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Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells.

De Filippis L, Halikere A, McGowan H, Moore JC, Tischfield JA, Hart RP, Pang ZP - Mol Brain (2016)

Bottom Line: Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway.This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution.In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs.

View Article: PubMed Central - PubMed

Affiliation: Child Health Institute of New Jersey, Rutgers University-Robert Wood Johnson Medical School, room 3233D, 89 French Street, New Brunswick, NJ, 08901, USA. ld473@rwjms.rutgers.edu.

ABSTRACT

Background: Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse.

Results: Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover, a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells.

Conclusions: This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models.

No MeSH data available.


Related in: MedlinePlus

Early ethanol exposure leads to a decrease of NPC-derived neurons. NPCs were pretreated with ethanol for 24hr or 7d and differentiated to neurons for 26 days. a Phase contrast pictures of treated and untreated NPCs after 7 days of differentiation toward the neuronal lineage, and immunofluorescence analysis of neurons differentiated from NPCs showing the synaptic marker synapsin, and glutamatergic marker vGlut. Scale bars: 200 μm (top), 10 μm (middle, bottom). b Graph showing the relative percentage of MAP2+ cells over the total number of DAPI+ nuclei. c Number of synapses quantified by synapsin+ puncta per 100 μm dendrite length under control or ethanol pre-exposure conditions. d Electrophysiological analysis. e Immunofluorescence analysis showing the expression of Casp1, MAP2, and NLRP3. Scale bar: 50 μm. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments
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Fig3: Early ethanol exposure leads to a decrease of NPC-derived neurons. NPCs were pretreated with ethanol for 24hr or 7d and differentiated to neurons for 26 days. a Phase contrast pictures of treated and untreated NPCs after 7 days of differentiation toward the neuronal lineage, and immunofluorescence analysis of neurons differentiated from NPCs showing the synaptic marker synapsin, and glutamatergic marker vGlut. Scale bars: 200 μm (top), 10 μm (middle, bottom). b Graph showing the relative percentage of MAP2+ cells over the total number of DAPI+ nuclei. c Number of synapses quantified by synapsin+ puncta per 100 μm dendrite length under control or ethanol pre-exposure conditions. d Electrophysiological analysis. e Immunofluorescence analysis showing the expression of Casp1, MAP2, and NLRP3. Scale bar: 50 μm. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments

Mentions: Since ethanol is teratogenic, and long-term effects may only become evident with time, we investigated whether pre-exposure to ethanol affects the differentiation of NPCs into neurons. Functional, mature neurons expressing MAP2, vGlut1, and synapsin were derived from the NPCs with or without ethanol pre-exposure (Fig. 3). Fewer mature neurons, identified by MAP2 expression, were generated from ethanol pre-exposed NPCs compared to untreated NPCs (Fig. 3a and b). Moreover, synaptic density appears to be reduced in neurons generated from ethanol treated NPCs when compared to neurons derived from control NPCs (Fig. 3a–c). However, in neurons derived from control and ethanol pre-exposed NPCs exhibited both repetitive action potentials as well as synaptic responses (Fig. 3d). Quite remarkably, it appears that inflammasome markers Casp1 and NLRP3 were prominent in neurons derived from ethanol pre-exposed NPCs (Fig. 3e). These findings suggest that neuronal differentiation is compromised (lower efficiency in maturation and less synapse formation) in NPCs pre-exposed to ethanol, and that neurons maintain certain cellular memory of neuroinflammation (i.e. with or without ethanol exposure) from the NPC stage.Fig. 3


Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells.

De Filippis L, Halikere A, McGowan H, Moore JC, Tischfield JA, Hart RP, Pang ZP - Mol Brain (2016)

Early ethanol exposure leads to a decrease of NPC-derived neurons. NPCs were pretreated with ethanol for 24hr or 7d and differentiated to neurons for 26 days. a Phase contrast pictures of treated and untreated NPCs after 7 days of differentiation toward the neuronal lineage, and immunofluorescence analysis of neurons differentiated from NPCs showing the synaptic marker synapsin, and glutamatergic marker vGlut. Scale bars: 200 μm (top), 10 μm (middle, bottom). b Graph showing the relative percentage of MAP2+ cells over the total number of DAPI+ nuclei. c Number of synapses quantified by synapsin+ puncta per 100 μm dendrite length under control or ethanol pre-exposure conditions. d Electrophysiological analysis. e Immunofluorescence analysis showing the expression of Casp1, MAP2, and NLRP3. Scale bar: 50 μm. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862119&req=5

Fig3: Early ethanol exposure leads to a decrease of NPC-derived neurons. NPCs were pretreated with ethanol for 24hr or 7d and differentiated to neurons for 26 days. a Phase contrast pictures of treated and untreated NPCs after 7 days of differentiation toward the neuronal lineage, and immunofluorescence analysis of neurons differentiated from NPCs showing the synaptic marker synapsin, and glutamatergic marker vGlut. Scale bars: 200 μm (top), 10 μm (middle, bottom). b Graph showing the relative percentage of MAP2+ cells over the total number of DAPI+ nuclei. c Number of synapses quantified by synapsin+ puncta per 100 μm dendrite length under control or ethanol pre-exposure conditions. d Electrophysiological analysis. e Immunofluorescence analysis showing the expression of Casp1, MAP2, and NLRP3. Scale bar: 50 μm. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments
Mentions: Since ethanol is teratogenic, and long-term effects may only become evident with time, we investigated whether pre-exposure to ethanol affects the differentiation of NPCs into neurons. Functional, mature neurons expressing MAP2, vGlut1, and synapsin were derived from the NPCs with or without ethanol pre-exposure (Fig. 3). Fewer mature neurons, identified by MAP2 expression, were generated from ethanol pre-exposed NPCs compared to untreated NPCs (Fig. 3a and b). Moreover, synaptic density appears to be reduced in neurons generated from ethanol treated NPCs when compared to neurons derived from control NPCs (Fig. 3a–c). However, in neurons derived from control and ethanol pre-exposed NPCs exhibited both repetitive action potentials as well as synaptic responses (Fig. 3d). Quite remarkably, it appears that inflammasome markers Casp1 and NLRP3 were prominent in neurons derived from ethanol pre-exposed NPCs (Fig. 3e). These findings suggest that neuronal differentiation is compromised (lower efficiency in maturation and less synapse formation) in NPCs pre-exposed to ethanol, and that neurons maintain certain cellular memory of neuroinflammation (i.e. with or without ethanol exposure) from the NPC stage.Fig. 3

Bottom Line: Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway.This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution.In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs.

View Article: PubMed Central - PubMed

Affiliation: Child Health Institute of New Jersey, Rutgers University-Robert Wood Johnson Medical School, room 3233D, 89 French Street, New Brunswick, NJ, 08901, USA. ld473@rwjms.rutgers.edu.

ABSTRACT

Background: Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse.

Results: Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover, a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells.

Conclusions: This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models.

No MeSH data available.


Related in: MedlinePlus