Joaquin N. Lugo

Rapamycin suppresses seizures and neuronal hypertrophy in a mouse model of cortical dysplasia.

SUMMARY Malformations of the cerebral cortex known as cortical dysplasia account for the majority of cases of intractable childhood epilepsy. With the exception of the tuberous sclerosis complex, the molecular basis of most types of cortical dysplasia is completely unknown. Currently, there are no good animal models available that recapitulate key features of the disease, such as structural cortical abnormalities and seizures, hindering progress in understanding and treating cortical dysplasia. At the neuroanatomical level, cortical abnormalities may include dyslamination and the presence of abnormal cell types, such as enlarged and misoriented neurons and neuroglial cells. Recent studies in resected human brain tissue suggested that a misregulation of the PI3K (phosphoinositide 3-kinase)-Akt-mTOR (mammalian target of rapamycin) signaling pathway might be responsible for the excessive growth of dysplastic cells in this disease. Here, we characterize neuronal subset (NS)-Pten mutant mice as an animal model of cortical dysplasia. In these mice, the Pten gene, which encodes a suppressor of the PI3K pathway, was selectively disrupted in a subset of neurons by using Cre-loxP technology. Our data indicate that these mutant mice, like cortical dysplasia patients, exhibit enlarged cortical neurons with increased mTOR activity, and abnormal electroencephalographic activity with spontaneous seizures. We also demonstrate that a short-term treatment with the mTOR inhibitor rapamycin strongly suppresses the severity and the duration of the seizure activity. These findings support the possibility that this drug may be developed as a novel antiepileptic treatment for patients with cortical dysplasia and similar disorders.

Altered phosphorylation and localization of the A-type channel, Kv4.2 in status epilepticus.

Extracelluar signal‐regulated kinase (ERK) pathway activation has been demonstrated following convulsant stimulation; however, little is known about the molecular targets of ERK in seizure models. Recently, it has been shown that ERK phosphorylates Kv4.2 channels leading to down‐regulation of channel function, and substantially alters dendritic excitability. In the kainate model of status epilepticus (SE), we investigated whether ERK phosphorylates Kv4.2 and whether the changes in Kv4.2 were evident at a synaptosomal level during SE. Western blotting was performed on rat hippocampal whole cell, membrane, synaptosomal, and surface biotinylated extracts following systemic kainate using an antibody generated against the Kv4.2 ERK sites and for Kv4.2, ERK, and phospho‐ERK. ERK activation was associated with an increase in Kv4.2 phosphorylation during behavioral SE. During SE, ERK activation and Kv4.2 phosphorylation were evident at the whole cell and synaptosomal levels. In addition, while whole‐cell preparations revealed no alterations in total Kv4.2 levels, a decrease in synaptosomal and surface expression of Kv4.2 was evident after prolonged SE. These results demonstrate ERK pathway coupling to Kv4.2 phosphorylation. The finding of decreased Kv4.2 levels in hippocampal synaptosomes and surface membranes suggest additional mechanisms for decreasing the dendritic A‐current, which could lead to altered intrinsic membrane excitability during SE.

Deletion of PTEN produces autism-like behavioral deficits and alterations in synaptic proteins.

Many genes have been implicated in the underlying cause of autism but each gene accounts for only a small fraction of those diagnosed with autism. There is increasing evidence that activity-dependent changes in neuronal signaling could act as a convergent mechanism for many of the changes in synaptic proteins. One candidate signaling pathway that may have a critical role in autism is the PI3K/AKT/mTOR pathway. A major regulator of this pathway is the negative repressor phosphatase and tensin homolog (PTEN). In the current study we examined the behavioral and molecular consequences in mice with neuron subset-specific deletion of PTEN. The knockout (KO) mice showed deficits in social chamber and social partition test. KO mice demonstrated alterations in repetitive behavior, as measured in the marble burying test and hole-board test. They showed no changes in ultrasonic vocalizations emitted on postnatal day 10 or 12 compared to wildtype (WT) mice. They exhibited less anxiety in the elevated-plus maze test and were more active in the open field test compared to WT mice. In addition to the behavioral alterations, KO mice had elevation of phosphorylated AKT, phosphorylated S6, and an increase in S6K. KO mice had a decrease in mGluR but an increase in total and phosphorylated fragile X mental retardation protein. The disruptions in intracellular signaling may be why the KO mice had a decrease in the dendritic potassium channel Kv4.2 and a decrease in the synaptic scaffolding proteins PSD-95 and SAP102. These findings demonstrate that deletion of PTEN results in long-term alterations in social behavior, repetitive behavior, activity, and anxiety. In addition, deletion of PTEN significantly alters mGluR signaling and many synaptic proteins in the hippocampus. Our data demonstrates that deletion of PTEN can result in many of the behavioral features of autism and may provide insights into the regulation of intracellular signaling on synaptic proteins.

Early-life seizures result in deficits in social behavior and learning.

Children with epilepsy show a high co-morbidity with psychiatric disorders and autism. One of the critical determinants of a childs behavioral outcome with autism and cognitive dysfunction is the age of onset of seizures. In order to examine whether seizures during postnatal days 7-11 result in learning and memory deficits and behavioral features of autism we administered the inhalant flurothyl to induce seizures in C57BL/6J mice. Mice received three seizures per day for five days starting on postnatal day 7. Parallel control groups consisted of similarly handled animals that were not exposed to flurothyl and naïve mice. Subjects were then processed through a battery of behavioral tests in adulthood: elevated-plus maze, nose-poke assay, marble burying, social partition, social chamber, fear conditioning, and Morris water maze. Mice with early-life seizures had learning and memory deficits in the training portion of the Morris water maze (p<0.05) and probe trial (p<0.01). Mice with seizures showed no differences in marble burying, the nose-poke assay, or elevated plus-maze testing compared to controls. However, they showed a significant difference in the social chamber and social partition tests. Mice with seizures during postnatal days 7-11 showed a significant decrease in social interaction in the social chamber test and had a significant impairment in social behavior in the social partition test. Together, these results indicate that early life seizures result in deficits in hippocampal-dependent memory tasks and produce long-term disruptions in social behavior.

