Tau isoform-specific enhancement of L-type calcium current and augmentation of afterhyperpolarization in rat hippocampal neurons.

Accumulation of tau is observed in dementia, with human tau displaying 6 isoforms grouped by whether they display either 3 or 4 C-terminal repeat domains (3R or 4R) and exhibit no (0N), one (1N) or two (2N) N terminal repeats. Overexpression of 4R0N-tau in rat hippocampal slices enhanced the L-type calcium (Ca2+) current-dependent components of the medium and slow afterhyperpolarizations (AHPs). Overexpression of both 4R0N-tau and 4R2N-tau augmented CaV1.2-mediated L-type currents when expressed in tsA-201 cells, an effect not observed with the third 4R isoform, 4R1N-tau. Current enhancement was only observed when the pore-forming subunit was co-expressed with CaVß3 and not CaVß2a subunits. Non-stationary noise analysis indicated that enhanced Ca2+ channel current arose from a larger number of functional channels. 4R0N-tau and CaVß3 were found to be physically associated by co-immunoprecipitation. In contrast, the 4R1N-tau isoform that did not augment expressed macroscopic L-type Ca2+ current exhibited greatly reduced binding to CaVß3. These data suggest that physical association between tau and the CaVß3 subunit stabilises functional L-type channels in the membrane, increasing channel number and Ca2+ influx. Enhancing the Ca2+-dependent component of AHPs would produce cognitive impairment that underlie those seen in the early phases of tauopathies.

Oral (-)-Epicatechin Inhibits Progressive Tau Pathology in rTg4510 Mice Independent of Direct Actions at GSK3ß.

Aggregation of the microtubule-associated protein tau into paired helical filaments (PHFs) and neurofibrillary tangles is a defining characteristic of Alzheimer's Disease. Various plant polyphenols disrupt tau aggregation in vitro but display poor bioavailability and low potency, challenging their therapeutic translation. We previously reported that oral administration of the flavonoid (-)-epicatechin (EC) reduced Amyloid-ß (Aß) plaque pathology in APP/PS1 transgenic mice. Here, we investigated whether EC impacts on tau pathology, independent of actions on Aß, using rTg4510 mice expressing P301L mutant tau. 4 and 6.5 months old rTg4510 mice received EC (∼18 mg/day) or vehicle (ethanol) via drinking water for 21 days and the levels of total and phosphorylated tau were assessed. At 4 months, tau appeared as two bands of ∼55 kDa, phosphorylated at Ser262 and Ser396 and was unaffected by exposure to EC. At 6.5 months an additional higher molecular weight form of tau was detected at ∼64 kDa which was phosphorylated at Ser262, Ser396 and additionally at the AT8 sites, indicative of the presence of PHFs. EC consumption reduced the levels of the ∼64 kDa tau species and inhibited phosphorylation at Ser262 and AT8 phosphoepitopes. Regulation of the key tau kinase glycogen synthase kinase 3ß (GSK3ß) by phosphorylation at Ser9 was not altered by exposure to EC in mice or primary neurons. Furthermore, EC did not significantly inhibit GSK3ß activity at physiologically-relevant concentrations in a cell free assay. Therefore, a 21-day intervention with EC inhibits or reverses the development of tau pathology in rTg4510 mice independently of direct inhibition of GSK3ß.

EEG microstate complexity for aiding early diagnosis of Alzheimer's disease.

The dynamics of the resting brain exhibit transitions between a small number of discrete networks, each remaining stable for tens to hundreds of milliseconds. These functional microstates are thought to be the building blocks of spontaneous consciousness. The electroencephalogram (EEG) is a useful tool for imaging microstates, and EEG microstate analysis can potentially give insight into altered brain dynamics underpinning cognitive impairment in disorders such as Alzheimer's disease (AD). Since EEG is non-invasive and relatively inexpensive, EEG microstates have the potential to be useful clinical tools for aiding early diagnosis of AD. In this study, EEG was collected from two independent cohorts of probable AD and cognitively healthy control participants, and a cohort of mild cognitive impairment (MCI) patients with four-year clinical follow-up. The microstate associated with the frontoparietal working-memory/attention network was altered in AD due to parietal inactivation. Using a novel measure of complexity, we found microstate transitioning was slower and less complex in AD. When combined with a spectral EEG measure, microstate complexity could classify AD with sensitivity and specificity > 80%, which was tested on an independent cohort, and could predict progression from MCI to AD in a small preliminary test cohort of 11 participants. EEG microstates therefore have potential to be a non-invasive functional biomarker of AD.

