increased as a function of time. Nucleocytoplasmic transport of RNA in cultured rat hippocampal neurons showed a comparable temperature sensitivity. The inhibition of nucleocytoplasmic transport of RNA at room temperature provides an opportunity to evaluate neuronal function when no new RNA molecules can reach the cytoplasm.
It has previously been reported that a single dose of amphetamine paired with training on a beam ... more It has previously been reported that a single dose of amphetamine paired with training on a beam walking task can enhance locomotor recovery following brain injury (Feeney et al., 1982). Here, we investigated whether this same drug/training regimen could enhance functional recovery following either thoracic (T9) or cervical (C5) spinal cord injury. Different groups of female Sprague-Dawley rats were trained on a beam walking task, and in a straight alley for assessment of hindlimb locomotor recovery using the BBB locomotor scale. For rats that received C5 hemisections, forelimb grip strength was assessed using a grip strength meter. Three separate experiments assessed the consequences of training rats on the beam walking task 24 h following a thoracic lateral hemisection with administration of either amphetamine or saline. Beginning 1 h following drug administration, rats either received additional testing/retraining on the beam hourly for 6 h, or they were returned to their home cages without further testing/retraining. Rats with thoracic spinal cord injuries that received amphetamine in conjunction with testing/retraining on the beam at 1 day post injury (DPI) exhibited significantly impaired recovery on the beam walking task and BBB. Rats with cervical spinal cord injuries that received training with amphetamine also exhibited significant impairments in beam walking and locomotion, as well as impairments in gripping and reaching abilities. Even when administered at 14 DPI, the drug/training regimen significantly impaired reaching ability in cervical spinal cord injured rats. Impairments were not seen in rats that received amphetamine without training. Histological analyses revealed that rats that received training with amphetamine had significantly larger lesions than saline controls. These data indicate that an amphetamine/training regimen that improves recovery after cortical injury has the opposite effect of impairing recovery following spinal cord injury because early training with amphetamine increases lesion severity.
In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several i... more In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and 8-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that isselectivelydistributed indendritesand cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.
Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) an... more Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) and cell discharge (population spike) were analyzed in the dentate gyrus of the rat following repetitive low-frequency stimulation of the medial and lateral entorhinal cortex. Stimulation of either subdivision of the entorhinal projection system resulted in comparable habituation-like response decrements with similar stimulation regimens. However, habituating stimulation of one subdivision did not result in decreased responsiveness to stimulation of the other. Repetitive lowfrequency stimulation or even a single pulse delivered to either subdivision did, however, result in a potentiation of granule cell discharge in response to stimulation of the other subdivision (a form of heterosynaptic potentiation). This heterosynaptic potentiation of granule cell discharge was not accompanied by any increase in the extracellular EPSP. Comparisons of the relationship between the population EPSP and population spike before and during habituating stimulation revealed changes in cell discharge in response to the habituating stimulus which could not be accounted for by changes in synaptic activation alone. The results suggest that repetitive activation of the temporodentate pathway alters granule cell output as a result of two processes, a habituation-like decrement in synaptic activation, and a potentiation of granule cell discharge as a consequence of prior activation.
Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylatio... more Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/ 236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.
ABSTRACTInjury responses require communication between different cell types in the skin. Sensory ... more ABSTRACTInjury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of privileged translation in vitro and in vivo. Inflammatory cues promote local translation of Arc in the skin. Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found...
While interest toward caloric restriction (CR) in various models of brain injury has increased in... more While interest toward caloric restriction (CR) in various models of brain injury has increased in recent decades, studies have predominantly focused on the benefits of chronic or intermittent CR. The effects of ultra-short, including overnight, CR on acute ischemic brain injury, however, are not well studied. Here, we show that overnight caloric restriction (75% over 14 h) prior to asphyxial cardiac arrest and resuscitation (CA) improves survival and neurological recovery, including complete prevention of neurodegeneration in multiple regions of the brain. We also show that overnight CR normalizes stress-induced hyperglycemia, while significantly decreasing insulin and glucagon production and increasing corticosterone and ketone body production. The benefits seen with ultra-short CR appear independent of Sirtuin 1 (SIRT-1) and brain-derived neurotrophic factor (BDNF) expression, which have been strongly linked to neuroprotective benefits seen in chronic CR. Further exploration into ...
