Rimante Minkeviciene

Amyloid β-Induced Neuronal Hyperexcitability Triggers Progressive Epilepsy

Alzheimers disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid β (Aβ) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Aβ1–42 or Aβ25–35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Aβ (proto-)fibrils. Overall, these data identify fibrillar Aβ as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice

We assessed baseline and KCl‐stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle‐aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild‐type littermates. In addition, we assessed the age‐related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17‐month‐old APdE9 mice as compared to 7‐month‐old APdE9 mice. This age‐dependent decrease in stimulated glutamate release was also evident in wild‐type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild‐type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age‐dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT‐1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle‐aged APdE9 mice. Blunted pre‐synaptic glutamate response may contribute to memory deficit in middle‐aged APdE9 mice.

Cognition-enhancing and anxiolytic effects of memantine.

Memantine, a moderate-affinity NMDA receptor antagonist, is clinically used for the treatment of Alzheimers disease (AD). Both clinical and preclinical studies have shown that memantine, at doses producing a steady-state plasma level of 0.5-1 microM, is well tolerated and improves cognition. Here we tested the effects of chronic oral administration of memantine (10, 30 and 100mg/kg per day) producing steady state plasma drug levels ranging between approximately 0.5 and 6 microM on motor, social, emotional and cognitive behavior in normal C57BL/6J mice. Memantine dose-dependently reduced escape latency (hidden platform) and decreased wall swimming tendency in the Morris water maze test, increased time spent in open arms in the elevated plus-maze test, and reduced the number of isolation-induced aggressive attacks, but did not affect exploratory activity in the open field. These data indicate that high, stable doses of memantine improved cognition and exhibited a potential anxiolytic response in normal mice.

MIM-Induced Membrane Bending Promotes Dendritic Spine Initiation

Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.

Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity

Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance. SIGNIFICANCE STATEMENT Dendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for regulating neuronal actin cytoskeleton that explains the specific organization and dynamics of actin in spines. The better we understand the regulation of the dendritic spine morphology, the better we understand what goes wrong in neurological diseases.

Cytoskeletal Organization: Actin

The regulation of cytoskeletal organization is key to the establishment of proper neuronal morphology. The neuronal cytoskeleton comprises three different elements: microtubules, intermediate filaments, and actin filaments. Actin cytoskeleton dynamics play a key role in the establishment and maintenance of both dendritic arborization and spines, as well as in spine shape changes associated with synaptic plasticity. Not surprisingly, abnormalities in dendritic arborization and dendritic spine morphology arising from the faulty regulation of the cytoskeleton have been linked to several neurological diseases. In this review, we describe the basic mechanisms regulating the actin cytoskeleton in spinogenesis and discuss examples of mutations in actin regulators associated with neurological disorders.

Short- and long-term habituation of auditory event-related potentials in the rat

An auditory oddball paradigm in humans generates a long-duration cortical negative potential, often referred to as mismatch negativity. Similar negativity has been documented in monkeys and cats, but it is controversial whether mismatch negativity also exists in awake rodents. To this end, we recorded cortical and hippocampal evoked responses in rats during alert immobility under a typical passive oddball paradigm that yields mismatch negativity in humans. The standard stimulus was a 9 kHz tone and the deviant either 7 or 11 kHz tone in the first condition. We found no evidence of a sustained potential shift when comparing evoked responses to standard and deviant stimuli. Instead, we found repetition-induced attenuation of the P60 component of the combined evoked response in the cortex, but not in the hippocampus. The attenuation extended over three days of recording and disappeared after 20 intervening days of rest. Reversal of the standard and deviant tones resulted is a robust enhancement of the N40 component not only in the cortex but also in the hippocampus. Responses to standard and deviant stimuli were affected similarly. Finally, we tested the effect of scopolamine in this paradigm. Scopolamine attenuated cortical N40 and P60 as well as hippocampal P60 components, but had no specific effect on the deviant response. We conclude that in an oddball paradigm the rat demonstrates repetition-induced attenuation of mid-latency responses, which resembles attenuation of the N1-component of human auditory evoked potential, but no mismatch negativity.

P1-109: Age-dependent changes in hippocampal glutamate levels parallel learning impairment in APP/PS1 transgenic mice

Ts65Dn mice, but not Ts1Cje, exhibited significantly reduced T2 relaxation times selectively in the medial septal area, from where BFCN originate, and in brain regions normally innervated by BFCN, including hippocampus and cingulate cortex. BFCN projections to these areas, identified by choline acetyltransferase (CHAT) immunocytochemistry or acetylcholinesterase histochemistry, were selectively and markedly reduced in Ts65Dn mice and numbers of detectable BFCN in the medial septal area, identified by CHAT or p75NGFR immunolabeling, were significantly below normal. In BFCN projection areas of the cortex, dendrites in Ts65Dn brain displayed morphological changes and markedly increased levels of MAP-2, a cytoskeletal protein regulated by cholinergic innervation. Using electron microscopy and antibody markers for apoptosis pathway activation, we detected a higher than normal frequency of neurons undergoing apoptosis in affected brain regions. Conclusions: MRI revealed more widespread neuropathology in Ts65Dn mice than previously appreciated, involving postsynaptic as well as presynaptic elements of the BFCN circuit. The cholinergic deficits, MRI T2 changes, and AD-related endosomal phenotype in Ts65Dn mice, but not Ts1Cje, support growing evidence linking APP triplication to endosome dysfunction (A.Boyer-Boiteau; et al., this meeting), retrograde signaling deficits, and increased vulnerability of neurons to degeneration.