Neurophysiological alterations in the nucleus reuniens of a mouse model of Alzheimer's disease.

Recently, increased neuronal activity in nucleus reuniens (Re) has been linked to hyperexcitability within hippocampal-thalamo-cortical networks in the J20 mouse model of amyloidopathy. Here in vitro whole-cell patch clamp recordings were used to compare old pathology-bearing J20 mice and wild-type controls to examine whether altered intrinsic electrophysiological properties could contribute to the amyloidopathy-associated Re hyperactivity. A greater proportion of Re neurons display hyperpolarized membrane potentials in J20 mice without changes to the incidence or frequency of spontaneous action potentials. Re neurons recorded from J20 mice did not exhibit increased action potential generation in response to depolarizing current stimuli but an increased propensity to rebound burst following hyperpolarizing current stimuli. Increased rebound firing did not appear to result from alterations to T-type Ca2+ channels. Finally, in J20 mice, there was an ~8% reduction in spike width, similar to what has been reported in CA1 pyramidal neurons from multiple amyloidopathy mice. We conclude that alterations to the intrinsic properties of Re neurons may contribute to hippocampal-thalmo-cortical hyperexcitability observed under pathological beta-amyloid load.

Network substrates of cognitive impairment in Alzheimer's Disease.

OBJECTIVES: Functional and structural disconnection of the brain is a prevailing hypothesis to explain cognitive impairment in Alzheimer's Disease (AD). We aim to understand the link between alterations to networks and cognitive impairment using functional connectivity analysis and modelling. METHODS: EEG was recorded from 21 AD patients and 26 controls, mapped into source space using eLORETA, and functional connectivity was calculated using phase locking factor. The mini-mental state exam (MMSE) was used to assess cognitive impairment. A computational model was used to uncover mechanisms of altered functional connectivity. RESULTS: Small-worldness (SW) of functional networks decreased in AD and was positively correlated with MMSE score and the language sub-score. Reduced SW was a result of increased path lengths, predominantly localized to the temporal lobes. Combining observed differences in local oscillation frequency with reduced temporal lobe effective connectivity in the model could account for observed functional network differences. CONCLUSIONS: Temporal lobe disconnection plays a key role in cognitive impairment in AD. SIGNIFICANCE: We combine electrophysiology, neuropsychological scores, and computational modelling to provide novel insight into the relationships between the disconnection hypothesis and cognitive decline in AD.

ß3-Adrenergic receptor-dependent modulation of the medium afterhyperpolarization in rat hippocampal CA1 pyramidal neurons.

Action potential firing in hippocampal pyramidal neurons is regulated by generation of an afterhyperpolarization (AHP). Three phases of AHP are recognized, with the fast AHP regulating action potential firing at the onset of a burst and the medium and slow AHPs supressing action potential firing over hundreds of milliseconds and seconds, respectively. Activation of ß-adrenergic receptors suppresses the slow AHP by a protein kinase A-dependent pathway. However, little is known regarding modulation of the medium AHP. Application of the selective ß-adrenergic receptor agonist isoproterenol suppressed both the medium and slow AHPs evoked in rat CA1 hippocampal pyramidal neurons recorded from slices maintained in organotypic culture. Suppression of the slow AHP was mimicked by intracellular application of cAMP, with the suppression of the medium AHP by isoproterenol still being evident in cAMP-dialyzed cells. Suppression of both the medium and slow AHPs was antagonized by the ß-adrenergic receptor antagonist propranolol. The effect of isoproterenol to suppress the medium AHP was mimicked by two ß3-adrenergic receptor agonists, BRL37344 and SR58611A. The medium AHP was mediated by activation of small-conductance calcium-activated K+ channels and deactivation of H channels at the resting membrane potential. Suppression of the medium AHP by isoproterenol was reduced by pretreating cells with the H-channel blocker ZD7288. These data suggest that activation of ß3-adrenergic receptors inhibits H channels, which suppresses the medium AHP in CA1 hippocampal neurons by utilizing a pathway that is independent of a rise in intracellular cAMP. This finding highlights a potential new target in modulating H-channel activity and thereby neuronal excitability. NEW & NOTEWORTHY The noradrenergic input into the hippocampus is involved in modulating long-term synaptic plasticity and is implicated in learning and memory. We demonstrate that activation of functional ß3-adrenergic receptors suppresses the medium afterhyperpolarization in hippocampal pyramidal neurons. This finding provides an additional mechanism to increase action potential firing frequency, where neuronal excitability is likely to be crucial in cognition and memory.

