Kelly T. Dineley

The A‐Type Potassium Channel Kv4.2 Is a Substrate for the Mitogen‐Activated Protein Kinase ERK

Abstract: The mitogen‐activated protein kinase ERK has recentlybecome a focus of studies of synaptic plasticity and learning and memory. Dueto the prominent role of potassium channels in regulating the electricalproperties of membranes, modulation of these channels by ERK could play animportant role in mediating learning‐related synaptic plasticity in the CNS.Kv4.2 is a Shal‐type potassium channel that passes an A‐type current and islocalized to dendrites and cell bodies in the hippocampus. The sequence ofKv4.2 contains several consensus sites for ERK phosphorylation. In the presentstudies, we tested the hypothesis that Kv4.2 is an ERK substrate. Wedetermined that the Kv4.2 C‐terminal cytoplasmic domain is an effective ERK2substrate, and that it is phosphorylated at three sites: Thr602,Thr607, and Ser616. We used this information to developantibodies that recognize Kv4.2 phosphorylated by ERK2. One of ourphospho‐site‐selective antibodies was generated using a triply phosphorylatedpeptide as the antigen. We determined that this antibody recognizesERK‐phosphorylated Kv4.2 in COS‐7 cells transfected with Kv4.2 and nativeERK‐phosphorylated Kv4.2 in the rat hippocampus. These observations indicatethat Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERKmay regulate potassium‐channel function by direct phosphorylation of thepore‐forming α subunit.

Presenilin 1 familial Alzheimer's disease mutation leads to defective associative learning and impaired adult neurogenesis.

Alzheimers disease is a learning and memory disorder pathologically characterized by the deposition of beta-amyloid plaques and loss of neurons and synapses in affected areas of the brain. Mutations in presenilin 1 (PS1) lead to the most aggressive form of familial Alzheimers disease (FAD), and are associated with accelerated plaque deposition. However, since the function of PS1 is pleiotropic, we reasoned that the FAD mutations may alter multiple PS1-mediated pathways, and the combination of which may account for the early onset nature of the disease phenotype. Using the PS1M146V knockin mice in which the M146V mutation was incorporated into the endogenous mouse PS1 gene, we report here that the FAD mutation results in impaired hippocampus-dependent associative learning, as measured by a contextual fear conditioning paradigm, at 3 months of age. This is correlated with reduced adult neurogenesis in the dentate gyrus. However, short-term and long-term synaptic plasticity in both area CA1 and dentate gyrus are not affected. Our results suggest that impaired adult neurogenesis may contribute to the memory deficit associated with FAD.

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimers disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain.

Loss of α7 Nicotinic Receptors Enhances β-Amyloid Oligomer Accumulation, Exacerbating Early-Stage Cognitive Decline and Septohippocampal Pathology in a Mouse Model of Alzheimer's Disease

Early Alzheimers disease (AD) is marked by cholinergic hypofunction, neuronal marker loss, and decreased nicotinic acetylcholine receptor (nAChR) density from the cortex and hippocampus. α7 nAChRs expressed on cholinergic projection neurons and target regions have been implicated in neuroprotection against β-amyloid (Aβ) toxicity and maintenance of the septohippocampal phenotype. We tested the role that α7 nAChRs perform in the etiology of early AD by genetically deleting the α7 nAChR subunit from the Tg2576 mouse model for AD and assessing animals for cognitive function and septohippocampal integrity. Thus, Tg2576 mice transgenic for mutant human amyloid precursor protein (APP) were crossed with α7 nAChR knock-out mice (A7KO) to render an animal with elevated Aβ in the absence of α7 nAChRs (A7KO–APP). We found that learning and memory deficits seen in 5-month-old APP mice are more severe in the A7KO–APP animals. Analyses of animals in early-stage preplaque cognitive decline revealed signs of neurodegeneration in A7KO–APP hippocampus as well as loss of cholinergic functionality in the basal forebrain and hippocampus. These changes occurred concomitant with the appearance of a dodecameric oligomer of Aβ that was absent from all other genotypic groups, generating the hypothesis that increased soluble oligomeric Aβ may underlie additional impairment of A7KO–APP cognitive function. Thus, α7 nAChRs in a mouse model for early-stage AD appear to serve a neuroprotective role through maintenance of the septohippocampal cholinergic phenotype and preservation of hippocampal integrity possibly through influences on Aβ accumulation and oligomerization.

Passive Immunization with Tau Oligomer Monoclonal Antibody Reverses Tauopathy Phenotypes without Affecting Hyperphosphorylated Neurofibrillary Tangles

Recent findings suggest that tau oligomers, which form before neurofibrillary tangles (NFTs), are the true neurotoxic tau entities in neurodegenerative tauopathies, including Alzheimers disease (AD). Studies in animal models of tauopathy suggest that tau oligomers play a key role in eliciting behavioral and cognitive impairments. Here, we used a novel tau oligomer-specific monoclonal antibody (TOMA) for passive immunization in mice expressing mutant human tau. A single dose of TOMA administered either intravenously or intracerebroventricularly was sufficient to reverse both locomotor and memory deficits in a mouse model of tauopathy for 60 d, coincident with rapid reduction of tau oligomers but not phosphorylated NFTs or monomeric tau. Our data demonstrate that antibody protection is mediated by extracellular and rapid peripheral clearance. These findings provide the first direct evidence in support of a critical role for tau oligomers in disease progression and validate tau oligomers as a target for the treatment of AD and other neurodegenerative tauopathies.

