Randy Peterson - Academia.edu (original) (raw)

Papers by Randy Peterson

<p>(A) Standard curves with R² demonstrate a dose-dependent response... more <p>(A) Standard curves with R² demonstrate a dose-dependent response of Shh-LTII cells to human recombinant Shh (rShh) and purmorphamine (Purm) (0.3 μM). Shh-LTII cells were cultured with rShh and Purm and developed with Promega Dual-Glo Luciferase Assay System, assaying for firefly (FFL) and Renilla (RL) luciferase-driven luminescence measured as luminescence light unit (LLU). Data are expressed as mean ± SD and shown as arbitrary units (AU) where FFL was normalized to RL (LLU FFL/RL). * and ** indicate significant differences from media alone for rShh and Purm, correspondingly, based on a one way ANOVA analysis with Tukey post-<i>hoc</i> test (<i>p</i> < 0.05). (B) The inhibitory effect of ALS CSF on Shh biological activity measured in Shh-LTII cells is shown. Fully confluent Shh-LTII cells were incubated either with CSF alone, half-diluted with reduced-serum medium as a baseline (white bar), or in the presence of rShh (0.3 μg/ml) (grey bar) or Purm (0.3 μM) (black bar) and assayed with Promega. Data are expressed as mean ± SD and shown as AU. * and *** indicate significant differences between a baseline, rShh, and Purm in neurological controls (Neuro) or normal controls (Controls), respectively, using one way ANOVA with Tukey or Newman-Keuls post-<i>hoc</i> tests (<i>p</i> < 0.05). (C) The inhibitory effect of Shh antagonist cyclopamine (Cycl) is shown in Shh-LTII cells cultured with hrShh and Purm and developed with Promega as described in detail. Data are expressed as mean ± SD and shown as AU with significance value illustrated. (D) The inhibitory effect of ALS CSF shown in (B) is expressed as fold increase over baseline for each sample (AU stimulated/AU unstimulated), demonstrating a significantly increased luminescence in response to rShh and Purm treatment in presence of CSF from controls, but not ALS patients. Data analysis was performed using one way ANOVA with Tukey post-<i>hoc</i> test. Displayed <i>p</i> values indicate significant differences between groups, and N.S. indicates non-significant differences. (E) The inhibitory effect of ALS CSF observed in Shh-LTII cells (B, C) was repeated in NSC-34-Gli cells cultured in half-diluted CSF from controls and ALS patients. Data are expressed as mean ± SD and shown as AU with significance indicated on a graph. (F) Nuclear expression of Gli1, Sufu, and Gli2 proteins is shown in immunofluorescently stained NSC-34-Gli cells cultured for 72 hours in a serum-reduced medium with a half-diluted pooled CSF from Neuro and ALS groups. Data are expressed as mean ± SEM, with significance value included, and shown as a relative nuclear intensity analyzed per cell (ImageJ) over manually contoured nuclear (DAPI) area. Reduced Gli1 and Sufu nuclear translocation in ALS CSF-treated cells are shown, in contrast to elevated Gli2 nuclear expression. (G) Representative immunofluorescence images demonstrating Gli1 (green), Sufu (red), and Gli2 (red) distribution in NSC-34-Gli cells treated either with CSF from neurological controls (Neuro panel) or ALS group (ALS panel) using DAPI (blue) as a nuclear stain. Single or merged images are shown with a scale bar depicted in a bottom right image.</p

Neuroreport, Jan 15, 2016

The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult ... more The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult motor neurons, of potential relevance to motor neuron diseases including amyotrophic lateral sclerosis. The Shh signaling pathway is incompletely understood and interactions with other signaling pathways are possible. We focus here on Notch, and first show that there is an almost linear reduction in light output from a Gli reporter in Shh Light II cells in the presence of increasing concentrations of the Notch inhibitor DAPT (r=0.982). Second, in the spinal cord of mutant superoxide dismutase mice, but not control mice, a key marker of Notch signaling changes with age. Before the onset of clinical signs, the Notch intracellular domain is expressed predominantly in motor neurons, but by 125 days of age, Notch intracellular domain expression is markedly reduced in motor neurons and increased in neighboring astroglia. Third, there is a parallel reduction in Gli protein expression in mutant ...

