Accumulation of high molecular weight kininogen in the brains of Alzheimer's disease patients may affect microglial function by altering phagocytosis and lysosomal cathepsin activity.

Increased activation of the contact system protein high molecular weight kininogen (HK) has been shown in plasma and cerebrospinal fluid of Alzheimer's disease (AD) patients, but its potential role in the brain has not been explored. We assessed HK levels in brain tissue from 20 AD patients and controls and modeled the effects of HK on microglia-like cells in culture. We show increased levels of HK in the hippocampus of AD patients, which colocalized with amyloid beta (Aß) deposits and activated microglia. Treatment of microglia with HK led to cell clustering and elevated levels of phagocytosed Aß. We demonstrate that microglia internalize HK and traffic it to lysosomes, which is accompanied by reduced activity of lysosomal cathepsins L and S. Our results suggest that HK accumulation in the AD hippocampus may alter microglial uptake and degradation of Aß fibrils, possibly contributing to microglial dysfunction in AD.

Transport rate of EAAT2 is regulated by amino acid located at the interface between the scaffolding and substrate transport domains.

Excitatory Amino Acid Transporters (EAATs) are plasma membrane proteins responsible for maintenance of low extracellular concentrations of glutamate in the CNS. Dysfunction in their activity is implicated in various neurological disorders. Glutamate transport by EAATs occurs through the movement of the central transport domain relative to the scaffold domain in the EAAT membrane protein. Previous studies suggested that residues located within the interface of these two domains in EAAT2, the main subtype of glutamate transporter in the brain, are involved in regulating transport rates. We used mutagenesis, structure-function relationship, surface protein expression and electrophysiology studies, in transfected COS-7 cells and oocytes, to examine residue glycine at position 298, which is located within this interface. Mutation G298A results in increased transport rate without changes in surface expression, suggesting a more hydrophobic and larger alanine results in facilitated transport movement. The increased transport rate does not involve changes in sodium affinity. Electrophysiological currents show that G298A increase both transport and anion currents, suggesting faster transitions through the transport cycle. This work identifies a region critically involved in setting the glutamate transport rate.

A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP ß-CTFs, Not Aß.

Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aß production but similar effects on APP ß C-terminal fragments (ß-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of ß-CTFs, not Aß, and could be rescued by pharmacological modulation of ß-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that ß-CTF may be critical in AD pathogenesis.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1-/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions.

Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling.

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to ß-Cell Mitochondrial Dysfunction in Type 2 Diabetes.

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic ß-cell dysfunction. Reduced mitochondrial function is thought to be central to ß-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in ß-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D ß-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D ß-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their ß-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of ß-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D ß-cells where we had little knowledge of which changes cause ß-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to ß-cell mitochondrial dysfunction in T2D.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Homozygous mutation of STXBP5L explains an autosomal recessive infantile-onset neurodegenerative disorder.

We report siblings of consanguineous parents with an infantile-onset neurodegenerative disorder manifesting a predominant sensorimotor axonal neuropathy, optic atrophy and cognitive deficit. We used homozygosity mapping to identify an ∼12-Mbp interval identical by descent (IBD) between the affected individuals on chromosome 3q13.13-21.1 with an LOD score of 2.31. We combined family-based whole-exome and whole-genome sequencing of parents and affected siblings and, after filtering of likely non-pathogenic variants, identified a unique missense variant in syntaxin-binding protein 5-like (STXBP5L c.3127G>A, p.Val1043Ile [CCDS43137.1]) in the IBD interval. Considering other modes of inheritance, we also found compound heterozygous variants in FMNL3 (c.114G>C, p.Phe38Leu and c.1372T>G, p.Ile458Leu [CCDS44874.1]) located on chromosome 12. STXBP5L (or Tomosyn-2) is expressed in the central and peripheral nervous system and is known to inhibit neurotransmitter release through inhibition of the formation of the SNARE complexes between synaptic vesicles and the plasma membrane. FMNL3 is expressed more widely and is a formin family protein that is involved in the regulation of cell morphology and cytoskeletal organization. The STXBP5L p.Val1043Ile variant enhanced inhibition of exocytosis in comparison with wild-type (WT) STXBP5L. Furthermore, WT STXBP5L, but not variant STXBP5L, promoted axonal outgrowth in manipulated mouse primary hippocampal neurons. However, the FMNL3 p.Phe38Leu and p.Ile458Leu variants showed minimal effects in these cells. Collectively, our clinical, genetic and molecular data suggest that the IBD variant in STXBP5L is the likely cause of the disorder.

Identification of different types of spinal afferent nerve endings that encode noxious and innocuous stimuli in the large intestine using a novel anterograde tracing technique.

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Huntingtin-associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells.

