Transient blockade of ERK phosphorylation in the critical period causes autistic phenotypes as an adult in mice

SL327 administration at P6 caused social deficits later in adulthood.

“resulting in social deficits, impaired memory and reduced long-term potentiation (LTP).”

SL327 administration at P6 caused reduced LTP later in adulthood.

“resulting in social deficits, impaired memory and reduced long-term potentiation (LTP).”

Blockade of ERK phosphorylation at P14 did not induce reduced LTP.

“Conversely, blockade of ERK phosphorylation at P14 no longer induced apoptosis, nor behavioral deficits, nor the reduced LTP.”

Transient blockade of ERK phosphorylation at P6 by intraperitoneal injection of SL327 caused a significant increase of apoptosis in the forebrain.

“Transient blockade of ERKs phosphorylation at postnatal day 6 (P6) by intraperitoneal injection of blood-brain barrier-penetrating MEK inhibitor, α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327) caused significant increase of apoptosis in the forebrain.”

There was no concurrent decrease in total ERK levels for both isoforms at P6.

“There was no concurrent decrease in total ERK levels for both isoforms.”

SL327 administration at P6 caused impaired memory later in adulthood.

“resulting in social deficits, impaired memory and reduced long-term potentiation (LTP).”

Blockade of ERK phosphorylation at P14 did not induce apoptosis.

“Conversely, blockade of ERK phosphorylation at P14 no longer induced apoptosis, nor behavioral deficits, nor the reduced LTP.”

Blockade of ERK phosphorylation at P14 did not induce behavioral deficits.

“Conversely, blockade of ERK phosphorylation at P14 no longer induced apoptosis, nor behavioral deficits, nor the reduced LTP.”

Single-dose administration of SL327 at 50 mg/kg effectively attenuated phosphorylation levels for ERK1 and ERK2 in the mouse forebrain at P6 compared with vehicle.

“We observed that single-dose administration of SL327 at this concentration effectively attenuated phosphorylation levels for ERK1 and ERK2 in the mouse forebrain at P6, compared with the vehicle group, by using an antibody for dually phosphorylated, and thus activated, ERKs (Fig. 2a).”

Mice administered SL327 at P14 did not exhibit significant alteration in grooming compared with controls.

“Conversely, mice administered SL327 at P14 did not exhibit significant alteration compared with controls (Fig 11c, d; t-test, P > 0.05).”

In the presence of SL327, the level of apoptosis was increased in Erk1 KO mice to the extent comparable to control mice.

“In the presence of SL327, the level of apoptosis was increased in Erk1 KO mice to the extent that was comparable to those in control mice (Fig. 12a).”

Numbers of activated caspase-3+ cells were significantly increased in mice treated with SL327 at P6 compared with natural apoptosis in vehicle controls.

“Immunohistochemical analysis confirmed that the numbers of activated (cleaved) caspase-3+(AC3+) cells were significantly increased in mice treated with SL327 at P6 compared with those by natural apoptosis in vehicle controls (Fig. 3).”

Mice administered SL327 at P6 performed Y-maze spontaneous alternation task significantly worse compared with vehicle controls, as assessed by ratio of correct choice.

“Mice administered SL327 at P6 performed this task significantly worse compared with vehicle controls, as assessed by the ratio of correct choice (Fig. 9c; t-test, t = 3.36, P = 0.0023).”

Mice administered SL327 at P6 exhibited a significant increase in time spent in the open arm compared with vehicle controls in EPM test.

“Mice administered SL327 at P6 exhibited a significant increase in time spent in the open arm compared with vehicle controls, indicating reduced anxiety-related behavior (Fig. 9b; t-test, t = 2.85, P = 0.0087).”

Apoptosis was induced in oligodendrocytes as well as neurons in the cortex and white matter after SL327 administration.

“Double staining for AC3 and the oligodendrocyte-specific marker 2’,3’cyclic nucleotide 3’ phosphodiesterase (CNPase) indicated that apoptosis was induced in oligodendrocyte as well as neurons in the cortex and white matter (Fig. 6e,f), although apoptosis was not observed in oligodendrocytes in the subiculum (Fig. 6g).”

Mice administered SL327 at P6 exhibited decreased interaction with the social target compared with controls.

“However, mice administered SL327 at P6 exhibited decreased interaction with the social target compared with controls.”

At P14, ERK phosphorylation levels were attenuated significantly by SL327.

