Elgersma, Y.

Rheb Regulates Mitophagy Induced by Mitochondrial Energetic Status (Article)

2013-04-19T00:00:00Z

Mitophagy has been recently described as a mechanism of elimination of damaged organelles. Although the regulation of the amount of mitochondria is a core issue concerning cellular energy homeostasis, the relationship between mitochondrial degradation and energetic activity has not yet been considered. Here, we report that the stimulation of mitochondrial oxidative phosphorylation enhances mitochondrial renewal by increasing its degradation rate. Upon high oxidative phosphorylation activity, we found that the small GTPase Rheb is recruited to the mitochondrial outer membrane. This mitochondrial localization of Rheb promotes mitophagy through a physical interaction with the mitochondrial autophagic receptor Nix and the autophagosomal protein LC3-II. Thus, Rheb-dependent mitophagy contributes to the maintenance of optimal mitochondrial energy production. Our data suggest that mitochondrial degradation contributes to a bulk renewal of the organelle in order to prevent mitochondrial aging and to maintain the efficiency of oxidative phosphorylation.

Accelerated age-related cognitive decline and neurodegeneration, caused by deficient DNA repair (Article)

2011-08-31T00:00:00Z

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage of Ercc1Δ/-mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an agedependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.

Rheb: Enrichment beyond the brain (Article)

2011-08-01T00:00:00Z

Interval timing is intact in arrhythmic cry1/Cry2-deficient mice (Article)

2011-08-01T00:00:00Z

Localizing the self in time is fundamental for daily life functioning and is lacking in severe disabling neuropsychiatric disorders like schizophrenia. Brains keep track of time across an impressive range of scales. Great progress has been made in identifying the molecular machinery of the circadian clock, the brain's master clock that operates on the 24-hour scale and allows animals to know the "time of the day" that important events occur, without referring to external cues. However, the biology of interval timing, the mechanism responsible for durations in the seconds-to-minutes-to-hours range, remains a mystery, and an obvious question is whether there is a common biological solution for keeping track of time across these 2 time scales. To address this, we trained Cry1/Cry2 double knockout mice on an interval timing task with durations that ranged between 3 and 27 seconds. The mice were kept under constant light conditions to avoid any exogenously induced form of daily rhythmicity. We observed that the homozygous knockouts displayed as accurate and precise a temporal memory as the control mice. This suggests that the Cry1 and Cry2 genes are not an important component of the interval timer. Furthermore, proper calibration of the interval timer does not depend on a functional circadian clock. Thus, these 2 timing systems likely rely on different and independent biological mechanisms.

βCaMKII plays a nonenzymatic role in hippocampal synaptic plasticity and learning by targeting α;CaMKII to synapses (Article)

2011-07-13T00:00:00Z

The calcium/calmodulin-dependent kinase type II (CaMKII) holoenzyme of the forebrain predominantly consists of heteromeric complexes of the αCaMKII andβCaMKII isoforms. Yet, in contrast toβCaMKII, the role of βCaMKII in hippocampal synaptic plasticity and learning has not been investigated. Here, we compare two targeted Camk2b mouse mutants to study the role of αCaMKII in hippocampal function. Using a Camk2b-1-mutant, in which βCaMKII is absent, we show that both hippocampal-dependent learning and Schaffer collateralβCA1 long-term potentiation (LTP) are highly dependent upon the presence of β Ca MKII. We further show that αCaMKII is required for proper targeting of β Ca MKII to the synapse, indicating that βCaMKII regulates the distribution of αCa M KII between the synaptic pool and the adjacent dendritic shaft. In contrast, localization of β Ca MKII, hippocampal synaptic plasticity and learning were unaffected in the Camk2bA303Rmutant, in which the calcium/calmodulin-dependent activation of βCaMKII is prevented, while the F-actin binding and bundling property is preserved. This indicates that the calcium/calmodulin-dependent kinase activity of αCaMKII is fully dispensable for hippocampal learning, LTP, and targeting of βCaMKII, but implies a critical role for the F-actin binding and bundling properties of βCaMKII in synaptic function. Together, our data provide compelling support for a model of CaMKII function in which βCaMKII and βCaMKII act in concert, but with distinct functions, to regulate hippocampal synaptic plasticity and learning.