Control of clustered action potential firing in a mathematical model of entorhinal cortex stellate cells.

The entorhinal cortex is a crucial component of our memory and spatial navigation systems and is one of the first areas to be affected in dementias featuring tau pathology, such as Alzheimer's disease and frontotemporal dementia. Electrophysiological recordings from principle cells of medial entorhinal cortex (layer II stellate cells, mEC-SCs) demonstrate a number of key identifying properties including subthreshold oscillations in the theta (4-12 Hz) range and clustered action potential firing. These single cell properties are correlated with network activity such as grid firing and coupling between theta and gamma rhythms, suggesting they are important for spatial memory. As such, experimental models of dementia have revealed disruption of organised dorsoventral gradients in clustered action potential firing. To better understand the mechanisms underpinning these different dynamics, we study a conductance based model of mEC-SCs. We demonstrate that the model, driven by extrinsic noise, can capture quantitative differences in clustered action potential firing patterns recorded from experimental models of tau pathology and healthy animals. The differential equation formulation of our model allows us to perform numerical bifurcation analyses in order to uncover the dynamic mechanisms underlying these patterns. We show that clustered dynamics can be understood as subcritical Hopf/homoclinic bursting in a fast-slow system where the slow sub-system is governed by activation of the persistent sodium current and inactivation of the slow A-type potassium current. In the full system, we demonstrate that clustered firing arises via flip bifurcations as conductance parameters are varied. Our model analyses confirm the experimentally suggested hypothesis that the breakdown of clustered dynamics in disease occurs via increases in AHP conductance.

Minocycline reduces microgliosis and improves subcortical white matter function in a model of cerebral vascular disease.

Chronic cerebral hypoperfusion is a key mechanism associated with white matter disruption in cerebral vascular disease and dementia. In a mouse model relevant to studying cerebral vascular disease, we have previously shown that cerebral hypoperfusion disrupts axon-glial integrity and the distribution of key paranodal and internodal proteins in subcortical myelinated axons. This disruption of myelinated axons is accompanied by increased microglia and cognitive decline. The aim of the present study was to investigate whether hypoperfusion impairs the functional integrity of white matter, its relation with axon-glial integrity and microglial number, and whether by targeting microglia these effects can be improved. We show that in response to increasing durations of hypoperfusion, the conduction velocity of myelinated fibres in the corpus callosum is progressively reduced and that paranodal and internodal axon-glial integrity is disrupted. The number of microglial cells increases in response to hypoperfusion and correlates with disrupted paranodal and internodal integrity and reduced conduction velocities. Further minocycline, a proposed anti-inflammatory and microglia inhibitor, restores white matter function related to a reduction in the number of microglia. The study suggests that microglial activation contributes to the structural and functional alterations of myelinated axons induced by cerebral hypoperfusion and that dampening microglia numbers/proliferation should be further investigated as potential therapeutic benefit in cerebral vascular disease.

Hippocampal neurophysiology is modified by a disease-associated C-terminal fragment of tau protein.

The accumulation of cleaved tau fragments in the brain is associated with several tauopathies. For this reason, we recently developed a transgenic mouse that selectively accumulates a C-Terminal 35 kDa human tau fragment (Tau35). These animals develop progressive motor and spatial memory impairment, paralleled by increased hippocampal glycogen synthase kinase 3ß activity. In this neurophysiological study, we focused on the CA1 subfield of the hippocampus, a brain area involved in memory encoding. The accumulation of Tau35 results in a significant increase of short-term facilitation of the synaptic response in the theta frequency range (10 Hz), without affecting basal synaptic transmission and long-term synaptic plasticity. Tau35 expression also alters the intrinsic excitability of CA1 pyramidal neurons. Thus, Tau35 presence is associated with increased and decreased excitability at hyperpolarized and depolarized potentials, respectively. These observations are paralleled by a hyperpolarization of the voltage-sensitivity of noninactivating K+ currents. Further investigation is needed to assess the causal link between such functional alterations and the cognitive and motor impairments previously observed in this model.

Aging-Associated Changes to Intrinsic Neuronal Excitability in the Bed Nucleus of the Stria Terminalis Is Cell Type-Dependent.