“We found no difference between the SL327-administered and vehicle group in levels of immunoreactivity for cleaved PARP, although the ERK phosphorylation levels were attenuated significantly by SL327 (Fig. 2b).”

In open field test at 11 weeks of age, no aberrant activity was observed in SL327-treated mice compared with vehicle controls as assessed by total distance traveled.

“In the open field test at 11 weeks of age, no aberrant activity was observed in SL327-treated mice compared with vehicle controls as assessed by the total distance traveled (Fig. 9d; t-test, P > 0.05).”

Transient blockade of ERKs activation caused robust apoptosis in the mouse brain at P6, but not at P14.

“In the current study, we found that transient blockade of ERKs activation caused robust apoptosis in the mouse brain at P6, but not at P14.”

No aberrant activity was observed in SL327-treated mice compared with vehicle controls in open-field test as assessed by total distance traveled.

“No aberrant activity was observed in SL327-treated mice compared with vehicle controls as assessed by the total distance traveled (Fig. 9a; t-test, P > 0.05).”

NMDA/AMPA ratio was reduced in mice administered SL327 at P6 compared with vehicle controls at 4-5 weeks of age.

“When recorded at 4-5 weeks of age, the ratio of NMDAR current to AMPAR current (NMDA/AMPA ratio) was reduced in mice administered SL327 at P6 compared with vehicle controls (Fig. 8; t-test, t = 3.85, P = 0.0007)”

Analysis of arterial blood before and 6 h after SL327 administration revealed no significant differences in pH, PaO2, and PaCO2 between groups.

“Analysis of arterial blood before and 6 h after SL327 administration revealed no significant differences in pH, partial pressure of arterial oxygen (PaO2), and partial pressure of arterial carbon dioxide (PaCO2) between the various groups (Table 1).”

In Y-maze test, no significant differences were observed in ratio of correct choice between mice administered SL327 at P14 and vehicle controls.

“In the Y-maze test, no significant differences were observed in the ratio of correct choice between mice administered SL327 at P14 and vehicle controls (Fig. 9f; t-test, P > 0.05).”

Apoptosis was not induced in astrocytes after SL327 administration.

“On the other hand, double staining for AC3 and the astrocyte-specific marker glial fibrillary acidic protein (GFAP) indicated that apoptosis was not induced in astrocytes after SL327 administration (Fig. 6c,d).”

LTP was not impaired at 4 weeks of age in mice administered SL327 at P14 compared with vehicle controls.

“Conversely, we observed that LTP was not impaired at 4 weeks of age in mice administered SL327 at P14 compared with vehicle controls (Fig. 7d; two-way repeated-measures ANOVA, P > 0.05).”

Social behavior was not impaired in mice administered SL327 at P14.

“Conversely, we observed that social behavior was not impaired in mice administered SL327 at P14.”

In SL327-administered mice at P6, there was no significant difference between interacting with a social target and interacting with an inanimate target.

“In SL327-administered mice, there was no significant difference between interacting with a social target and interacting with an inanimate target (Fig. 10a).”

Mice administered SL327 at P6 showed increased frequency of grooming bouts.

“and showed increased frequency of grooming bouts (Fig 11b; t-test, t = 4.18, P = 0.0006).”

Apoptosis was not observed in oligodendrocytes in the subiculum after SL327 administration.

“Double staining for AC3 and the oligodendrocyte-specific marker 2’,3’cyclic nucleotide 3’ phosphodiesterase (CNPase) indicated that apoptosis was induced in oligodendrocyte as well as neurons in the cortex and white matter (Fig. 6e,f), although apoptosis was not observed in oligodendrocytes in the subiculum (Fig. 6g).”

SL327 administration at P6 induced a significant increase in apoptosis in the brain compared with vehicle.

“Using an antibody for cleaved PARP, we found that SL327 administration induced a significant increase in apoptosis in the brain compared with the vehicle group (Fig. 2a).”

LTP was impaired in mice administered SL327 at P6.

“We found that LTP was impaired in mice administered SL327 at P6.”

Mice administered SL327 at P6 spent almost twice as much time grooming themselves as controls.

“Mice administered SL327 at P6 spent almost twice as much time grooming themselves as controls (Fig 11a; t-test, t = 2.60, P = 0.018)”

Neonatal administration of SL327 at P6 caused a profound suppression of LTP at 4 weeks of age compared with vehicle controls.

“Figure 7a indicates that neonatal administration of SL327 at P6 caused a profound suppression of LTP at 4 weeks of age compared with vehicle controls, despite the presence of robust short-term potentiation.”