BMC neuroscience, Jan 11, 2013

The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the suppo... more The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis. Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival ...

Journal of Molecular Neuroscience, 2011

We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SO... more We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SOD1 (mSOD) mice, a mouse model of amyotrophic lateral sclerosis (ALS). Sonic hedgehog (Shh) signaling involves the primary cilium and Shh has been shown to be cytoprotective in models of other neurodegenerative diseases. Thus, the Shh signaling pathway may bear further study in ALS. Accordingly, we established that interference with the Shh pathway (with the Shh antagonist cyclopamine or with miRNA 3245p) sensitized HT22 cells, while augmentation of the Shh pathway (with Shh or the Shh agonist purmorphamine) protected cells against hydrogen peroxide (H 2 O 2) challenge. We ectopically expressed mSOD, human wild-type SOD1 (wtSOD), or an empty vector in HT22 cells. Compared to empty vector, wtSOD decreased cell death and mSOD increased cell death in response to H 2 O 2 challenge. Treatment with cyclopamine or miRNA 3245p sensitized all three transfections to H 2 O 2 challenge. Treatment with recombinant human Shh or purmorphamine decreased cell death after H 2 O 2 challenge, an effect more pronounced in mSOD cells. Compared with empty vector, overexpression of wtSOD increased Shh and Gli transcript levels and increased activity in a Gliresponsive reporter assay. Overexpression of mSOD did not change Shh transcript levels, but decreased Gli transcript levels, especially Gli3, and reduced activity in a Gli reporter assay. These results suggest that overexpression of mSOD but not wtSOD reduces signaling in the Shh pathway and renders mSOD cells more susceptible to H 2 O 2 challenge, and that treatment with Shh or Shh agonists is cytoprotective to mSOD cells. Shh or Shh agonists merit further consideration as potential therapy in ALS.

PloS one, 2017

Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to ... more Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest i...

BMC Neuroscience, 2011

Background The primary cilium is a solitary organelle important in cellular signaling, that proje... more Background The primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord. Results At 7 days in culture there is no difference in the proportion of G93A SOD1 and WT motor neurons staining for the cilial marker ACIII. However, at 21 days there is a large relative drop in the proportion of ciliated G93A SOD1 motor neurons. In situ, at 40 days there was a slight relative drop in the proportion of ciliated motor neurons in G93A SOD1 mice. At 98 days of age there was no change in motor ne...

Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease with no know... more Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease with no known cause. Despite the efforts of investigators over the past 150 years, there remains no effective cure which substantially prolongs life. Therapeutic strategies have explored all of the proposed underlying pathological pathways of the disease from increased oxidative damage to impaired axonal transport, with little to no success. In the following pages, a novel perspective will be presented outlining the preliminary investigations of a new line of research demonstrating that Sonic hedgehog (Shh) protein and its agonists have cytoprotective effects on motor neurons. To begin these investigations, initial experiments were conducted in vitro utilizing a mouse hippocampal cell-line (HT-22) which served as a model for transient transfection and oxidative challenge assays. The results are reported in Chapter 2. Building upon these introductory findings, further investigations were conducted exploiting the SOD1G93A mouse model of ALS. Chapter 3 summarizes key observations pertaining to the abundance of a key cellular organelle in the sensing of Shh signalling, the primary cilium, in the spinal cord of SOD1G93A mice. In Chapter 4, a semi-quantitative analysis of the effects of Shh and Shh agonists pre-treatment in vitro on primary mixed spinal cord cultures are described. Subsequent challenge with an excitotoxic NMDA treatment was also conducted, as well as an in vivo survival study exploring the potential therapeutic effects of chronic Shh administration on SOD1G93A mice. The cumulative research presented here represents the very first investigation into the unique application of Shh and its agonists as potential therapeutic agents for the treatment of ALS, and our findings indicate that Shh has the potential of becoming a novel therapeutic agent for the treatment of ALS.

