Anna G. Orr

Reversing EphB2 depletion rescues cognitive functions in Alzheimer model

Amyloid-β oligomers may cause cognitive deficits in Alzheimer’s disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer’s disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-β-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer’s disease.

Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive deficits in an Alzheimer’s disease model

In light of the rising prevalence of Alzheimer’s disease (AD), new strategies to prevent, halt, and reverse this condition are needed urgently. Perturbations of brain network activity are observed in AD patients and in conditions that increase the risk of developing AD, suggesting that aberrant network activity might contribute to AD-related cognitive decline. Human amyloid precursor protein (hAPP) transgenic mice simulate key aspects of AD, including pathologically elevated levels of amyloid-β peptides in brain, aberrant neural network activity, remodeling of hippocampal circuits, synaptic deficits, and behavioral abnormalities. Whether these alterations are linked in a causal chain remains unknown. To explore whether hAPP/amyloid-β–induced aberrant network activity contributes to synaptic and cognitive deficits, we treated hAPP mice with different antiepileptic drugs. Among the drugs tested, only levetiracetam (LEV) effectively reduced abnormal spike activity detected by electroencephalography. Chronic treatment with LEV also reversed hippocampal remodeling, behavioral abnormalities, synaptic dysfunction, and deficits in learning and memory in hAPP mice. Our findings support the hypothesis that aberrant network activity contributes causally to synaptic and cognitive deficits in hAPP mice. LEV might also help ameliorate related abnormalities in people who have or are at risk for AD.

Adenosine A2A receptor mediates microglial process retraction

Cell motility drives many biological processes, including immune responses and embryonic development. In the brain, microglia are immune cells that survey and scavenge brain tissue using elaborate and motile cell processes. The motility of these processes is guided by the local release of chemoattractants. However, most microglial processes retract during prolonged brain injury or disease. This hallmark of brain inflammation remains unexplained. We identified a molecular pathway in mouse and human microglia that converted ATP-driven process extension into process retraction during inflammation. This chemotactic reversal was driven by upregulation of the A2A adenosine receptor coincident with P2Y12 downregulation. Thus, A2A receptor stimulation by adenosine, a breakdown product of extracellular ATP, caused activated microglia to assume their characteristic amoeboid morphology during brain inflammation. Our results indicate that purine nucleotides provide an opportunity for context-dependent shifts in receptor signaling. Thus, we reveal an unexpected chemotactic switch that generates a hallmark feature of CNS inflammation.

Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

Astrocytes express a variety of G protein–coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimers disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.

A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors

NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and have a role in neurological disorders. In this article, we identify a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone) enhances receptor responses two-fold with an EC(50) of 3 μM by increasing channel opening frequency without altering mean open time or EC(50) values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and on the linker between the N-terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1.

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC50 64 nm) and Fura-2/AM fluorescence (195 ± 6.7% above base line, EC50 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the increase in phospho-ERK1/2 levels were diminished in PAR1-/- astrocytes and were blocked by 1 μm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca2+]i signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-d-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on α-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or γ-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 ± 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 μm; 1.02 ± 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca2+]i in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.

Differential regulation of microglial motility by ATP/ADP and adenosine.

Microglia are motile immune-competent cells of the central nervous system. They assume a highly branched morphology and monitor the brain parenchyma under physiological conditions. In the presence of injury, microglia retract their branching processes, migrate to the site of injury, and help clear cellular debris by phagocytosis. This response appears to be mediated in part by ATP released at the site of injury. Here, we review the evidence for the involvement of ATP and the purinergic P2Y(12) receptor in microglial process extension and chemoattraction to injury. We subsequently discuss recent findings regarding a switch of this chemotactic response to ATP in activated, or proinflammatory, microglia. Specifically, in LPS-activated microglia, ATP induces process retraction and repulsive migration, effects opposite to those seen in unstimulated cells. These repulsive effects of ATP are mediated by the G(s)-coupled adenosine A(2A) receptor and depend on the breakdown of ATP to adenosine. Thus, ATP-induced repulsion by activated microglia involves upregulation of the adenosine A(2A) receptor and coincident downregulation of the P2Y(12) receptor. The roles of the A(2A) receptor in brain pathologies such as Parkinsons disease and ischemia are also examined. We propose that the effects of A(2A) receptor antagonists on brain injury may be in part due to the inactivation of A(2A) on activated microglia.

Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

N-Methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

Istradefylline reduces memory deficits in aging mice with amyloid pathology

Adenosine A2A receptors are putative therapeutic targets for neurological disorders. The adenosine A2A receptor antagonist istradefylline is approved in Japan for Parkinsons disease and is being tested in clinical trials for this condition elsewhere. A2A receptors on neurons and astrocytes may contribute to Alzheimers disease (AD) by impairing memory. However, it is not known whether istradefylline enhances cognitive function in aging animals with AD-like amyloid plaque pathology. Here, we show that elevated levels of Aβ, C-terminal fragments of the amyloid precursor protein (APP), or amyloid plaques, but not overexpression of APP per se, increase astrocytic A2A receptor levels in the hippocampus and neocortex of aging mice. Moreover, in amyloid plaque-bearing mice, low-dose istradefylline treatment enhanced spatial memory and habituation, supporting the conclusion that, within a well-defined dose range, A2A receptor blockers might help counteract memory problems in patients with Alzheimers disease.