At P14, there was no difference between SL327-administered and vehicle group in levels of immunoreactivity for cleaved PARP.

“We found no difference between the SL327-administered and vehicle group in levels of immunoreactivity for cleaved PARP, although the ERK phosphorylation levels were attenuated significantly by SL327 (Fig. 2b).”

Genetic abrogation of Erk1 or Erk2 per se does not cause increased apoptosis at P6.

“Our results indicate that genetic abrogation of Erk1 or Erk2 per se does not cause increased apoptosis at P6.”

No significant difference was observed in the level of cleaved PARP between Erk2 CKO and control mice when vehicle was administered.

“However, no significant difference was observed in the level of cleaved PARP between Erk2 CKO and control mice when vehicle was administered (Fig. 12c), indicating that genetic abrogation of Erk2 per se did not cause increased apoptosis at P6, contrary to our expectation.”

Vehicle control group spent significantly more time interacting with social target than with inanimate target in sociability test.

“The vehicle control group spent significantly more time interacting with the social target than with the inanimate target (Fig. 10a).”

Apoptosis was induced in neurons after SL327 administration.

“Double staining for AC3 and the neuron-specific marker neuronal nuclear antigen (NeuN) revealed that apoptosis was induced in neurons (Fig. 6a,b).”

When vehicle was administered, there was no significant difference in levels of cleaved PARP between Erk1 KO and control mice.

“When vehicle was administered, there was no significant difference in levels of cleaved PARP between Erk1 KO and control mice, indicating that the genetic abrogation of Erk1 per se does not cause increased apoptosis at P6 (Fig. 12a).”

Increased apoptosis was observed mainly in neurons and oligodendrocytes, but not in astrocytes, when SL327 was administered at P6.

“We found that increased apoptosis was observed mainly in neurons and oligodendrocytes, but not in astrocytes, when SL327 was administered at P6.”

In EPM test, no significant differences were observed in time spent in open arms between mice administered SL327 at P14 and vehicle controls.

“In the EPM test, no significant differences were observed in time spent in the open arms between mice administered SL327 at P14 and vehicle controls, indicating normal anxiety-related behavior (Fig. 9e; t-test, P > 0.05).”

In Erk2 CKO mice, the apoptosis level was significantly decreased compared with control mice in the presence of SL327.

“In Erk2 CKO mice, the apoptosis level was significantly decreased when compared with control mice in the presence of SL327.”

Mice deficient for Erk2 in the central nervous system exhibited learning impairment and social deficits similar to those seen in ASD.

“We previously reported that mice deficient for Erk2 in the central nervous system (CNS) exhibited learning impairment and social deficits similar to those seen in ASD10.”

• cites [10] Satoh, Y. et al. ERK2 contributes to the control of social behaviors in mice. J. Neurosci. 31, 11953–11967 (2011). sourceNot checked yet

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Various neurodevelopment disorders might be generally linked to defects in ERKs signaling during the critical period.

“various neurodevelopment disorders might be generally linked to defects in ERKs signaling during the critical period.”

• Also stated in: § Discussion · hedged

  “Our results provide novel insight into the mechanistic basis for neurodevelopment disorders, and suggest that various neurodevelopment disorders might be generally linked to defects in ERKs signaling during the critical period.”

• AssumptionRests on an assumption: SL327 is a blood-brain barrier-penetrating MEK inhibitor that inhibits ERK phosphorylation.

The effects of ERKs are strongly age-dependent.

“Thus, surprisingly, these effects of ERKs are strongly age-dependent”

This neuro-apoptosis was attenuated by the counteractive effect of lithium on the ERKs suppression.

“Furthermore, this neuro-apoptosis was attenuated by the counteractive effect of lithium on the ERKs suppression8.”

• cites [8] Straiko, M. M. et al. Lithium protects against anesthesia-induced developmental neuroapoptosis. Anesthesiology 110, 862–868 (2009). sourceNot checked yet

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ERKs play important roles in proper formation of neural circuits during the critical period.

“Herein we show that extracellular signal-regulated protein kinases (ERKs) play important roles in proper formation of neural circuits during the critical period.”

Phosphorylation of ERKs during the critical period is absolutely required for proper development of brain functioning.

“indicating that phosphorylation of ERKs during the critical period is absolutely required for proper development of brain functioning.”

Exposure to NMDA antagonist, ethanol, or general anesthetics causes a significant increase in neuronal degeneration at P6-P7.