Motor deficits in neurofibromatosis type 1 mice: The role of the cerebellum (Article)

2011-06-01T00:00:00Z

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disease, characterized by various neurocutaneous symptoms, cognitive impairments and problems in fine and gross motor performance. Although cognitive deficits in NF1 have been attributed to increased release of the inhibitory neurotransmitter γ-amino butyric acid (GABA) in the hippocampus, the origin of the motor deficits is unknown. Cerebellar Purkinje cells, the sole output neurons of the cerebellar cortex, are GABAergic neurons and express neurofibromin at high levels, suggesting an important role for the cerebellum in the observed motor deficits in NF1. To test this, we determined the cerebellar contribution to motor problems in Nf1+/-mice, a validated mouse model for NF1. Using the Rotarod, a non-specific motor performance test, we confirmed that, like NF1 patients, Nf1+/-mice have motor deficits. Next, to evaluate the role of the cerebellum in these deficits, mice were subjected to cerebellum-specific motor performance and learning tests. Nf1+/-mice showed no impairment on the Erasmus ladder, as step time and number of missteps were not different. Furthermore, when compensatory eye movements were tested, no performance deficits were found in the optokinetic reflex and vestibulo-ocular reflex in the dark (VOR) or in the light (VVOR). Finally, Nf1+/-mice successfully completed short- and long-term VOR adaptation paradigms, tests that both depend on cerebellar function. Thus, despite the confirmed presence of motor performance problems in Nf1+/-mice, we found no indication of a cerebellar component. These results, combined with recent clinical data, suggest that cerebellar function is not overtly affected in NF1 patients. © 2011 The Authors. Genes, Brain and Behavior

Rheb is essential for murine development (Article)

2011-04-01T00:00:00Z

Ras homolog enriched in brain (Rheb) couples growth factor signaling to activation of the target of rapamycin complex 1 (TORC1). To study its role in mammals, we generated a Rheb knockout mouse. In contrast to mTOR or regulatory-associated protein of mTOR (Raptor) mutants, the inner cell mass of Rheb-/-embryos differentiated normally. Nevertheless, Rheb-/-embryos died around midgestation, most likely due to impaired development of the cardiovascular system. Rheb-/-embryonic fibroblasts showed decreased TORC1 activity, were smaller, and showed impaired proliferation. Rheb heterozygosity extended the life span of tuberous sclerosis complex 1-deficient (Tsc1-/-) embryos, indicating that there is a genetic interaction between the Tsc1 and Rheb genes in mouse.

Motor learning in children with neurofibromatosis type I (Article)

2011-03-01T00:00:00Z

The aim of this study was to quantify the frequently observed problems in motor control in Neurofibromatosis type 1 (NF1) using three tasks on motor performance and motor learning. A group of 70 children with NF1 was compared to age-matched controls. As expected, NF1 children showed substantial problems in visuo-motor integration (Beery VMI). Prism-induced hand movement adaptation seemed to be mildly affected. However, no significant impairments in the accuracy of simple eye or hand movements were observed. Also, saccadic eye movement adaptation, a cerebellum dependent task, appeared normal. These results suggest that the motor problems of children with NF1 in daily life are unlikely to originate solely from impairments in motor learning. Our findings, therefore, do not support a general dysfunction of the cerebellum in children with NF1.

