Synaptic Elimination in Neurological Disorders.

Synapses are well known as the main structures responsible for transmitting information through the release and recognition of neurotransmitters by pre- and post-synaptic neurons. These structures are widely formed and eliminated throughout the whole lifespan via processes termed synaptogenesis and synaptic pruning, respectively. Whilst the first process is needed for ensuring proper connectivity between brain regions and also with the periphery, the second phenomenon is important for their refinement by eliminating weaker and unnecessary synapses and, at the same time, maintaining and favoring the stronger ones, thus ensuring proper synaptic transmission. It is well-known that synaptic elimination is modulated by neuronal activity. However, only recently the role of the classical complement cascade in promoting this phenomenon has been demonstrated. Specifically, microglial cells recognize activated complement component 3 (C3) bound to synapses targeted for elimination, triggering their engulfment. As this is a highly relevant process for adequate neuronal functioning, disruptions or exacerbations in synaptic pruning could lead to severe circuitry alterations that could underlie neuropathological alterations typical of neurological and neuropsychiatric disorders. In this review, we focus on discussing the possible involvement of excessive synaptic elimination in Alzheimer's disease, as it has already been reported dendritic spine loss in post-synaptic neurons, increased association of complement proteins with its synapses and, hence, augmented microglia-mediated pruning in animal models of this disorder. In addition, we briefly discuss how this phenomenon could be related to other neurological disorders, including multiple sclerosis and schizophrenia.

Identification of new phosphorylation sites of CD23 in B-cells of patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common lymphoproliferative disorder in adults. Patients with B-CLL strongly express the CD23 - C type of lectin (low affinity IgE receptor, Fc epsilon RII), which is linked to B cell activation and proliferation. Phosphorylation in lymphocytes is tightly associated with regulation of protein activities, functional regulation and cell signaling, and may thus affect initiation and/or progression of the disease. Here we report changes in the phosphorylation of CD23 on threonine (pThr314) and two serine residues (pSer254, pSer265) in B lymphocytes of B-CLL patients, using a flow cytometry approach. The majority of tested patients with active forms of B-CLL presented a notable overexpression of CD23 along with pThr314, pSer254, and pSer265 CD23 phosphorylation positivity. Moreover, we have experimentally stimulated the CD23 phosphorylations in a subset of peripheral blood lymphocytes of healthy controls by phorbol-12-myristate-13-acetate treatment. This affects the activation of competent phosphorylation mediating kinases, resulting in the enhanced phosphorylation pattern. Together, these data confirm that CD23 protein is phosphorylated in B cells of B-CLL patients, report the identification of new CD23 phosphorylation sites, and suggest a possible role(s) of such phosphorylations in the activation of CD23 during the process of lymphocytic activation in B-CLL.

N-type Ca2+ channels are affected by full-length mutant huntingtin expression in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin (htt) protein. In addition to facilitating neurodegeneration, mutant htt is implicated in HD-related alterations of neurotransmission. Previous data showed that htt can modulate N-type voltage-gated Ca2+ channels (Cav2.2), which are essential for presynaptic neurotransmitter release. Thus, to elucidate the mechanism underlying mutant htt-mediated alterations in neurotransmission, we investigated how Cav2.2 is affected by full-length mutant htt expression in a mouse model of HD (BACHD). Our data indicate that young BACHD mice exhibit increased striatal glutamate release, which is reduced to wild type levels following Cav2.2 block. Cav2.2 Ca2+ current-density and plasma membrane expression are increased in BACHD mice, which could account for increased glutamate release. Moreover, mutant htt affects the interaction between Cav2.2 and 2 major channel regulators, namely syntaxin 1A and Gßγ protein. Notably, 12-month old BACHD mice exhibit decreased Cav2.2 cell surface expression and glutamate release, suggesting that Cav2.2 alterations vary according to disease stage.

82-kDa choline acetyltransferase is in nuclei of cholinergic neurons in human CNS and altered in aging and Alzheimer disease.

Cholinergic neurons express choline acetyltransferase (ChAT) which synthesizes acetylcholine. We show here for the first time that primate-specific 82-kDa ChAT is expressed in nuclei of cholinergic neurons in human brain and spinal cord; isoform-specific antibodies were used to compare localization patterns and temporal expression of the more abundant 69-kDa ChAT and primate-specific 82-kDa ChAT in necropsy tissues. The 82-kDa ChAT co-localizes with 69-kDa ChAT in well-characterized cholinergic areas, but is also found in the claustrum which does not contain 69-kDa ChAT. Cholinergic neuron function changes with increasing age and are targeted in neurodegenerative diseases such as AD, thus we compared expression and subcellular localization of 69- and 82-kDa ChAT in necropsy brain samples from control subjects of varying ages and from Alzheimer disease (AD) subjects. The 82-kDa ChAT protein was expressed in cholinergic neurons in brain from birth until the eighth decade of life and in AD, but the subcellular staining pattern and proportion of neurons that were immunopositive changed with increasing age and in AD.

Surface-entropy reduction used in the crystallization of human choline acetyltransferase.

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

A model for dynamic regulation of choline acetyltransferase by phosphorylation.

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) and is a phenotypic marker for cholinergic neurons. Cholinergic neurons in brain are involved in cognitive function, attentional processing and motor control, and decreased ChAT activity is found in several neurological disorders including Alzheimer's disease. Dysregulation of ChAT and cholinergic communication is also associated with some spontaneous point-mutations in ChAT that alter its substrate binding kinetics, or by disruption of signaling pathways that could regulate protein kinases for which ChAT is a substrate. It has been identified recently that the catalytic activity and subcellular distribution of ChAT, and its interaction with other cellular proteins, can be modified by phosphorylation of the enzyme by protein kinase-C and Ca2+/calmodulin-dependent protein kinase II; these kinases appear also to mediate some of the effects of beta-amyloid peptides on cholinergic neuron functions, including the effects on ChAT. This review outlines a new model for the regulation of cholinergic transmission at the level of the presynaptic terminal that is mediated by hierarchically-regulated, multi-site phosphorylation of ChAT.

Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity.

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.

Functional regulation of choline acetyltransferase by phosphorylation.

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.

Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42.

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.

Beta-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization.

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.