“For instance, exposure to N-methyl-D-aspartate (NMDA) antagonist, ethanol, or general anesthetics, causes a significant increase in neuronal degeneration at P6–P7456.”

• cites [4] Ikonomidou, C., Mosinger, J. L., Salles, K. S., Labruyere, J. & Olney, J. W. Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity. J. Neurosci. 9, 2809–2818 (1989). sourceNot checked yet

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• cites [5] Ikonomidou, C. et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70–74 (1999). sourceNot checked yet

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• cites [6] Jevtovic-Todorovic, V., et al. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23, 876–882 (2003). sourceNot checked yet

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Anesthetic-induced apoptosis is greatest if exposure occurs at P6-P7, with little or no increase in apoptosis at P14 in rodents.

“Studies about anesthetic-induced toxicity in brain development indicate that anesthetic-induced apoptosis is the greatest if exposure occurs at P6–P745611, with little or no increase in apoptosis at P1411 in rodents.”

• cites [11] Yon, J. H., Daniel-Johnson, J., Carter, L. B. & Jevtovic-Todorovic, V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135, 815–827 (2005). sourceNot checked yet

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Interruption during the critical period could cause permanent and irreversible problems.

“The critical period is a time of learning opportunity but also of vulnerability for interruption: interruption during the critical period could cause permanent and irreversible problems.”

Inhibition of both ERK1 and ERK2 may be required to induce increased apoptosis at P6.

“Additionally, inhibition of both ERK1 and ERK2 may be required to induce increased apoptosis at P6.”

Our results do not contradict the hypothesis that ERK pathway is, at least partly, involved in the mechanisms underlying apoptotic action of general anesthetics in the developing brain.

“Thus, our results do not contradict the hypothesis that ERK pathway is, at least partly, involved in the mechanisms underlying apoptotic action of general anesthetics in the developing brain.”

SL327 administration at P6 induced abnormal social behavior.

“Therefore, it can be concluded that SL327 administration at P6 induced abnormal social behavior.”

Basal synaptic transmissions appear to be unaltered in mice administered SL327 at P6.

“Thus, basal synaptic transmissions appear to be unaltered in mice administered SL327 at P6.”

ERK phosphorylation is required to ensure the proper development of neural functions during the critical period.

“These results indicated that ERK phosphorylation should be required to ensure the proper development of neural functions during the critical period.”

Toxicity caused by inhibition of ERK phosphorylation is strongly age-dependent.

“These results indicate that the toxicity caused by the inhibition of ERK phosphorylation is strongly age-dependent.”

Deranged function of NMDA and/or AMPA receptor subunits at postsynaptic sites in hippocampal neurons is suggested by reduced NMDA/AMPA ratio.

“suggesting deranged function of NMDA and/or AMPA receptor subunits at the postsynaptic sites in hippocampal neurons.”

Genetic abrogation of Erk2 causes significant developmental changes.

“Consistent with this hypothesis, previous reports have indicated significant developmental changes by genetic abrogation of Erk2222324, but not in Erk1 KO mice25.”

• cites [22] Satoh, Y. et al. Deletion of ERK1 and ERK2 in the CNS causes cortical abnormalities and neonatal lethality: Erk1 deficiency enhances the impairment of neurogenesis in Erk2-deficient mice. J. Neurosci. 31, 1149–1155 (2011). sourceNot checked yet

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• cites [23] Samuels, I. S. et al. Deletion of ERK2 mitogen-activated protein kinase identifies its key roles in cortical neurogenesis and cognitive function. J. Neurosci. 28, 6983–6995 (2008). sourceNot checked yet

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• cites [24] Pucilowska, J., Puzerey, P. A., Karlo, J. C., Galan, R. F. & Landreth, G. E. Disrupted ERK signaling during cortical development leads to abnormal progenitor proliferation, neuronal and network excitability and behavior, modeling human neuro-cardio-facial-cutaneous and related syndromes. J. Neurosci. 32, 8663–8677 (2012). sourceNot checked yet

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Genetic abrogation of Erk1 per se does not cause increased apoptosis at P6.

“When vehicle was administered, there was no significant difference in levels of cleaved PARP between Erk1 KO and control mice, indicating that the genetic abrogation of Erk1 per se does not cause increased apoptosis at P6 (Fig. 12a).”

SL327 at P6 reduced anxiety-related behavior.

“Mice administered SL327 at P6 exhibited a significant increase in time spent in the open arm compared with vehicle controls, indicating reduced anxiety-related behavior (Fig. 9b; t-test, t = 2.85, P = 0.0087).”