Intrinsic plasticity complements long-term potentiation in parallel fiber input gain control in cerebellar Purkinje cells (Article)

2010-10-13T00:00:00Z

Synaptic gain control and information storage in neural networks are mediated by alterations in synaptic transmission, such as in long-term potentiation (LTP). Here,weshowusingboth in vitroandin vivo recordingsfromthe rat cerebellum that tetanization protocols for the induction of LTP at parallel fiber (PF)-to-Purkinje cell synapsescanalsoevokeincreases in intrinsic excitability. Thisformof intrinsic plasticity shares with LTP a requirement for the activation of protein phosphatases 1, 2A, and 2B for induction. Purkinje cell intrinsic plasticity resembles CA1 hippocampal pyramidal cell intrinsic plasticity in that it requires activity of protein kinase A(PKA) and casein kinase 2 (CK2) and is mediated by a downregulation of SK-type calcium-sensitive K conductances. In addition, Purkinje cell intrinsic plasticity similarly results in enhanced spine calcium signaling. However, there are fundamental differences: first, while in the hippocampus increases in excitability result in a higher probability for LTP induction, intrinsic plasticity in Purkinje cells lowers the probability for subsequent LTP induction. Second, intrinsic plasticity raises the spontaneous spike frequency of Purkinje cells. The latter effect does not impair tonic spike firing in the target neurons of inhibitory Purkinje cell projections in the deep cerebellar nuclei, but lowers the Purkinje cell signal-to-noise ratio, thus reducing the PF readout. These observations suggest that intrinsic plasticity accompanies LTP of active PF synapses, while it reduces at weaker, nonpotentiated synapses the probability for subsequent potentiation and lowers the impact on the Purkinje cell output. Copyright

Age-related motor neuron degeneration in DNA repair-deficient Ercc1 mice (Article)

2010-10-01T00:00:00Z

Degeneration of motor neurons contributes to senescence-associated loss of muscle function and underlies human neurodegenerative conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors contributing to motor neuron vulnerability and degenerative phenotypes in vivo are therefore important for our understanding of the neuromuscular system in health and disease. Here, we analyzed neurodegenerative abnormalities in the spinal cord of progeroid Ercc1Δ/- mice that are impaired in several DNA repair systems, i.e. nucleotide excision repair, interstrand crosslink repair, and double strand break repair. Ercc1Δ/- mice develop age-dependent motor abnormalities, and have a shortened life span of 6-7 months. Pathologically, Ercc1Δ/- mice develop widespread astrocytosis and microgliosis, and motor neuron loss and denervation of skeletal muscle fibers. Degenerating motor neurons in many occasions expressed genotoxic-responsive transcription factors p53 or ATF3, and in addition, displayed a range of Golgi apparatus abnormalities. Furthermore, Ercc1Δ/- motor neurons developed perikaryal and axonal intermediate filament abnormalities reminiscent of cytoskeletal pathology observed in aging spinal cord. Our findings support the notion that accumulation of DNA damage and genotoxic stress may contribute to neuronal aging and motor neuron vulnerability in human neuromuscular disorders.

Purkinje cell-specific knockout of the protein phosphatase PP2B impairs potentiation and cerebellar motor learning (Article)

2010-08-01T00:00:00Z

Cerebellar motor learning is required to obtain procedural skills. Studies have provided supportive evidence for a potential role of kinase-mediated long-term depression (LTD) at the parallel fiber to Purkinje cell synapse in cerebellar learning. Recently, phosphatases have been implicated in the induction of potentiation of Purkinje cell activities in vitro, but it remains to be shown whether and how phosphatase-mediated potentiation contributes to motor learning. Here, we investigated its possible role by creating and testing a Purkinje cell-specific knockout of calcium/calmodulin-activated protein-phosphatase-2B (L7-PP2B). The selective deletion of PP2B indeed abolished postsynaptic long-term potentiation in Purkinje cells and their ability to increase their excitability, whereas LTD was unaffected. The mutants showed impaired "gain-decrease" and "gain-increase" adaptation of their vestibulo-ocular reflex (VOR) as well as impaired acquisition of classical delay conditioning of their eyeblink response. Thus, our data indicate that PP2B may indeed mediate potentiation in Purkinje cells and contribute prominently to cerebellar motor learning.

betaCaMKII controls the direction of plasticity at parallel fiber–Purkinje cell synapses (Article)

2009-07-01T00:00:00Z

Abstract We found that betaCaMKII, the predominant CaMKII isoform of the cerebellum, is important for controlling the direction of plasticity at the parallel fiber-Purkinje cell synapse; a protocol that induced synaptic depression in wild-type mice resulted in synaptic potentiation in Camk2b knockout mice and vice versa. These findings provide us with unique experimental insight into the mechanisms that transduce graded calcium signals into either synaptic depression or potentiation.

