R. Jane Rylett

β-Arrestins regulate a Ral-GDS–Ral effector pathway that mediates cytoskeletal reorganization

β-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 β-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 β-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, β-arrestin–Ral-GDS protein complexes dissociate and Ral-GDS translocates with β-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 β-arrestin–Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, β-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

Role of neurotrophins in cholinergic-neurone function in the adult and aged CNS

Cholinergic neurones in the CNS undergo complex changes during normal aging. In recent years, considerable attention has focussed on the neurotrophins and, in particular, nerve growth factor, as potential maintenance factor for cholinergic-neurone function, and as therapeutic agents for use in a variety of neurodegenerative disorders including Alzheimers disease. While brain cholinergic neurones from the neonate to the aged respond to nerve growth factor with enhanced expression of transmitter phenotype, there appears to be an age-related, region-specific decline in responsiveness. This age-related decrement in neurotrophin action might play a role in dysfunction of cholinergic neurones, and cognitive loss, and could limit the use of these factors as therapeutic agents.

Exogenous Nerve Growth Factor Increases the Activity of High-Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats

Abstract: The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium‐sensitive high‐affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats, in 24‐month‐old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4‐month‐old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4‐month‐old animals, NGF treatment at 1.2 μg/ day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle‐treated 4‐month‐old animals; vehicle treatment had no effect on the activity of either marker. In 24‐month‐old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24‐month‐old control animals. Synaptosomal HACU in 24‐month‐old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values. Treatment of 24‐month‐old rats with NGF for 4 weeks produced further increases in ChAT activity and HACU in striatum and frontal cortex; in hippocampus, the 4‐week treatment did increase HACU, but ChAT activity was not further increased over that produced by 2 weeks of NGF administration. These data indicate an age‐related differential regulation of ChAT activity and HACU between specific brain areas. In addition, NGF stimulated phenotypic expression of these cholinergic markers in both young adult and aged rat brain; there might be an age‐associated differential sensitivity of particular brain regions to exogenous NGF.

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys150–Cys169 and Cys196–His215 consisting of CX2-3CX11CX2C and CX4–6CX10–16-CX2(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn2+ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

ABSTRACT The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

The “ins” and “outs” of the high‐affinity choline transporter CHT1

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high‐affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin‐coated pits in a mechanism dependent on a dileucine‐like motif present in the carboxyl‐terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimers disease.

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrPC) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrPC extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrPC engagement induces an increase in intracellular Ca2+ levels. This effect was not detected in PrPC-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrPC. Using a best candidate approach to test for potential channels involved in Ca2+ influx evoked by STI1-PrPC, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrPC-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrPC and allowed reconstitution of signaling by PrPC-STI1 interaction. These results indicate that STI1 can interact with the PrPC·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Amyloid‐beta oligomers increase the localization of prion protein at the cell surface

J. Neurochem. (2011) 117, 538–553.

An Evaluation of Irreversible Inhibition of Synaptosomal High-Affinity Choline Transport by Choline Mustard Aziridinium Ion

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium‐dependent, high‐affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low‐affinity transport of choline. The IC50 value for high‐affinity transport was 0.94 μM, compared to 29 μM for low‐affinity uptake. The inhibitory action of choline mustard aziridinium ion on high‐affinity transport of choline was graded with respect to time; a 12‐fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high‐affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high‐affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10−2 s−1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high‐affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high‐affinity choline carriers.

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl‐CoA in cholin‐ergic neurons. The present study examined conditions for modulation of kinase‐mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti‐human choline acetyltransferase antibody to immunoprecipi‐tate cytosolic and membrane‐associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium‐free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane‐associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane‐associated choline acetyltransferase, although the specific activity of the membrane‐associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.