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Sera from children with autism induce autistic features which can be rescued with a CNTF small peptide mimetic in rats.

Kazim SF, Cardenas-Aguayo Mdel C, Arif M, Blanchard J, Fayyaz F, Grundke-Iqbal I, Iqbal K - PLoS ONE (2015)

Bottom Line: The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression.Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth.Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment.

View Article: PubMed Central - PubMed

Affiliation: Inge Grundke-Iqbal Research Floor, Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities (NYSIBR), Staten Island, New York, United States of America; Neural and Behavioral Science Graduate Program, State University of New York (SUNY) Downstate Medical Center, Brooklyn, New York, United States of America; SUNY Downstate/NYSIBR Center for Developmental Neuroscience (CDN), Staten Island, New York, United States of America.

ABSTRACT
Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism.

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Effect of autism and control sera treatment with or without P6 on neuronal death/viability and oxidative stress in mouse primary cultured cortical neurons.(A) Representative images of phase contrast microscopy and β-III-tubulin (mature neuronal marker) staining of DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data is based on evaluation of the effect of 22 pairs of autism/control sera in 3 independent set of experiments. Autism sera markedly reduced the length of the neuritis and the number of cells and showed increased number of cell spheres and P6 could rescue these changes (B and C) Quantification of LDH cytotoxicity assay for evaluation of cell death (LDH release) and neuronal viability in DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data are shown as mean±S.E.M. based on the effect of 3 pairs of autism/control sera in 3 independent sets of experiments. (D and E) Data for DCF-DA assay for free radical production and TBARS assay for lipid peroxidation 3 days after treatment (DIV7) with sera from autistic or control children (0.2%) with or without P6 pre-treatment (0.005 μM, 0.05 μM, and 1 μM) is shown. Data are shown as mean±S.E.M. based on two independent sets of experiments evaluating 3 pairs of autism/control sera. *p<0.05, **p<0.01, and ***p<0.001. ANOVA with Bonferroni’s post-hoc test and/or Student’s t-test. Scale bar = 100 μm.
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pone.0118627.g001: Effect of autism and control sera treatment with or without P6 on neuronal death/viability and oxidative stress in mouse primary cultured cortical neurons.(A) Representative images of phase contrast microscopy and β-III-tubulin (mature neuronal marker) staining of DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data is based on evaluation of the effect of 22 pairs of autism/control sera in 3 independent set of experiments. Autism sera markedly reduced the length of the neuritis and the number of cells and showed increased number of cell spheres and P6 could rescue these changes (B and C) Quantification of LDH cytotoxicity assay for evaluation of cell death (LDH release) and neuronal viability in DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data are shown as mean±S.E.M. based on the effect of 3 pairs of autism/control sera in 3 independent sets of experiments. (D and E) Data for DCF-DA assay for free radical production and TBARS assay for lipid peroxidation 3 days after treatment (DIV7) with sera from autistic or control children (0.2%) with or without P6 pre-treatment (0.005 μM, 0.05 μM, and 1 μM) is shown. Data are shown as mean±S.E.M. based on two independent sets of experiments evaluating 3 pairs of autism/control sera. *p<0.05, **p<0.01, and ***p<0.001. ANOVA with Bonferroni’s post-hoc test and/or Student’s t-test. Scale bar = 100 μm.