βCaMKII controls the direction of plasticity at parallel fiber–Purkinje cell synapses (Article)

2009-06-07T00:00:00Z

We found that betaCaMKII, the predominant CaMKII isoform of the cerebellum, is important for controlling the direction of plasticity at the parallel fiber-Purkinje cell synapse; a protocol that induced synaptic depression in wild-type mice resulted in synaptic potentiation in Camk2b knockout mice and vice versa. These findings provide us with unique experimental insight into the mechanisms that transduce graded calcium signals into either synaptic depression or potentiation.

Health-Related Quality of Life in Children with Neurofibromatosis Type 1: Contribution of Demographic Factors, Disease-Related Factors, and Behavior (Article)

2009-03-01T00:00:00Z

Objective: To investigate health-related quality of life (HR-QOL) in children with neurofibromatosis type 1 (NF1) with parental reports and children's self-reports, and to investigate the potential contribution of demographic factors, disease-specific factors, and problems in school performance or behavior. Study design: In a prospective observational study, parents of 58 children with NF1 (32 boys, 26 girls, age 12.2 ± 2.5 years) visiting a university clinic, and their 43 children 10 years or older were assessed with the Child Health Questionnaire (CHQ). Potential determinants of domain scores were assessed in 3 explorative regression models. Results: Parents reported a significant impact of NF1 on 9/13 CHQ scales, with moderate effect sizes on 8 (general health perceptions, physical functioning, general behavior, mental health, self esteem, family activities, role functioning emotional/behavioral, and parent emotional impact). Children report an impact on bodily pain, and an above average general behavior. Multiple CHQ scales were sensitive to demographic factors and behavioral problems, and 1 to NF1 severity. NF1 visibility and school problems did not influence HR-QOL. Conclusions: Parents, but not the children with NF1, report a profound impact of NF1 on physical, social, behavioral, and emotional aspects of HR-QOL. Multiple HR-QOL domains were most sensitive to behavioral problems, which points to an exciting potential opportunity to improve HR-QOL in children with NF1 by addressing these behavioral problems.

Spred1 is required for synaptic plasticity and hippocampus-dependent learning (Article)

2008-12-31T00:00:00Z

Germline mutations in SPRED1, a negative regulator of Ras, have been described in a neurofibromatosis type 1 (NF1)-like syndrome (NFLS) that included learning difficulties in some affected individuals. NFLS belongs to the group of phenotypically overlapping neurocardio-facial-cutaneous syndromes that are all caused by germ line mutations in genes of the Ras/mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway and that present with some degree of learning difficulties or mental retardation. We investigated hippocampus-dependent learning and memory as well as synaptic plasticity in Spred1-/-mice, an animal model of this newly discovered human syndrome. Spred1-/-mice show decreased learning and memory performance in the Morris water maze and visual-discrimination T-maze, but normal basic neuromotor and sensory abilities. Electrophysiological recordings on brain slices from these animals identified defects in short- and long-term synaptic hippocampal plasticity, including a disequilibrium between long-term potentiation (LTP) and long-term depression in CA1 region. Biochemical analysis, 4 h after LTP induction, demonstrated increased ERK-phosphorylation in Spred1-/-slices compared with those of wild-type littermates. This indicates that deficits in hippocampusdependent learning and synaptic plasticity induced by SPRED1 deficiency are related to hyperactivation of the Ras/ERK pathway. Copyright

Statin therapy and cognitive deficits associated with neurofibromatosis type 1: Reply (Article)

2008-11-26T00:00:00Z

Neurofibromin Regulation of ERK Signaling Modulates GABA Release and Learning (Article)