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1(-/-) and HAP1(+/+) littermate mice. Numbers of Ca(2+)-dependent and Ca(2+)-independent full fusion events in HAP1(-/-) cells are significantly decreased compared with those in HAP1(+/+) cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca(2+) entry. Whereas release per full fusion event is unchanged in HAP1(-/-) cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1(-/-) cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.

Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells.

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca(2+) imaging, immunocytochemistry and 3D modelling. Ca(2+) enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca(2+) channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.

Copper modulates the large dense core vesicle secretory pathway in PC12 cells.

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

Repetitive common peroneal nerve stimulation increases ankle dorsiflexor motor evoked potentials in incomplete spinal cord lesions.

Plasticity of corticospinal tract (CST) activity likely plays a key role in motor function recovery after central nervous system (CNS) lesions. In non-injured adults, 30 min of repetitive common peroneal nerve stimulation (rCPnS) increases CST excitability by 40-50% and the effect persists for at least 30 min. The present study evaluated with transcranial magnetic stimulation (TMS) the changes in CST excitability after 30 min of rCPnS in people with foot drop due to incomplete SCI. Suprathreshold rCPnS (25 Hz, alternating 1 s on 1 s off stimulation cycle) was given for two 15-min periods, while the subject sat at rest with ankle and knee joints fixed. Before, between, and after the periods of stimulation, the tibialis anterior (TA) motor evoked potentials (MEPs) to TMS were measured at a TMS intensity that originally produced a half-maximum MEP (typically 10-20% above threshold) while the sitting subject provided 25-30% maximum voluntary TA contraction. In 10 subjects with SCI, the peak-to-peak TA MEP increased by 14 ± 3% after rCPnS and the peak increase (+21 ± 7%) occurred 15 min after the cessation of rCPnS. The TA H-reflex, measured in separate experiments in 7 subjects, did not increase after rCPnS. The results indicate that rCPnS can increase CST excitability for the TA in people with incomplete SCI, although its effects appear smaller and shorter lasting than those found in non-injured control subjects. Such short-term plasticity in the CST excitability induced by rCPnS may contribute to long-term therapeutic effects of functional electrical stimulation previously reported in people with CNS lesions.

Living donor liver transplantation--adult donor outcomes: a systematic review.

The objective of this study was to evaluate the safety and efficacy of adult-to-adult living donor liver transplantation, specifically donor outcomes. A systematic review, with searches of the literature up to January 2004, was undertaken. Two hundred and fourteen studies provided information on donor outcomes. The majority of these were case series studies, although there were also studies comparing living donor liver transplantation with deceased donor liver transplantation. Both underreporting and duplicate reporting is likely to have occurred, and so caution is required in interpretation of these results. Overall reported donor mortality was 12 to 13 in about 6,000 procedures (0.2%) (117 studies). Mortality for right lobe donors to adult recipients is estimated to be 2 to 8 out of 3,800 (0.23 to 0.5%). The donor morbidity rate ranged from 0% to 100% with a median of 16% (131 studies). Biliary complications and infections were the most commonly reported donor morbidities. Nearly all donors had returned to normal function by 3 to 6 months (18 studies). In conclusion, there are small, but real, risks for living liver donors. Due to the short history of adult-to-adult living donor liver transplantation, the long-term risks for donors are unknown.

Zinc inhibits human ClC-1 muscle chloride channel by interacting with its common gating mechanism.

Transition metals block the muscle Cl- channel ClC-1, which belongs to a large family of double-barreled Cl- channels and transporters. In the Torpedo Cl- channel ClC-0, Zn2+ block is closely related to the common gating mechanism that opens and closes both pores of the channel simultaneously, and the mutation C212S, which locks the common gate open, also eliminates the block. In ClC-1, however, previous results suggested that Zn2+ block is independent of gating, and that the cysteine residues involved in Zn2+ binding are in different positions to those that confer Zn2+ sensitivity on ClC-0. In this work, we show that Zn2+ block of ClC-1 is faster at hyperpolarized potentials where the channel is more likely to be in the closed state. Mutation C277S, equivalent to C212S in ClC-0, which locks the common gate in ClC-1 open, virtually eliminates Zn2+ block. A mutation, V321A, which reduces open probability of the common gate, facilitated Zn2+ block. These results demonstrate that Zn2+ block is state dependent, acting on the common gate. The extent of the block, however, is not a simple function of the open probability of the common gate. The Q10 of approximately 13 of the time course of Zn2+ block, which is significantly higher than the Q10 of common gating transitions in WT ClC-1, suggests that Zn2+ binds to a very high temperature-dependent low-probability closed substate of the common gate, which has not yet been characterized in this channel.

Involvement of helices at the dimer interface in ClC-1 common gating.

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.