Intermediate-and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition

The Tg2576 transgenic mouse is an extensively characterized animal model for Alzheimers disease (AD). Similar to AD, these mice suffer from progressive decline in several forms of declarative memory including contextual fear conditioning and novel object recognition (NOR). Recent work on this and other AD animal models suggests that initial cognitive deficits are due to synaptic dysfunction that, with the correct intervention, are fully treatable. We recently reported that acute calcineurin (CaN) inhibition with FK506 ameliorates one form of declarative memory (contextual fear conditioning) impairment in 5 months old Tg2576. This study tested whether acute CaN inhibition rescues deficits in an additional form of declarative memory, spontaneous object recognition, by employing the NOR paradigm. Furthermore, we determined whether FK506 rescue of NOR deficits depends on the retention interval employed and therefore is restricted to short-term, intermediate-term, or long-term memory (STM, ITM or LTM, respectively). In object recognition, Tg2576 are unimpaired when NOR is tested as a STM task and CaN inhibition with FK506 does not influence NOR STM performance in Tg2576 or WT mice. Tg2576 were impaired in NOR compared to WT mice when a 4 or 24h retention interval was employed to model ITM and LTM, respectively. Acute CaN inhibition prior to and during the training session reversed these deficits in Tg2576 mice with no effect on WT performance. Our findings demonstrate that aberrant CaN activity mediates object recognition deficits in 5 months old Tg2576 when NOR is employed as a test for ITM and LTM. In human AD, CaN inhibition may lead the way for therapeutics to improve declarative memory performance as demonstrated in a mouse model for AD.

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer’s disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain‐enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin‐dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5‐mediated) is increased in Alzheimer’s disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin‐1 (PS1)M146VKI; Thy1.2‐amyloid precursor protein (APP)swe; Thy1.2tauP301L] that develops AD‐like plaques and tangles, as well as the double (PS1M146VKI; Thy1.2‐APPswe) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid β peptide levels (Tg2576 and APP/amyloid β‐binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over‐expression and/or processing defect.

Research update: Alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease

Aberrant amyloid-β peptide (Aβ) accumulation along with altered expression and function of nicotinic acetylcholine receptors (nAChRs) stand prominently in the etiology of Alzheimers disease (AD). Since the discovery that Aβ is bound to α7 nAChRs under many experimental settings, including post-mortem AD brain, much effort has been expended to understand the implications of this interaction in the disease milieu. This research update will review the current literature on the α7 nAChR-Aβ interaction in vitro and in vivo, the functional consequences of this interaction from sub-cellular to cognitive levels, and discuss the implications these relationships might have for AD therapies.

Acute inhibition of calcineurin restores associative learning and memory in Tg2576 APP transgenic mice

Misfolded amyloid beta peptide (Abeta) is a pathological hallmark of Alzheimers disease (AD), a neurodegenerative illness characterized by cognitive deficits and neuronal loss. Transgenic mouse models of Abeta over-production indicate that Abeta-induced cognitive deficits occur in the absence of overt neuronal death, suggesting that while extensive neuronal death may be associated with later stages of the human disease, subtle physiological changes may underlie initial cognitive deficits. Therefore, identifying signaling elements involved in those Abeta-induced cognitive impairments that occur prior to loss of neurons may reveal new potential pharmacological targets. Here, we report that the enzymatic activity of calcineurin, a key protein phosphatase involved in phosphorylation-dependent kinase activity crucial for synaptic plasticity and memory function, is upregulated in the CNS of the Tg2576 animal model for Abeta over-production. Furthermore, acute treatment of Tg2576 mice with the calcineurin inhibitor FK506 (10mg/kg i.p.) improves memory function. These results indicate that calcineurin may mediate some of the cognitive effects of excess Abeta such that inhibition of calcineurin shall be further explored as a potential treatment to reverse cognitive impairments in AD.

MAPK recruitment by β-amyloid in organotypic hippocampal slice cultures depends on physical state and exposure time

Elevated β‐amyloid is thought to trigger the onset of Alzheimers disease. Alzheimers disease is marked by progressive loss of cognitive function, an early symptom of which is episodic memory deficits. Impairment of episodic memory is linked to hippocampal pathology. We investigated the signal transduction consequences of exposure to nanomolar to low micromolar concentrations of aggregate forms of β‐amyloid in the hippocampus. We found that, in addition to activation of ERK MAPK and its downstream target ribosomal S6 kinase in hippocampal slice cultures following acute exposure to oligomeric β‐amyloid1−42, ERK activation also requires phosphoinositide‐3 kinase activity. These effects were contingent on the α7 subtype of nicotinic acetylcholine receptor. Hippocampal slice cultures treated acutely with oligomeric β‐amyloid1−42 did not exhibit JNK MAPK activation; however, chronic exposure to oligomers or high molecular weight aggregates of β‐amyloid1−42 led to JNK MAPK activation coincident with ERK MAPK down‐regulation. In contrast to the effects of acute application of oligomeric β‐amyloid1−42, nicotine activated ERK MAPK via α7 nicotinic acetylcholine receptors utilizing protein kinase A as an intermediate. In conclusion, we found that both the physical state and duration of exposure to β‐amyloid are determinants of MAPK recruitment in hippocampus. We also found that nicotine and β‐amyloid activate ERK MAPK via α7 nicotinic acetylcholine receptors but use distinct intermediate kinases. These data indicate the existence of differential coupling of α7 to downstream targets depending on the type of ligand that leads to receptor activation.