2008-10-31T00:00:00Z

We uncovered a role for ERK signaling in GABA release, long-term potentiation (LTP), and learning, and show that disruption of this mechanism accounts for the learning deficits in a mouse model for learning disabilities in neurofibromatosis type I (NF1). Our results demonstrate that neurofibromin modulates ERK/synapsin I-dependent GABA release, which in turn modulates hippocampal LTP and learning. An Nf1 heterozygous null mutation, which results in enhanced ERK and synapsin I phosphorylation, increased GABA release in the hippocampus, and this was reversed by pharmacological downregulation of ERK signaling. Importantly, the learning deficits associated with the Nf1 mutation were rescued by a subthreshold dose of a GABAAantagonist. Accordingly, Cre deletions of Nf1 showed that only those deletions involving inhibitory neurons caused hippocampal inhibition, LTP, and learning abnormalities. Importantly, our results also revealed lasting increases in GABA release triggered by learning, indicating that the mechanisms uncovered here are of general importance for learning.

Oncogenes on my mind: ERK and MTOR signaling in cognitive diseases (Article)

2008-10-01T00:00:00Z

Defects in rat sarcoma viral oncogene homolog (RAS)-extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (MTOR) signaling pathways have recently been shown to cause several genetic disorders classified as neuro-cardio-facial-cutaneous (NCFC) and Hamartoma syndromes. Although these pathways are well-known players in cell proliferation and cancer, their role in cognitive function is less appreciated. Here, we focus on the cognitive problems associated with mutations in the RAS-ERK and PI3K-MTOR signaling pathways and on the underlying mechanisms revealed by recent animal studies. Cancer drugs have been shown to reverse the cognitive deficits in mouse models of NCFC and Hamartoma syndromes, raising hopes for clinical trials.

Impact of neurofibromatosis type 1 on school performance (Article)

2008-09-03T00:00:00Z

School functioning of 86 Dutch neurofibromatosis type 1 children (7-17 years) using teacher questionnaires was analyzed to determine the impact of neurofibromatosis type 1 on school performance. In all, 75% of the neurofibromatosis type 1 children performed more than 1 standard deviation below grade peers in at least one of the domains of spelling, mathematics, technical reading or comprehensive reading. Furthermore, neurofibromatosis type 1 children had a 4-fold increased risk for attending special education and a 6-fold increased risk for receiving remedial teaching for learning, behavior, speech, or motor problems. Children without apparent learning disabilities still frequently displayed neuropsychological deficits. Only 10% of the children did not show any school-functioning problems. Finally, it was found that the clinical severity of neurofibromatosis type 1 correlated with the cognitive deficits. Taken together, it was shown that neurofibromatosis type 1 has profound impact on school performance. Awareness of these problems may facilitate timely recognition and appropriate support.

Effect of simvastatin on cognitive functioning in children with neurofibromatosis type 1: A randomized controlled trial (Article)

2008-07-16T00:00:00Z

Context: Neurofibromatosis type 1 (NF1) is among the most common genetic disorders that cause learning disabilities. Recently, it was shown that statin-mediated inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase restores the cognitive deficits in an NF1 mouse model. Objective: To determine the effect of simvastatin on neuropsychological, neurophysiological, and neuroradiological outcome measures in children with NF1. Design, Setting, and Participants: Sixty-two of 114 eligible children (54%) with NF1 participated in a randomized, double-blind, placebo-controlled trial conducted between January 20, 2006, and February 8, 2007, at an NF1 referral center at a Dutch university hospital. Intervention: Simvastatin or placebo treatment once daily for 12 weeks. Main Outcome Measures: Primary outcomes were scores on a Rey complex figure test (delayed recall), cancellation test (speed), prism adaptation, and the mean brain apparent diffusion coefficient based on magnetic resonance imaging. Secondary outcome measures were scores on the cancellation test (standard deviation), Stroop color word test, block design, object assembly, Rey complex figure test (copy), Beery developmental test of visual-motor integration, and judgment of line orientation. Scores were corrected for baseline performance, age, and sex. Results: No significant differences were observed between the simvastatin and placebo groups on any primary outcome measure: Rey complex figure test (β=0.10; 95% confidence interval [CI], -0.36 to 0.56); cancellation test (β=-0.19; 95% CI, -0.67 to 0.29); prism adaptation (odds ratio=2.0; 95% CI, 0.55 to 7.37); and mean brain apparent diffusion coefficient (β=0.06; 95% CI, -0.07 to 0.20). In the secondary outcome measures, we found a significant improvement in the simvastatin group in object assembly scores (β=0.54; 95% CI, 0.08 to 1.01), which was specifically observed in children with poor baseline performance (β=0.80; 95% CI, 0.29 to 1.30). Other secondary outcome measures revealed no significant effect of simvastatin treatment. Conclusion: In this 12-week trial, simvastatin did not improve cognitive function in children with NF1. Trial Registration: isrctn.org Identifier: ISRCTN14965707