Cover: A hippocampal neuron grown in culture. The neuron is immunostained with an antibody to the... more Cover: A hippocampal neuron grown in culture. The neuron is immunostained with an antibody to the microtubule associated protein MAP2. Courtesy G.A. Banker. Library of Congress Cataloging-in-Publication Data Steward, Oswald. Principles of cellular. molecular, and developmental neuroscience/ Oswald Steward. p. cm. Includes bibliographies and index.
Demyelination contributes to loss of function after spinal cord injury, and thus a potential ther... more Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.
Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part ... more Previous studies have provided evidence that a quantitative trait locus (QTL) on the distal part of chromosome 18 (chr18) is a major determinant of vulnerability to hippocampal neurodegeneration following kainic acid (KA)-induced seizures in inbred mouse strains. We assessed excitotoxic vulnerability in two congenic, "genome tagged" mouse strains carrying segments of either distal or proximal/medial chr18 from vulnerable DBA/2J mice on a resistant C57BL/6 background. Systemic KA injections triggered brain-wide neurodegeneration in the distal chr18 congenic strain, and specifically in the hilus of the dentate gyrus, but not in CA3. In contrast, the proximal/medial chr18 congenic strain exhibited enhanced degeneration in CA1 and CA3, but little neurodegeneration elsewhere. Both strains exhibited low levels of QUIN-induced striatal neurodegeneration comparable to what is seen in C57BL/6 mice. These results suggest that gene(s) on distal chr18 are important determinants of vulnerability to KA-induced hippocampal neurodegeneration, but not QUIN-induced striatal neurodegeneration.
Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of in... more Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. Adult rats received unilateral intra-cortical injections of AAV expressing shRNA against PTEN and were allowed to survive for 2 months. Immunostaining for c-fos under resting conditions (home cage, HC) and after 1hr of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphosphorylation of ERK1/2.
et al., Regulatory T cells promote remyelination in the murine experimental autoimmune encephalom... more et al., Regulatory T cells promote remyelination in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis following human neural stem cell transplant, Neurobiology of Disease (2019),
In mice, the genetic background determines susceptibility to hippocampal neurodegeneration induce... more In mice, the genetic background determines susceptibility to hippocampal neurodegeneration induced by the excitotoxin kainic acid (KA). If genetic background plays as significant a role in the striatum, the area most affected in Huntington's disease (HD), it is important to characterize intrinsic differences in neuronal susceptibility in mouse strains used in HD models. This study was performed to investigate whether strain differences of different HD mouse models are determinants of striatal resistance to excitotoxicity. We conducted a survey of the susceptibility of striatal neurons to neurodegeneration induced by quinolinic acid and KA in six inbred, two outbred and two F1 hybrid (resistant*vulnerable) strains. These are the same strains in which we have assessed vulnerability to KA-induced hippocampal neurodegeneration. We found significant strain differences in response to both excitotoxins and, for the most part, the strain-dependent patterns of susceptibility to quinolinic acid and KA were similar and comparable to those previously found with KA-induced hippocampal neurodegeneration. There were some incongruities, suggesting that the genetic determinants may be different for the two forms of excitotoxicity or that there are important interacting factors. For example, the F1 hybrid strains exhibited neurodegeneration similar to their vulnerable parent, indicating that the vulnerable phenotype is dominant. This is in contrast to KA-induced hippocampal neurodegeneration, where F1 hybrids exhibit the resistant phenotype. These results are also of significance with regard to the issue of region-specific vulnerability in the context of different diseases in which genetic modifiers affect age of onset and ⁄ or disease progression.
Over the past century, the question of whether the cells of origin of the corticospinal tract (CS... more Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent study reported an approximately 20% loss of retrogradely labeled cortical motoneurons following damage to their axons resulting from SCI at T9 (Hains et al. [2003] J. Comp. Neurol. 462:328-341). In follow-up studies, however, we failed to find any evidence of loss of CST axons in the medullary pyramid, which must occur if CST neurons die. Here, we seek to resolve the discrepancy by re-evaluating possible loss of CST neurons using the same techniques as Hains et al. (quantitative analysis of retrograde labeling and staining for cell death markers including TUNEL and Hoechst labeling of the nuclei). Following either dorsal funiculus lesions at thoracic level 9 (T9) or lateral hemisection at cervical level 5 (C5), our results reveal no evidence for a loss of retrogradely labeled neurons and no evidence for TUNEL staining of axotomized cortical motoneurons. These results indicate that CST cell bodies do not undergo retrograde cell death following SCI, and therefore targeting such cell death is not a valid therapeutic target.