<p>(A) Standard curves with R² demonstrate a dose-dependent response... more <p>(A) Standard curves with R² demonstrate a dose-dependent response of Shh-LTII cells to human recombinant Shh (rShh) and purmorphamine (Purm) (0.3 μM). Shh-LTII cells were cultured with rShh and Purm and developed with Promega Dual-Glo Luciferase Assay System, assaying for firefly (FFL) and Renilla (RL) luciferase-driven luminescence measured as luminescence light unit (LLU). Data are expressed as mean ± SD and shown as arbitrary units (AU) where FFL was normalized to RL (LLU FFL/RL). * and ** indicate significant differences from media alone for rShh and Purm, correspondingly, based on a one way ANOVA analysis with Tukey post-<i>hoc</i> test (<i>p</i> < 0.05). (B) The inhibitory effect of ALS CSF on Shh biological activity measured in Shh-LTII cells is shown. Fully confluent Shh-LTII cells were incubated either with CSF alone, half-diluted with reduced-serum medium as a baseline (white bar), or in the presence of rShh (0.3 μg/ml) (grey bar) or Purm (0.3 μM) (black bar) and assayed with Promega. Data are expressed as mean ± SD and shown as AU. * and *** indicate significant differences between a baseline, rShh, and Purm in neurological controls (Neuro) or normal controls (Controls), respectively, using one way ANOVA with Tukey or Newman-Keuls post-<i>hoc</i> tests (<i>p</i> < 0.05). (C) The inhibitory effect of Shh antagonist cyclopamine (Cycl) is shown in Shh-LTII cells cultured with hrShh and Purm and developed with Promega as described in detail. Data are expressed as mean ± SD and shown as AU with significance value illustrated. (D) The inhibitory effect of ALS CSF shown in (B) is expressed as fold increase over baseline for each sample (AU stimulated/AU unstimulated), demonstrating a significantly increased luminescence in response to rShh and Purm treatment in presence of CSF from controls, but not ALS patients. Data analysis was performed using one way ANOVA with Tukey post-<i>hoc</i> test. Displayed <i>p</i> values indicate significant differences between groups, and N.S. indicates non-significant differences. (E) The inhibitory effect of ALS CSF observed in Shh-LTII cells (B, C) was repeated in NSC-34-Gli cells cultured in half-diluted CSF from controls and ALS patients. Data are expressed as mean ± SD and shown as AU with significance indicated on a graph. (F) Nuclear expression of Gli1, Sufu, and Gli2 proteins is shown in immunofluorescently stained NSC-34-Gli cells cultured for 72 hours in a serum-reduced medium with a half-diluted pooled CSF from Neuro and ALS groups. Data are expressed as mean ± SEM, with significance value included, and shown as a relative nuclear intensity analyzed per cell (ImageJ) over manually contoured nuclear (DAPI) area. Reduced Gli1 and Sufu nuclear translocation in ALS CSF-treated cells are shown, in contrast to elevated Gli2 nuclear expression. (G) Representative immunofluorescence images demonstrating Gli1 (green), Sufu (red), and Gli2 (red) distribution in NSC-34-Gli cells treated either with CSF from neurological controls (Neuro panel) or ALS group (ALS panel) using DAPI (blue) as a nuclear stain. Single or merged images are shown with a scale bar depicted in a bottom right image.</p

Neuroreport, Jan 15, 2016

The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult ... more The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult motor neurons, of potential relevance to motor neuron diseases including amyotrophic lateral sclerosis. The Shh signaling pathway is incompletely understood and interactions with other signaling pathways are possible. We focus here on Notch, and first show that there is an almost linear reduction in light output from a Gli reporter in Shh Light II cells in the presence of increasing concentrations of the Notch inhibitor DAPT (r=0.982). Second, in the spinal cord of mutant superoxide dismutase mice, but not control mice, a key marker of Notch signaling changes with age. Before the onset of clinical signs, the Notch intracellular domain is expressed predominantly in motor neurons, but by 125 days of age, Notch intracellular domain expression is markedly reduced in motor neurons and increased in neighboring astroglia. Third, there is a parallel reduction in Gli protein expression in mutant ...