Blockade of both isoforms of ERK is needed to induce apoptosis at P6.

“These results indicated that blockade of both isoforms of ERK is needed to induce apoptosis at P6.”

Genetic abrogation of Erk2 per se did not cause increased apoptosis at P6.

“However, no significant difference was observed in the level of cleaved PARP between Erk2 CKO and control mice when vehicle was administered (Fig. 12c), indicating that genetic abrogation of Erk2 per se did not cause increased apoptosis at P6, contrary to our expectation.”

The critical period closes between P6 and P14 in this mouse model.

“This strong age-dependency suggests that the critical period closes between P6 and P14 in this mouse model.”

Spatial working memory was normal in mice administered SL327 at P14.

“Thus, spatial working memory was normal in mice administration SL327 at P14.”

Apoptosis caused by SL327 is not attributable to an indirect effect such as hypoxia.

“suggesting that apoptosis caused by SL327 is not attributable to an indirect effect such as hypoxia.”

Inhibition of ERK pathway leaves short-term presynaptic plasticity intact.

“indicating that the inhibition of ERK pathway leaves short-term presynaptic plasticity intact.”

SL327 administration at P14 did not induce abnormal social behavior.

“Therefore, it can be concluded that SL327 administration at P14 did not induce abnormal social behavior.”

Deranged function of NMDA and/or AMPA receptor subunits at postsynaptic sites might contribute to the reduced LTP.

“indicating the deranged function of NMDA and/or AMPA receptor subunits at the postsynaptic sites might contribute to the reduced LTP, although the precise mechanism is still unknown.”

Erk1 KO mice do not show significant developmental changes.

“Consistent with this hypothesis, previous reports have indicated significant developmental changes by genetic abrogation of Erk2222324, but not in Erk1 KO mice25.”

• cites [25] Pages, G. et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286, 1374–1377 (1999). sourceNot checked yet

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SL327 administration during the critical period leads to impaired neuronal function in modulating synaptic transmission later in adulthood.

“These results indicate that SL327 administration during the critical period leads to impaired neuronal function in modulating synaptic transmission later in adulthood.”

Inhibition of ERK phosphorylation caused apoptosis in the developing brain in a dose-dependent manner.

“Thus, the inhibition of ERK phosphorylation caused apoptosis in the developing brain in a dose-dependent manner.”

Spatial working memory was impaired by SL327 administration at P6.

“Thus, spatial working memory was impaired by SL327 administration at P6.”

Regulating mechanism of ERK in relation to the formation of neural circuits during the critical period may be different from that in the adult stage.

“In this respect, regulating mechanism of ERK in relation to the formation of neural circuits during the critical period may be different from that in the adult stage.”

Genetic abrogation of Erk2 gene in the CNS results in ASD-like behavior in mice.

“We previously reported that genetic abrogation of Erk2 gene in the CNS results in ASD-like behavior in mice10.”

• cites [10] Satoh, Y. et al. ERK2 contributes to the control of social behaviors in mice. J. Neurosci. 31, 11953–11967 (2011). sourceNot checked yet

  Not checked in the free run.

Only transient inactivation of ERK phosphorylation during the critical period causes ASD-like behaviors later in adulthood.

“In the current study, we demonstrate that only transient inactivation of ERKs phosphorylation during the critical period cause ASD-like behaviors later in adulthood.”

• AssumptionRests on an assumption: SL327 is a blood-brain barrier-penetrating MEK inhibitor that inhibits ERK phosphorylation.

Altered expression of ERKs seems to be related to ASD-like symptoms.

“Furthermore, altered expression of ERKs seems to be related to ASD-like symptoms.”

ASD may result from disruption of proper formation of neural circuits during the critical period.

“Accumulating evidence suggests that ASD may result from disruption of proper formation of neural circuits during the critical period (see review29).”

• cites [29] LeBlanc, J. J. & Fagiolini, M. Autism: a “critical period” disorder? Neural. Plast. 2011, 921680 (2011). sourceNot checked yet

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Drugs that inhibit ERK activation might have the potential to cause developmental/mental retardation disorders.

“In this respect, drugs that can have an inhibitory effect on ERK activation, although long considered safe for pediatric use as with general anesthetics, might have potential to cause developmental/mental retardation disorders.”

• AssumptionRests on an assumption: SL327 is a blood-brain barrier-penetrating MEK inhibitor that inhibits ERK phosphorylation.