Previous studies in mice have demonstrated that forepaw gripping ability, as measured by a grip s... more Previous studies in mice have demonstrated that forepaw gripping ability, as measured by a grip strength meter (GSM), is dependent on the contralateral sensorimotor cortex, but this dependency changes after hemisection injury at cervical level 4 (C4). Initially, the mouse fails to grip with the forepaw ipsilateral to the hemisection but gripping recovers. Additionally, a mouse's gripping by the contralateral paw becomes independent of the sensorimotor cortex, indicating a reorganization of cortical control of gripping function (Blanco et al, 2007). Here we explore whether a similar reorganization occurs after cervical hemisection injuries in rats. We show that as in mice, unilateral lesions of the sensorimotor cortex impair rats' griping by the contralateral paw. We also confirm from previous studies that cervical hemisections in the impair rats' griping by the ipsilateral paw. In contrast to mice, however there is minimal recovery of gripping after complete lateral hemisections and secondary lesions of the sensorimotor cortex continue to impair rats' gripping by the contralateral paw. Thus, forelimb gripping ability as measured by the GSM is dependent on the contralateral sensorimotor cortex in rats even after a cervical hemisection.
In situ hybridization was used to assess the subcellular distribution of rRNA and poly(A) RNA in ... more In situ hybridization was used to assess the subcellular distribution of rRNA and poly(A) RNA in hippocampal neurons maintained in culture. Labeling produced with 35S-labeled probes to either rRNA or poly(A) was heaviest over the cell body with lighter, patchy labeling of proximal dendrites. In contrast, 3H-labeled probes labeled dendrites throughout their length, and the ratio of dendritic to cell body labeling was higher with 3H-labeled probes. There was no detectable labeling of axons of mature neurons with either probe. The pattern of hybridization produced by 35S-labeled oligonucleotide probes to rRNA varied depending on the concentration of the oligonucleotide. These studies provide the first detailed study of the subcellular distribution of rRNA and poly(A) RNA in neurons, and highlight technical issues to consider when evaluating results of hybridizations carried out with 35S- and 3H-labeled probes on cells in culture.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1993
This study evaluates whether protein synthesis plays a role in targeting RNA molecules to differe... more This study evaluates whether protein synthesis plays a role in targeting RNA molecules to different subcellular domains within neurons. Transport of newly synthesized RNA (labeled with [3HJuridine) was emined in the presence of the protein synthesis inhibitors puromycin and cycloheximide. In situ hybridization was used to determine whether inhibition of protein synthesis altered the subcellular distribution ofmRNAs. Transport ofrecently synthesized RNA was not disrupted after prolonged exposure to either inhibitor. However, inhibition ofprotein synthesis caused several mRNAs that are normally confined to the cell body to appear in dendrites. The distribution of mRNAs that are normally present in dendrites was unaffected. These findings suggest that protein synthesis is not required to translocate RNA into the dendrites but may play a role in restricting particular mRNAs to the neuronal cell body. Abbreviations: DMSO, dimethyl sulfoxide; MAP2, microtubuleassociated protein 2; GAP-43, growth-associated protein 43; cRNA, complementary RNA.
Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervou... more Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system (CNS) and for which there is no cure. Current treatments focus on suppression of the immune system but fail to repair the resulting damage to the CNS. Neural stem cell (NSC) transplantation is a promising therapeutic strategy for treating neurodegenerative diseases through cell replacement and repair however it is unclear how these cells would mediate repair in MS. We report that human NSCs promote CNS specific T regulatory cells (Tregs) which activate endogenous repair pathways and promote remyelination in a murine model of MS. We observed remyelination, decreased inflammation and an increase in (CNS)-infiltrating CD4+CD25+FoxP3+ Tregs in EAE mice receiving an intra-spinal transplant of hNSCs. Recovery was not a result of cell replacement, as hNSCs underwent xenograft rejection, and was Treg dependent, as ablation of Tregs abrogated histopathological improvement. Treg expansion is antigen driven as hNSCs expanded CD25+FoxP3+ Tregs in-vitro when cultured with neural antigen restricted RAG2−/−2D2+ (R2D2) splenocytes but not RAG2−/−OT-II+ splenocytes. When co-cultured with B6 splenocytes, hNSCs drove the expansion of unique TCRs when compared to controls. Additionally, hNSC-Tregs also appear to derive from the exTreg pool suggesting both antigen specific expansion and antigen dependent maintenance of FOXP3 in CNS-specific Tregs. hNSC Tregs also have a unique expression profile and express transglutimase-2 which is implicated in oligodendrocyte dependent repair in the CNS.