Intrinsic neuronal excitability has been reported to change during normal aging. The bed nucleus of the stria terminalis (BNST), a limbic forebrain structure, is involved in fear, stress and anxiety; behavioral features that exhibit age-dependent properties. To examine the effect of aging on intrinsic neuronal properties in BNST we compared patch clamp recordings from cohorts of female mice at two ages, 3-4 months (Young) and 29-30 months (Aged) focusing on 2 types of BNST neurons. Aged Type I neurons exhibited a hyperpolarized resting membrane potential (RMP) of circa -80 mV compared to circa -70 mV in the Young. A key finding in this study is a hyper-excitability of Type II neurons with age reflected in an increase in firing frequency in response to depolarizing current injections; activation of Type II neurons is believed to dampen anxiety like responses. Such age-related changes in intrinsic neurophysiological function are likely to modulate how the limbic system, acting via BNST, shapes function in the HPA-axis.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Altered intrinsic excitability of hippocampal CA1 pyramidal neurons in aged PDAPP mice.

Amyloidopathy involves the accumulation of insoluble amyloid ß (Aß) species in the brain's parenchyma and is a key histopathological hallmark of Alzheimer's disease (AD). Work on transgenic mice that overexpress Aß suggests that elevated Aß levels in the brain are associated with aberrant epileptiform activity and increased intrinsic excitability (IE) of CA1 hippocampal neurons. In this study we examined if similar changes could be observed in hippocampal CA1 pyramidal neurons from aged PDAPP mice (20-23 month old, Indiana mutation: V717F on APP gene) compared to their age-matched wild-type littermate controls. Whole-cell current clamp recordings revealed that sub-threshold intrinsic properties, such as input resistance, resting membrane potential and hyperpolarization activated "sag" were unaffected, but capacitance was significantly decreased in the transgenic animals. No differences between genotypes were observed in the overall number of action potentials (AP) elicited by 500 ms supra-threshold current stimuli. PDAPP neurons, however, exhibited higher instantaneous firing frequencies after accommodation in response to high intensity current injections. The AP waveform was narrower and shorter in amplitude in PDAPP mice: these changes, according to our in silico model of a CA1/3 pyramidal neuron, depended on the respective increase and reduction of K(+) and Na(+) voltage-gated channels maximal conductances. Finally, the after-hyperpolarization, seen after the first AP evoked by a +300 pA current injection and after 50 Hz AP bursts, was more pronounced in PDAPP mice. These data show that Aß-overexpression in aged mice altered the capacitance, the neuronal firing and the AP waveform of CA1 pyramidal neurons. Some of these findings are consistent with previous work on younger PDAPP; they also show important differences that can be potentially ascribed to the interaction between amyloidopathy and ageing. Such a change of IE properties over time underlies that the increased incidence of seizure observed in AD patients might rely on different mechanistic pathways during progression of the disease.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

Intrinsic excitability changes induced by acute treatment of hippocampal CA1 pyramidal neurons with exogenous amyloid ß peptide.

Accumulation of beta-amyloid (Aß) peptides in the human brain is a canonical pathological hallmark of Alzheimer's disease (AD). Recent work in Aß-overexpressing transgenic mice indicates that increased brain Aß levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2-5 h treatment with an oligomeric preparation of synthetic human Aß 1-42 peptide. Whole cell current clamp recordings were compared between Aß-(500 nM) and vehicle-(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aß treatment did not produce alterations in sub-threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated "sag". Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra-threshold stimuli. However, Aß 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aß at 500 nM depressed the after-hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aß oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients.

Disrupted in schizophrenia 1 and synaptic function in the mammalian central nervous system.

The disrupted in schizophrenia 1 (DISC1) gene is found at the breakpoint of an inherited chromosomal translocation, and segregates with major mental illnesses. Its potential role in central nervous system (CNS) malfunction has triggered intensive investigation of the biological roles played by DISC1, with the hope that this may shed new light on the pathobiology of psychiatric disease. Such work has ranged from investigations of animal behavior to detailed molecular-level analysis of the assemblies that DISC1 forms with other proteins. Here, we discuss the evidence for a role of DISC1 in synaptic function in the mammalian CNS.

Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease.

BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid ß peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the ß2-subunit of voltage-gated sodium channels (Navß2). Although increased BACE1 levels are associated with Navß2 cleavage in AD patients, whether Navß2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navß2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navß-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navß2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navß2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navß2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

Age-related changes to Na+ channel gating contribute to modified intrinsic neuronal excitability.