Rapid changes in hippocampal CA1 pyramidal cell function via pre- as well as postsynaptic membrane mineralocorticoid receptors (Article)

2008-05-01T00:00:00Z

Corticosterone (100 nm) rapidly increases the frequency of miniature excitatory postsynaptic currents in mouse CA1 pyramidal neurons via membrane-located mineralocorticoid receptors (MRs). We now show that a presynaptic ERK1/2 signalling pathway mediates the nongenomic effect, as it was blocked by the MEK inhibitors U0126 (10 μm) and PD098059 (40 μm) and occluded in H-RasG12V-mutant mice with constitutive activation of the ERK1/2 presynaptic pathway. Notably, the increase in mEPSC frequency was not mediated by retrograde signalling through endocannabinoids or nitric oxide, supporting presynaptic localization of the signalling pathway. Unexpectedly, corticosterone was also found to have a direct postsynaptic effect, rapidly decreasing the peak amplitude of IAcurrents. This effect takes place via postsynaptic membrane MRs coupled to a G protein-mediated pathway, as the effect of corticosterone on IAwas effectively blocked by 0.5 mm GDP-β-S administered via the recording pipette into the postsynaptic cell. Taken together, these results indicate that membrane MRs mediate rapid, nongenomic effects via pre- as well as postsynaptic pathways. Through these dual pathways, high corticosterone concentrations such as occur after stress could contribute to enhanced CA1 pyramidal excitability.

Quantitative differentiation between healthy and disordered brain matter in patients with neurofibromatosis type I using diffusion tensor imaging (Article)

2008-04-01T00:00:00Z

BACKGROUND AND PURPOSE: Hyperintensities on T2-weighted images are seen in the brains of most patients with neurofibromatosis type I (NF-1), but the origin of these unidentified bright objects (UBOs) remains obscure. In the current study, we examined the diffusion characteristics of brain tissue in children with NF-1 to test the hypothesis that a microstructural abnormality is present in NF-1. MATERIALS AND METHODS: Diffusion tensor imaging (DTI) was performed in 50 children with NF-1 and 8 controls. Circular regions of interest were manually placed in 7 standardized locations in both hemispheres, including UBO sites. Apparent diffusion coefficients (ADC), fractional anisotropy (FA), and axial anisotropy (Am) were used to differentiate quantitatively between healthy and disordered brain matter. Differences in eigenvalues (λ1, λ2, λ3) were determined to examine parenchymal integrity. RESULTS: We found higher ADC values for UBOs than for normal-appearing sites (P < .01) and higher ADC values for normal-appearing sites than for controls (P < .04 in 5 of 7 regions). In most regions, we found no differences in FA or Am. Eigenvalues λ2and λ3were higher at UBO sites than in normal-appearing sites (P < .04). CONCLUSION: With ADC, it was possible to differentiate quantitatively between normal- and abnormal-appearing brain matter in NF-1 and also between normal-appearing brain matter in NF-1 and healthy brain matter in controls, indicating subtle pathologic damage disrupting the tissue microstructure in the NF-1 brain. Higher diffusivity for λ1, λ2, and λ3indicates that this disturbance of microstructure is caused by accumulation of fluid or vacuolation.