BMC neuroscience, Jan 11, 2013

The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the suppo... more The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis. Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival ...

Journal of Molecular Neuroscience, 2011

We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SO... more We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SOD1 (mSOD) mice, a mouse model of amyotrophic lateral sclerosis (ALS). Sonic hedgehog (Shh) signaling involves the primary cilium and Shh has been shown to be cytoprotective in models of other neurodegenerative diseases. Thus, the Shh signaling pathway may bear further study in ALS. Accordingly, we established that interference with the Shh pathway (with the Shh antagonist cyclopamine or with miRNA 3245p) sensitized HT22 cells, while augmentation of the Shh pathway (with Shh or the Shh agonist purmorphamine) protected cells against hydrogen peroxide (H 2 O 2) challenge. We ectopically expressed mSOD, human wild-type SOD1 (wtSOD), or an empty vector in HT22 cells. Compared to empty vector, wtSOD decreased cell death and mSOD increased cell death in response to H 2 O 2 challenge. Treatment with cyclopamine or miRNA 3245p sensitized all three transfections to H 2 O 2 challenge. Treatment with recombinant human Shh or purmorphamine decreased cell death after H 2 O 2 challenge, an effect more pronounced in mSOD cells. Compared with empty vector, overexpression of wtSOD increased Shh and Gli transcript levels and increased activity in a Gliresponsive reporter assay. Overexpression of mSOD did not change Shh transcript levels, but decreased Gli transcript levels, especially Gli3, and reduced activity in a Gli reporter assay. These results suggest that overexpression of mSOD but not wtSOD reduces signaling in the Shh pathway and renders mSOD cells more susceptible to H 2 O 2 challenge, and that treatment with Shh or Shh agonists is cytoprotective to mSOD cells. Shh or Shh agonists merit further consideration as potential therapy in ALS.

PloS one, 2017

Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to ... more Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest i...

BMC Neuroscience, 2011

Background The primary cilium is a solitary organelle important in cellular signaling, that proje... more Background The primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord. Results At 7 days in culture there is no difference in the proportion of G93A SOD1 and WT motor neurons staining for the cilial marker ACIII. However, at 21 days there is a large relative drop in the proportion of ciliated G93A SOD1 motor neurons. In situ, at 40 days there was a slight relative drop in the proportion of ciliated motor neurons in G93A SOD1 mice. At 98 days of age there was no change in motor ne...

Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease with no know... more Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease with no known cause. Despite the efforts of investigators over the past 150 years, there remains no effective cure which substantially prolongs life. Therapeutic strategies have explored all of the proposed underlying pathological pathways of the disease from increased oxidative damage to impaired axonal transport, with little to no success. In the following pages, a novel perspective will be presented outlining the preliminary investigations of a new line of research demonstrating that Sonic hedgehog (Shh) protein and its agonists have cytoprotective effects on motor neurons. To begin these investigations, initial experiments were conducted in vitro utilizing a mouse hippocampal cell-line (HT-22) which served as a model for transient transfection and oxidative challenge assays. The results are reported in Chapter 2. Building upon these introductory findings, further investigations were conducted exploiting the SOD1G93A mouse model of ALS. Chapter 3 summarizes key observations pertaining to the abundance of a key cellular organelle in the sensing of Shh signalling, the primary cilium, in the spinal cord of SOD1G93A mice. In Chapter 4, a semi-quantitative analysis of the effects of Shh and Shh agonists pre-treatment in vitro on primary mixed spinal cord cultures are described. Subsequent challenge with an excitotoxic NMDA treatment was also conducted, as well as an in vivo survival study exploring the potential therapeutic effects of chronic Shh administration on SOD1G93A mice. The cumulative research presented here represents the very first investigation into the unique application of Shh and its agonists as potential therapeutic agents for the treatment of ALS, and our findings indicate that Shh has the potential of becoming a novel therapeutic agent for the treatment of ALS.