Kv4.2 knockout mice have hippocampal-dependent learning and memory deficits

Kv4.2 channels contribute to the transient, outward K(+) current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit in the learning phase of the Morris water maze (MWM) and significant impairment in the probe trial compared with wild type (WT). Kv4.2 KO mice also demonstrated a specific deficit in contextual learning in the fear-conditioning test, without impairment in the conditioned stimulus or new context condition. Kv4.2 KO mice had normal activity, anxiety levels, and prepulse inhibition compared with WT mice. A compensatory increase in tonic inhibition has been previously described in hippocampal slice recordings from Kv4.2 KO mice. In an attempt to decipher whether increased tonic inhibition contributed to the learning and memory deficits in Kv4.2 KO mice, we administered picrotoxin to block GABA(A) receptors (GABA(A)R), and thereby tonic inhibition. This manipulation had no effect on behavior in the WT or KO mice. Furthermore, total protein levels of the α5 or δ GABA(A)R subunits, which contribute to tonic inhibition, were unchanged in hippocampus. Overall, our findings add to the growing body of evidence, suggesting an important role for Kv4.2 channels in hippocampal-dependent learning and memory.

A critical review of the postulated role of the non-essential amino acid, β-N-methylamino-L-alanine, in neurodegenerative disease in humans

ABSTRACT The compound BMAA (β-N-methylamino-L-alanine) has been postulated to play a significant role in four serious neurological human diseases: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, Parkinsonism, and dementia that occur globally. ALS/PDC with symptoms of all three diseases first came to the attention of the scientific community during and after World War II. It was initially associated with cycad flour used for food because BMAA is a product of symbiotic cycad root-dwelling cyanobacteria. Human consumption of flying foxes that fed on cycad seeds was later suggested as a source of BMAA on Guam and a cause of ALS/PDC. Subsequently, the hypothesis was expanded to include a causative role for BMAA in other neurodegenerative diseases including Alzheimer’s disease (AD) through exposures attributed to proximity to freshwaters and/or consumption of seafood due to its purported production by most species of cyanobacteria. The hypothesis that BMAA is the critical factor in the genesis of these neurodegenerative diseases received considerable attention in the medical, scientific, and public arenas. This review examines the history of ALS/PDC and the BMAA-human disease hypotheses; similarities and differences between ALS/PDC and the other diseases with similar symptomologies; the relationship of ALS/PDC to other similar diseases, studies of BMAA-mediated effects in lab animals, inconsistencies and data gaps in the hypothesis; and other compounds and agents that were suggested as the cause of ALS/PDC on Guam. The review concludes that the hypothesis of a causal BMAA neurodegenerative disease relationship is not supported by existing data.

Deletion of PTEN produces deficits in conditioned fear and increases fragile X mental retardation protein

The phosphatase and tensin homolog detected on chromosome 10 (PTEN) gene product modulates activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The PI3K pathway has been found to be involved in the regulation of the fragile X mental retardation protein, which is important for long-term depression and in the formation of new memories. We used delayed fear conditioning and trace fear conditioning to determine learning and memory deficits in neuron subset-specific Pten (NS-Pten) conditional knockout (KO) mice. We found that NS-Pten KO mice had deficits in contextual learning and trace conditioning, but did not have deficits in the ability to learn a conditioned stimulus. Furthermore, we found increased levels in the total and phosphorylated forms of the fragile X mental retardation protein (FMRP) in the hippocampus of NS-Pten KO mice.

Testing for odor discrimination and habituation in mice.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animals response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

Sex-specific and genotype-specific differences in vocalization development in FMR1 knockout mice.

Fragile X syndrome is a neurodevelopmental disorder caused by a trinucleotide (CGG) hyperexpansion in the FMR1 gene, functionally silencing transcription of the fragile X mental retardation protein (FMRP). This disorder is characterized by impaired cognition, communication, and social behavior. The aim of this study was to investigate the development of ultrasonic vocalization (USV) behavior in a Fmr1-deficient mouse model. On postnatal days (PD) 9–14, separate cohorts of FVB/NJ pups were removed from their homecage and isolation-induced USVs were recorded. There were significant genotype-dependent and sex-dependent differences in USV behavior across the different testing days. Fmr1 knockout (KO) mice showed a significant reduction in vocalizations across all days. There was also a significant difference in vocalizations between male and female mice. We found a significant decrease in the total number of calls for KO males on PD9 and PD13 as well as an increase in the total number of calls for KO males on PD12. The KO males also showed a significant increase in the total call duration on PD12 and a reduction on PD13. The KO female showed a significant decrease in the total number of calls on PD9 and PD10. They also showed a significant decrease in the total call duration on PD9 and a marginal decrease in the total call duration on PD10. These results provide additional evidence for communication deficits in Fmr1 deficient mice and provide new insight suggesting sexually dimorphic vocalizations during the neonatal period.

Deletion of Fmr1 results in sex‐specific changes in behavior

In this study, we used a systemic Fmr1 knockout in order to investigate both genotype‐ and sex‐specific differences across multiple measures of sociability, repetitive behaviors, activity levels, anxiety, and fear‐related learning and memory.