Mentions: Previously, sera from individuals with autism which possess abnormal levels of various regulatory elements were shown to alter the development and proliferation of human neural progenitor cells (NPCs) and to possess autoantibodies against human NPCs [78–80]. In the present study, we observed that mouse primary cultured cortical neurons grown for 72 hours in medium supplemented with sera from autistic children either formed neurospheres like colonies of cells with sharp spinous processes or multiple small cells with markedly short processes and decreased cell density as compared to the untreated or control sera treated cell cultures both as observed by phase contrast microscopy and by immunostaining for neuronal marker, β-III-tubulin (Fig. 1A). Primary cultured neurons grown in the presence of sera from normal healthy controls revealed no gross morphological changes and only a few neurosphere like colonies were observed. Pretreatment with 1 μM P6 for 3 hours prevented the decrease in neurite length and cell density caused by the autism sera (Fig. 1A). These results were confirmed with 22 pairs of sera (autism and age-matched control, Table 1) in 3 different sets of primary cultures. Further analyses of cell death and oxidative stress were performed in 3 pairs of sera (Table 2) which showed the most marked consistent effect on neuronal morphology. A significant increase in cell death was found by LDH cytotoxicity assay in cultured neurons grown in the presence of sera from autistic children compared to untreated neurons (Fig. 1B; Bonferroni’s post-hoc test, p<0.05; Student’s t-test, p = 0.0039). Pretreatment with different doses of P6 resulted in a significant reduction in cell death in cultured neurons treated with sera from autistic children (Fig. 1B; P6 0.005 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p = 0.0183; P6 0.05 μM, Bonferroni’s post-hoc test, p<0.01; P6 1 μM, Bonferroni’s post-hoc test, p<0.01). The cell death was not significantly altered in cultured neurons treated with sera from normal healthy controls compared to untreated controls (Fig. 1B; Bonferroni’s post-hoc test, p>0.05). Also, the cell death was less in control sera treated neurons compared to neurons treated with sera from autistic children (Fig. 1B; Student’s t-test, p = 0.0642, marginal significance). The neuronal viability, measured as a percentage of viability in untreated cells, was also significantly decreased in autistic sera treated neurons compared to those treated with control sera (Fig. 1C; Bonferroni’s post hoc test, p<0.01, Student’s t-test, p = 0.0076). P6 pretreatment showed improvement in neuronal viability in autism sera treated cultured neurons (Fig. 1C; P6 0.005 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p = 0.5619; P6 0.05 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p<0.0765; P6 1 μM, Student’s t-test, p<0.0964). Thus, we found that primary cultured neurons grown in the presence of sera from autistic children showed neuronal loss which was rescued by pretreatment with P6.


Sera from children with autism induce autistic features which can be rescued with a CNTF small peptide mimetic in rats.

Kazim SF, Cardenas-Aguayo Mdel C, Arif M, Blanchard J, Fayyaz F, Grundke-Iqbal I, Iqbal K - PLoS ONE (2015)

Effect of autism and control sera treatment with or without P6 on neuronal death/viability and oxidative stress in mouse primary cultured cortical neurons.(A) Representative images of phase contrast microscopy and β-III-tubulin (mature neuronal marker) staining of DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data is based on evaluation of the effect of 22 pairs of autism/control sera in 3 independent set of experiments. Autism sera markedly reduced the length of the neuritis and the number of cells and showed increased number of cell spheres and P6 could rescue these changes (B and C) Quantification of LDH cytotoxicity assay for evaluation of cell death (LDH release) and neuronal viability in DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data are shown as mean±S.E.M. based on the effect of 3 pairs of autism/control sera in 3 independent sets of experiments. (D and E) Data for DCF-DA assay for free radical production and TBARS assay for lipid peroxidation 3 days after treatment (DIV7) with sera from autistic or control children (0.2%) with or without P6 pre-treatment (0.005 μM, 0.05 μM, and 1 μM) is shown. Data are shown as mean±S.E.M. based on two independent sets of experiments evaluating 3 pairs of autism/control sera. *p<0.05, **p<0.01, and ***p<0.001. ANOVA with Bonferroni’s post-hoc test and/or Student’s t-test. Scale bar = 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359103&req=5