Cognitive decline occurs during normal aging and is likely to be reflected in the neurophysiological properties of neural circuits with key roles in cognition, for example those of the limbic system. To identify candidate neurophysiological changes we used patch clamp methods to compare the intrinsic excitability properties of hippocampal CA1 pyramidal neurons of mature adult (8-10 month) and aged (22-24 month) mice. Resting potential, input resistance, and the "sag" observed on injection of hyperpolarizing current were not age-dependent. In contrast, the patterns of spike firing observed with depolarizing current injections demonstrated the presence of an age-related hypoexcitability. Action potential waveform analysis revealed that spike thresholds were approximately 3 mV more depolarized in aged animals. In line with this, voltage clamp recordings of Na(+) currents from nucleated macropatches exhibited an approximate 3 mV depolarizing shift in the voltage-dependence of activation gating. Inactivation curves, in contrast, were not different. These data indicate alterations in Na(+) channel activation gating contribute to neuronal hypoexcitability in aging, and therefore may be a factor in age-related cognitive decline.

Voltage- and Temperature-Dependent Allosteric Modulation of α7 Nicotinic Receptors by PNU120596.

Alpha7 nicotinic acetylcholine receptors (α7 nAChR) are widely distributed throughout the central nervous system and are found at particularly high levels in the hippocampus and cortex. Several lines of evidence indicate that pharmacological enhancement of α7 nAChRs function could be a potential therapeutic route to alleviate disease-related cognitive deficits. A recent pharmacological approach adopted to increase α7 nAChR activity has been to identify selective positive allosteric modulators (PAMs). α7 nAChR PAMs have been divided into two classes: type I PAMs increase agonist potency with only subtle effects on kinetics, whereas type II agents produce additional dramatic effects on desensitization and deactivation kinetics. Here we report novel observations concerning the pharmacology of the canonical type II PAM, PNU120596. Using patch clamp analysis of acetylcholine (ACh)-mediated currents through recombinant rat α7 nAChR we show that positive allosteric modulation measured in two different ways is greatly attenuated when the temperature is raised to near physiological levels. Furthermore, PNU120596 largely removes the strong inward rectification usually exhibited by α7 nAChR-mediated responses.

Profiles of SUMO and ubiquitin conjugation in an Alzheimer's disease model.

Alzheimer's disease (AD) is a major cause of disability in the elderly. The formation of senile plaques and neurofibrillary tangles are the main hallmarks of the disorder, whereas synaptic loss best correlates to the progressive cognitive decline. Interestingly, some of the proteins involved in these pathophysiological processes have been reported to be subject to posttranslational modification by ubiquitin and/or the small ubiquitin-like modifier (SUMO). Here we investigated global changes in protein SUMOylation and ubiquitination in vivo in a model of AD. We used Tg2576 transgenic mice, which overexpress a mutated human amyloid precursor protein (APP) gene implicated in familial AD. As expected, APP protein levels were dramatically increased in the hippocampus, cortex and cerebellum of Tg2576 mice. A significant increase in the global level of ubiquitinated proteins was observed in the hippocampus of Tg2576 mice. Significant or close to significant changes in individual bands of SUMO-1 or SUMO-2/3 conjugation were apparent in all brain regions investigated, although global levels were unaltered between wild-type and transgenic mice. Levels of SUMO-specific conjugating and deconjugating enzymes, UBC9 and SENP-1 were also unaltered in any of the brain regions analysed. Surprisingly, given the well-documented loss of synaptic function, total levels of the excitatory AMPA and kainate receptors were unaffected in the Tg2576 mice. These results suggest that alterations in SUMO substrate conjugation may occur and that global posttranslational modifications by ubiquitin may play an important role in the mechanisms underlying AD.

Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer's disease.

Transgenic mice that overproduce beta-amyloid (Aß) peptides can exhibit central nervous system network hyperactivity. Patch clamp measurements from CA1 pyramidal cells of PSAPP and wild type mice were employed to investigate if altered intrinsic excitability could contribute to such network hyperfunction. At approximately 10 months, when PSAPP mice have a substantial central nervous system Aß load, resting potential and input resistance were genotype-independent. However, PSAPP mice exhibited a substantially more prominent action potential (AP) burst close to the onset of weak depolarizing current stimuli. The spike afterdepolarization (ADP) was also larger in PSAPP mice. The rate of rise, width and height of APs were reduced in PSAPP animals; AP threshold was unaltered. Voltage-clamp recordings from nucleated macropatches revealed that somatic Na(+) current density was depressed by approximately 50% in PSAPP mice. K(+) current density was unaltered. All genotype-related differences were absent in PSAPP mice aged 5-7 weeks which lack a substantial Aß load. We conclude that intrinsic neuronal hyperexcitability and changes to AP waveforms may contribute to neurophysiological deficits that arise as a consequence of Aß accumulation.