It has been recognized since the time of Cajal that different parts of the neuron are specialized... more It has been recognized since the time of Cajal that different parts of the neuron are specialized for different functions; indeed, this is the basis for what Cajal called the “dynamic polarization of the neuron.” Axons are specialized for transmitting information over great distances, dendrites and cell bodies for receiving and integrating inputs from other cells, and synapses for communicating information from one neuron to the next. Each of these functional attributes depends upon two features: (1) the “molecular anatomy” of the neuron, which includes the composition and spatial distribution of receptors, channels, etc. in the neuronal plasma membrane and the intracellular distribution of organelles, second messenger systems, and enzyme systems; and (2) neuronal microstructure, i. e., the shape and spatial configuration of axons, dendrites, and the synaptic connections between them.
This study evaluates the temperature sensitivity of transport of recently synthesized RNA from th... more This study evaluates the temperature sensitivity of transport of recently synthesized RNA from the nucleus to the cytoplasm (nucleocytoplasmic transport) in CNS neurons. Rat hippocampal slices were incubated with [3H]uridine for 1 h to label recently synthesized RNA. Slices were then fixed immediately or maintained at 27°C or 37°C for chase intervals of 3, 4.5, and 6 h to allow for nucleocytoplasmic transport of recently synthesized RNA. The time-dependent translocation of recently synthesized RNA was evaluated autoradiographically. At the end of the i h pulse at either 27°C or 37°C, the label was localized exclusively over nuclei. In slices maintained at 37°C, labeling expanded to cover the cell body and proximal dendrites. However, in slices that were labeled and maintained at room temperature, labeling remained confined to the nucleus. In slices that were pulse-labeled at room temperature, and then transferred to 37°C medium, cytoplasmic labeling increased as a function of time. Nucleocytoplasmic transport of RNA in cultured rat hippocampal neurons showed a comparable temperature sensitivity. The inhibition of nucleocytoplasmic transport of RNA at room temperature provides an opportunity to evaluate neuronal function when no new RNA molecules can reach the cytoplasm.
It has previously been reported that a single dose of amphetamine paired with training on a beam ... more It has previously been reported that a single dose of amphetamine paired with training on a beam walking task can enhance locomotor recovery following brain injury (Feeney et al., 1982). Here, we investigated whether this same drug/training regimen could enhance functional recovery following either thoracic (T9) or cervical (C5) spinal cord injury. Different groups of female Sprague-Dawley rats were trained on a beam walking task, and in a straight alley for assessment of hindlimb locomotor recovery using the BBB locomotor scale. For rats that received C5 hemisections, forelimb grip strength was assessed using a grip strength meter. Three separate experiments assessed the consequences of training rats on the beam walking task 24 h following a thoracic lateral hemisection with administration of either amphetamine or saline. Beginning 1 h following drug administration, rats either received additional testing/retraining on the beam hourly for 6 h, or they were returned to their home cages without further testing/retraining. Rats with thoracic spinal cord injuries that received amphetamine in conjunction with testing/retraining on the beam at 1 day post injury (DPI) exhibited significantly impaired recovery on the beam walking task and BBB. Rats with cervical spinal cord injuries that received training with amphetamine also exhibited significant impairments in beam walking and locomotion, as well as impairments in gripping and reaching abilities. Even when administered at 14 DPI, the drug/training regimen significantly impaired reaching ability in cervical spinal cord injured rats. Impairments were not seen in rats that received amphetamine without training. Histological analyses revealed that rats that received training with amphetamine had significantly larger lesions than saline controls. These data indicate that an amphetamine/training regimen that improves recovery after cortical injury has the opposite effect of impairing recovery following spinal cord injury because early training with amphetamine increases lesion severity.
In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several i... more In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and 8-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that isselectivelydistributed indendritesand cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.
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Papers by Oswald Steward