pone.0118627.g001: Effect of autism and control sera treatment with or without P6 on neuronal death/viability and oxidative stress in mouse primary cultured cortical neurons.(A) Representative images of phase contrast microscopy and β-III-tubulin (mature neuronal marker) staining of DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data is based on evaluation of the effect of 22 pairs of autism/control sera in 3 independent set of experiments. Autism sera markedly reduced the length of the neuritis and the number of cells and showed increased number of cell spheres and P6 could rescue these changes (B and C) Quantification of LDH cytotoxicity assay for evaluation of cell death (LDH release) and neuronal viability in DIV7 primary cultured cortical neurons treated with 0.2% sera from autistic or control children with or without 1 μM P6 for 72 hours. Data are shown as mean±S.E.M. based on the effect of 3 pairs of autism/control sera in 3 independent sets of experiments. (D and E) Data for DCF-DA assay for free radical production and TBARS assay for lipid peroxidation 3 days after treatment (DIV7) with sera from autistic or control children (0.2%) with or without P6 pre-treatment (0.005 μM, 0.05 μM, and 1 μM) is shown. Data are shown as mean±S.E.M. based on two independent sets of experiments evaluating 3 pairs of autism/control sera. *p<0.05, **p<0.01, and ***p<0.001. ANOVA with Bonferroni’s post-hoc test and/or Student’s t-test. Scale bar = 100 μm.
Mentions: Previously, sera from individuals with autism which possess abnormal levels of various regulatory elements were shown to alter the development and proliferation of human neural progenitor cells (NPCs) and to possess autoantibodies against human NPCs [78–80]. In the present study, we observed that mouse primary cultured cortical neurons grown for 72 hours in medium supplemented with sera from autistic children either formed neurospheres like colonies of cells with sharp spinous processes or multiple small cells with markedly short processes and decreased cell density as compared to the untreated or control sera treated cell cultures both as observed by phase contrast microscopy and by immunostaining for neuronal marker, β-III-tubulin (Fig. 1A). Primary cultured neurons grown in the presence of sera from normal healthy controls revealed no gross morphological changes and only a few neurosphere like colonies were observed. Pretreatment with 1 μM P6 for 3 hours prevented the decrease in neurite length and cell density caused by the autism sera (Fig. 1A). These results were confirmed with 22 pairs of sera (autism and age-matched control, Table 1) in 3 different sets of primary cultures. Further analyses of cell death and oxidative stress were performed in 3 pairs of sera (Table 2) which showed the most marked consistent effect on neuronal morphology. A significant increase in cell death was found by LDH cytotoxicity assay in cultured neurons grown in the presence of sera from autistic children compared to untreated neurons (Fig. 1B; Bonferroni’s post-hoc test, p<0.05; Student’s t-test, p = 0.0039). Pretreatment with different doses of P6 resulted in a significant reduction in cell death in cultured neurons treated with sera from autistic children (Fig. 1B; P6 0.005 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p = 0.0183; P6 0.05 μM, Bonferroni’s post-hoc test, p<0.01; P6 1 μM, Bonferroni’s post-hoc test, p<0.01). The cell death was not significantly altered in cultured neurons treated with sera from normal healthy controls compared to untreated controls (Fig. 1B; Bonferroni’s post-hoc test, p>0.05). Also, the cell death was less in control sera treated neurons compared to neurons treated with sera from autistic children (Fig. 1B; Student’s t-test, p = 0.0642, marginal significance). The neuronal viability, measured as a percentage of viability in untreated cells, was also significantly decreased in autistic sera treated neurons compared to those treated with control sera (Fig. 1C; Bonferroni’s post hoc test, p<0.01, Student’s t-test, p = 0.0076). P6 pretreatment showed improvement in neuronal viability in autism sera treated cultured neurons (Fig. 1C; P6 0.005 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p = 0.5619; P6 0.05 μM, Bonferroni’s post-hoc test, p>0.05, Student’s t-test, p<0.0765; P6 1 μM, Student’s t-test, p<0.0964). Thus, we found that primary cultured neurons grown in the presence of sera from autistic children showed neuronal loss which was rescued by pretreatment with P6.

Bottom Line: The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression.Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth.Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment.

View Article: PubMed Central - PubMed

Affiliation: Inge Grundke-Iqbal Research Floor, Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities (NYSIBR), Staten Island, New York, United States of America; Neural and Behavioral Science Graduate Program, State University of New York (SUNY) Downstate Medical Center, Brooklyn, New York, United States of America; SUNY Downstate/NYSIBR Center for Developmental Neuroscience (CDN), Staten Island, New York, United States of America.

ABSTRACT
Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism.

Show MeSH
Related in: MedlinePlus