α-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus (Article)

2008-01-02T00:00:00Z

N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the α-isoform of calcium-calmodulin-dependent protein kinase II (αCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory αCaMKII Thr305 substituted with Asp in α-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor αCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and αCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and αCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.

Cognitive deficits in Tsc1+/- mice in the absence of cerebral lesions and seizures (Article)

2007-12-01T00:00:00Z

Objective: Tuberous sclerosis complex (TSC) is characterized by brain lesions, epilepsy, increased incidence of mental retardation and autism. The causal link between lesion load and epilepsy on cognitive disabilities has been debated, and these factors explain only part of the intelligence quotient variability. A Tsc2 rat model of the disease provided evidence that the TSC genes are directly involved in neuronal function. However, these lesion- and epilepsy-free animals did not show learning deficits, leaving open the possibility that the presence of brain lesions or epilepsy is a prerequisite for the cognitive deficits to fully develop. Here, we reinvestigated the relation among cerebral lesions, epilepsy, and cognitive function using Tsc1+/-mice. Methods: We used immunocytochemistry and high-resolution magnetic resonance imaging to study the presence of neuronal pathology in Tsc+/-mice. We used the Morris water maze, fear conditioning, social interaction, and nest building test to study the presence of cognitive and social deficits. Results: We observed no spontaneous seizures or cerebral lesions in the brains of Tsc1+/-mice. In addition, giant dysmorphic cells were absent, and spine number and dendritic branching appeared to be normal. Nevertheless, Tscl+/-mice showed impaired learning in the hippocampus-sensitive versions of the learning tasks and impaired social behavior. Interpretation: Tsc+/-mice show social and cognitive deficits in the absence of apparent cerebral pathology and spontaneous seizures. These findings support a model in which haploinsufficiency for the TSC genes leads to aberrations in neuronal functioning resulting in impaired learning and social behavior.

Kinase activity is not required for αCaMKII-dependent presynaptic plasticity at CA3-CA1 synapses (Article)

2007-09-01T00:00:00Z

Using targeted mouse mutants and pharmacologic inhibition of αCaMKII, we demonstrate that the αCaMKII protein, but not its activation, autophosphorylation or its ability to phosphorylate synapsin I, is required for normal short-term presynaptic plasticity. Furthermore, αCaMKII regulates the number of docked vesicles independent of its ability to be activated. These results indicate that αCaMKII has a nonenzymatic role in short-term presynaptic plasticity at hippocampal CA3-CA1 synapses.

Genetic engineering cures mice of neurological deficits: Prospects for treating Angelman syndrome (Article)

2007-06-01T00:00:00Z

Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of αCaMKII inhibitory phosphorylation (Article)

2007-03-01T00:00:00Z

Angelman syndrome (AS) is a severe neurological disorder characterized by mental retardation, motor dysfunction and epilepsy. We show that the molecular and cellular deficits of an AS mouse model can be rescued by introducing an additional mutation at the inhibitory phosphorylation site of αCaMKII. Moreover, these double mutants no longer show the behavioral deficits seen in AS mice, suggesting that these deficits are the direct result of increased inhibitory phosphorylation of αCaMKII.

Rescue of neurological deficits in a mouse model for Angelman Syndrome by reduction of αCaMKII inhibitory phosphorylation (Article)

2007-01-01T00:00:00Z

Angelman Syndrome (AS) is a severe neurological disorder characterized by mental retardation, motor dysfunction and epilepsy. We now show that the molecular and cellular deficits of an AS mouse model can be rescued by introducing an additional mutation at the inhibitory phosphorylation site of αCaMKII. Moreover, these double mutants do no longer show the behavioral deficits seen in AS mice, suggesting that these deficits are the direct result of increased αCaMKII inhibitory phosphorylation.

Mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase II function in plasticity and cognition (Article)

2004-01-01T00:00:00Z

DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2 (Article)

1991-01-01T00:00:00Z

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage (Article)

1991-01-01T00:00:00Z

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.