Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling.

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

Knock-in mouse model of alternating hemiplegia of childhood: behavioral and electrophysiologic characterization.

OBJECTIVES: Mutations in the ATP1α3 subunit of the neuronal Na+/K+-ATPase are thought to be responsible for seizures, hemiplegias, and other symptoms of alternating hemiplegia of childhood (AHC). However, the mechanisms through which ATP1A3 mutations mediate their pathophysiologic consequences are not yet understood. The following hypotheses were investigated: (1) Our novel knock-in mouse carrying the most common heterozygous mutation causing AHC (D801N) will exhibit the manifestations of the human condition and display predisposition to seizures; and (2) the underlying pathophysiology in this mouse model involves increased excitability in response to electrical stimulation of Schaffer collaterals and abnormal predisposition to spreading depression (SD). METHODS: We generated the D801N mutant mouse (Mashlool, Mashl+/-) and compared mutant and wild-type (WT) littermates. Behavioral tests, amygdala kindling, flurothyl-induced seizure threshold, spontaneous recurrent seizures (SRS), and other paroxysmal activities were compared between groups. In vitro electrophysiologic slice experiments on hippocampus were performed to assess predisposition to hyperexcitability and SD. RESULTS: Mutant mice manifested a distinctive phenotype similar to that of humans with AHC. They had abnormal impulsivity, memory, gait, motor coordination, tremor, motor control, endogenous nociceptive response, paroxysmal hemiplegias, diplegias, dystonias, and SRS, as well as predisposition to kindling, to flurothyl-induced seizures, and to sudden unexpected death. Hippocampal slices of mutants, in contrast to WT animals, showed hyperexcitable responses to 1 Hz pulse-trains of electrical stimuli delivered to the Schaffer collaterals and had significantly longer duration of K+-induced SD responses. SIGNIFICANCE: Our model reproduces the major characteristics of human AHC, and indicates that ATP1α3 dysfunction results in abnormal short-term plasticity with increased excitability (potential mechanism for seizures) and a predisposition to more severe SD responses (potential mechanism for hemiplegias). This model of the human condition should help in understanding the molecular pathways underlying these phenotypes and may lead to identification of novel therapeutic strategies of ATP1α3 related disorders and seizures.

Fragile X mental retardation protein regulates protein expression and mRNA translation of the potassium channel Kv4.2.

A prominent characteristic of the inherited intellectual impairment disease fragile X syndrome (FXS) is neuronal hyperexcitability, resulting in a variety of symptoms, such as hyperactivity, increased sensitivity to sensory stimuli, and a high incidence of epileptic seizures. These symptoms account for a significant part of the disease pattern, but the underlying molecular mechanisms of neuronal hyperexcitability in FXS remain poorly understood. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), which regulates synaptic protein synthesis and is a key player to limit signaling pathways downstream of metabotropic glutamate receptors 1/5 (mGlu1/5). Recent findings suggest that FMRP might also directly regulate voltage-gated potassium channels. Here, we show that total and plasma membrane protein levels of Kv4.2, the major potassium channel regulating hippocampal neuronal excitability, are reduced in the brain of an FXS mouse model. Antagonizing mGlu5 activity with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) partially rescues reduced surface Kv4.2 levels in Fmr1 knock-out (KO) mice, suggesting that excess mGlu1/5 signal activity contributes to Kv4.2 dysregulation. As an additional mechanism, we show that FMRP is a positive regulator of Kv4.2 mRNA translation and protein expression and associates with Kv4.2 mRNA in vivo and in vitro. Our results suggest that absence of FMRP-mediated positive control of Kv4.2 mRNA translation, protein expression, and plasma membrane levels might contribute to excess neuronal excitability in Fmr1 KO mice, and thus imply a potential mechanism underlying FXS-associated epilepsy.

Excess phosphoinositide 3-kinase subunit synthesis and activity as a novel therapeutic target in fragile X syndrome.

Fragile X syndrome (FXS) is an inherited neurologic disease caused by loss of fragile X mental retardation protein (FMRP), which is hypothesized to mediate negative regulation of mRNA translation at synapses. A prominent feature of FXS animal models is exaggerated signaling through group 1 metabotropic glutamate receptors (gp1 mGluRs), and therapeutic strategies to treat FXS are targeted mainly at gp1 mGluRs. Recent studies, however, indicate that a variety of receptor-mediated signal transduction pathways are dysregulated in FXS, suggesting that FMRP acts on a common downstream signaling molecule. Here, we show that deficiency of FMRP results in excess activity of phosphoinositide 3-kinase (PI3K), a downstream signaling molecule of many cell surface receptors. In Fmr1 knock-out neurons, excess synaptic PI3K activity can be reduced by perturbation of gp1 mGluR-mediated signaling. Remarkably, increased PI3K activity was also observed in FMRP-deficient non-neuronal cells in the absence of gp1 mGluRs. Here, we show that FMRP regulates the synthesis and synaptic localization of p110beta, the catalytic subunit of PI3K. In wild type, gp1 mGluR activation induces p110beta translation, p110beta protein expression, and PI3K activity. In contrast, both p110beta protein synthesis and PI3K activity are elevated and insensitive to gp1 mGluR stimulation in Fmr1 knock-out. This suggests that dysregulated PI3K signaling may underlie the synaptic impairments in FXS. In support of this hypothesis, we show that PI3K antagonists rescue three FXS-associated phenotypes: dysregulated synaptic protein synthesis, excess AMPA receptor internalization, and increased spine density. Targeting excessive PI3K activity might thus be a potent therapeutic strategy for FXS.

MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset.

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

[Relationship between nuclear factor-kappaB activity and kidney injury in rats with postburn sepsis].

OBJECTIVE: To investigate the relationship between nuclear factor-KappaB (NF-KappaB) activity and kidney injury in rats with postburn sepsis. METHODS: Rats subjected to 30% full-thickness scald injury, followed by intraperitoneal injection of lipopolysaccharide (LPS), were used in the present study. Fifty-four Wistar rats were randomly divided into normal control group, postburn sepsis 1, 2, 6, 12, 24 hours groups, and sepsis with NF-KappaB inhibitor pyrrolidine dithiocarbamate (PDTC) treatment 1, 2, and 6 hour groups. NF-KappaB activity, tumor necrosis factor-alpha (TNF-alpha) protein expression, and renal function were determined with electrophoretic mobility shift assay (EMSA), enzyme linked immunoadsorbent assay (ELISA), and automatic biochemistry analyzer, respectively. RESULTS: NF-KappaB activity in kidney was markedly enhanced and reached its peak 1 hour after scalding and injection of LPS (all P<0.01), and was decreased remarkably after the administration of PDTC. PDTC could suppress the elevated plasma TNF-alpha protein expression (both P<0.01), but not renal TNF-alpha levels. PDTC could not reduce blood urea nitrogen and blood creatinine contents, which were increased after scalding followed by LPS challenge. CONCLUSION: The results suggest that the treatment with NF-KappaB inhibitor PDTC could down-regulate the NF-KappaB activity in kidney, but it could not protect the renal function in rats with postburn sepsis.

Epileptic encephalopathy-causing mutations in DNM1 impair synaptic vesicle endocytosis.

OBJECTIVE: To elucidate the functional consequences of epileptic encephalopathy-causing de novo mutations in DNM1 (A177P, K206N, G359A), which encodes a large mechanochemical GTPase essential for neuronal synaptic vesicle endocytosis. METHODS: HeLa and COS-7 cells transfected with wild-type and mutant DNM1 constructs were used for transferrin assays, high-content imaging, colocalization studies, Western blotting, and electron microscopy (EM). EM was also conducted on the brain sections of mice harboring a middle-domain Dnm1 mutation (Dnm1 (Ftfl)). RESULTS: We demonstrate that the expression of each mutant protein decreased endocytosis activity in a dominant-negative manner. One of the G-domain mutations, K206N, decreased protein levels. The G359A mutation, which occurs in the middle domain, disrupted higher-order DNM1 oligomerization. EM of mutant DNM1-transfected HeLa cells and of the Dnm1 (Ftfl) mouse brain revealed vesicle defects, indicating that the mutations likely interfere with DNM1's vesicle scission activity. CONCLUSION: Together, these data suggest that the dysfunction of vesicle scission during synaptic vesicle endocytosis can lead to serious early-onset epilepsies.

Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.

We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.

Negative regulation of RhoA translation and signaling by hnRNP-Q1 affects cellular morphogenesis.

The small GTPase RhoA has critical functions in regulating actin dynamics affecting cellular morphogenesis through the RhoA/Rho kinase (ROCK) signaling cascade. RhoA signaling controls stress fiber and focal adhesion formation and cell motility in fibroblasts. RhoA signaling is involved in several aspects of neuronal development, including neuronal migration, growth cone collapse, dendrite branching, and spine growth. Altered RhoA signaling is implicated in cancer and neurodegenerative disease and is linked to inherited intellectual disabilities. Although much is known about factors regulating RhoA activity and/or degradation, little is known about molecular mechanisms regulating RhoA expression and the subsequent effects on RhoA signaling. We hypothesized that posttranscriptional control of RhoA expression may provide a mechanism to regulate RhoA signaling and downstream effects on cell morphology. Here we uncover a cellular function for the mRNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) Q1 in the control of dendritic development and focal adhesion formation that involves the negative regulation of RhoA synthesis and signaling. We show that hnRNP-Q1 represses RhoA translation and knockdown of hnRNP-Q1 induced phenotypes associated with elevated RhoA protein levels and RhoA/ROCK signaling. These morphological changes were rescued by ROCK inhibition and/or RhoA knockdown. These findings further suggest that negative modulation of RhoA mRNA translation can provide control over downstream signaling and cellular morphogenesis.

Automated 4D analysis of dendritic spine morphology: applications to stimulus-induced spine remodeling and pharmacological rescue in a disease model.

Uncovering the mechanisms that regulate dendritic spine morphology has been limited, in part, by the lack of efficient and unbiased methods for analyzing spines. Here, we describe an automated 3D spine morphometry method and its application to spine remodeling in live neurons and spine abnormalities in a disease model. We anticipate that this approach will advance studies of synapse structure and function in brain development, plasticity, and disease.